[108] Hara S, Mukae H, Sakamoto N, Ishimoto H, Amenomori M, Fujita H, et al. Plectasin has antibacterial activity and no affect on cell viability or IL-8 production. Biochemical

[109] Risso A, Zanetti M, Gennaro R. Cytotoxicity and apoptosis mediated by two peptides

[110] Lau YE, Bowdish DME, Cosseau C, Hancock REW, Davidson DJ. Apoptosis of airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology.

[111] Holle L, Song W, Holle E, Wei Y, Wagner T, Yu X. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo.

[112] Rawlins MD. Cutting the cost of drug development? Nat Rev Drug Discov.

[113] Merrifield R. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am

[114] Bray BL. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat

[115] Gesell J, Zasloff M, Opella SJ. Two-dimensional 1H NMR experiments show that the 23-residue magainin antibiotic peptide is an α-helix in dodecylphosphocholine micelles,

and cathelicidin (LL-37). Journal of Leukocyte Biology. 2001;69(5):691-7.

The Journal of Immunology. 2005;174(10):6257-65.

INFECTION AND IMMUNITY. 2005;73(1):583-91.

Discov. [10.1038/nrd1470]. 2004;3(8):711-6.

and Chemotherapy. 2009;53(4):1581-5.

2006;34(4):399-409.

Int J Oncol. 2003;22(1):93-8.

Chem Soc. 1963;85:2149-54.

[10.1038/nrd1347]. 2004;3(4):360-4.

science and market. Drug Discovery Today. 2010;15(1–2):40-56.

endocytosis. Journal of Biological Chemistry. 2004;279(17):17951-6.

and Biophysical Research Communications. 2008;374(4):709-13.

of innate immunity. Cellular Immunology. 1998;189(2):107-15.

Rev Drug Discov. [10.1038/nrd1133]. 2003;2(7):587-93.

[131] Andreu D, Rivas L. Animal antimicrobial peptides: An overview. Peptide Science. 1998;47(6):415-33.

**Chapter 7**

© 2012 Takahashi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**The Study of MASPs Knockout Mice**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50500

**1. Introduction**

knockout mice.

**2. Complement system**

Minoru Takahashi, Daisuke Iwaki, Yuichi Endo and Teizo Fujita

Plasma proteases, e.g. thrombin, factor X, complement factor D and C1s are responsible for the physiological activities, such as coagulation and complement system. These proteases circulate as their zymogen in blood and are activated by various stimulations. In this chapter, we focus on a family of plasma serine proteases, called MASP (MBL/ficolinassociated serine protease) that can activate the complement. Three distinct MASP, MASP-1, MASP-2 and MASP-3 have been identified in many species of vertebrates. Although the contribution of MASP-2 in activation of complement was well defined, the substrates for MASP-1 and MASP-3 were still obscure. We have generated MASP-1- and MASP-3-deficient mice (*Masp1/3*-/-) to verify roles of MASP-1 and MASP-3 proteases *in vivo*. One major finding is that MASP-1, considered being a lectin pathway component—also acts as a pro-factor D (Df) convertase, the initiator of the alternative pathway. Our results emphasize a unique feature of MASP-1, participating two complement pathways. We also generated MASP-2 deficient mice. In here, we would like to summarize the results obtained from these

The complement system is an important part of the innate immune system, mediating several major effector functions, such as directly killing pathogens, promoting phagocytosis, and clearance of immune complexes and apoptotic cells and modulating adaptive immune responses, as describing in some excellent reviews (Ricklin, et al., 2010) (Fujita, et al., 2004) (Carroll, 2004). On the other hand, inappropriate activation of complement affects the pathogenesis of inflammatory diseases (Holers, 2003). Therefore, well-understanding of the mechanisms of its activation is very important. More than 30 proteins in plasma consist of the complement system. The most abundant protein among them is the third component (C3). Once the complement system is activated, a chain of reactions involving restricted proteolysis and assembly occurs, resulting in cleavage of C3 into C3b and C3a. The cascade

and reproduction in any medium, provided the original work is properly cited.

## **The Study of MASPs Knockout Mice**

Minoru Takahashi, Daisuke Iwaki, Yuichi Endo and Teizo Fujita

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50500

## **1. Introduction**

164 Binding Protein

1998;47(6):415-33.

[131] Andreu D, Rivas L. Animal antimicrobial peptides: An overview. Peptide Science.

[132] Bellemare A, Vernoux N, Morin S, Gagne S, Bourbonnais Y. Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against

[133] Baranger K, Zani M-L, Chandenier J, Dallet-Choisy S, Moreau T. The antibacterial and antifungal properties of trappin-2 (pre-elafin) do not depend on its protease inhibitory

[134] Hong RW, Shchepetov M, Weiser JN, Axelsen PH. Transcriptional profile of the *Escherichia coli* response to the antimicrobial insect peptide cecropin A. Antimicrobial

[135] Chan YR, Gallo RL. PR-39, a syndecan-inducing antimicrobial peptide, binds and

[136] Woodley J. Enzymatic barriers for GI peptide and protein delivery. Crit Rev Ther Drug

affects p130Cas. Journal of Biological Chemistry. 1998;273(44):28978-85.

*Pseudomonas aeruginosa*. BMC Microbiol. 2010;10(1):253.

function. FEBS Journal. 2008;275(9):2008-20.

Agents and Chemotherapy. 2003;47(1):1-6.

Carrier Syst. 1994;11(2-3):61-95.

Plasma proteases, e.g. thrombin, factor X, complement factor D and C1s are responsible for the physiological activities, such as coagulation and complement system. These proteases circulate as their zymogen in blood and are activated by various stimulations. In this chapter, we focus on a family of plasma serine proteases, called MASP (MBL/ficolinassociated serine protease) that can activate the complement. Three distinct MASP, MASP-1, MASP-2 and MASP-3 have been identified in many species of vertebrates. Although the contribution of MASP-2 in activation of complement was well defined, the substrates for MASP-1 and MASP-3 were still obscure. We have generated MASP-1- and MASP-3-deficient mice (*Masp1/3*-/-) to verify roles of MASP-1 and MASP-3 proteases *in vivo*. One major finding is that MASP-1, considered being a lectin pathway component—also acts as a pro-factor D (Df) convertase, the initiator of the alternative pathway. Our results emphasize a unique feature of MASP-1, participating two complement pathways. We also generated MASP-2 deficient mice. In here, we would like to summarize the results obtained from these knockout mice.

## **2. Complement system**

The complement system is an important part of the innate immune system, mediating several major effector functions, such as directly killing pathogens, promoting phagocytosis, and clearance of immune complexes and apoptotic cells and modulating adaptive immune responses, as describing in some excellent reviews (Ricklin, et al., 2010) (Fujita, et al., 2004) (Carroll, 2004). On the other hand, inappropriate activation of complement affects the pathogenesis of inflammatory diseases (Holers, 2003). Therefore, well-understanding of the mechanisms of its activation is very important. More than 30 proteins in plasma consist of the complement system. The most abundant protein among them is the third component (C3). Once the complement system is activated, a chain of reactions involving restricted proteolysis and assembly occurs, resulting in cleavage of C3 into C3b and C3a. The cascade

© 2012 Takahashi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

up to C3 cleavage is called the activation pathway. There are three distinct activation pathways of the complement cascade; the classical, alternative, and lectin pathways, that all converge on factor C3 and lead to activation of complement effector functions as above (Walport, 2001a)(Fig. 1).

The Study of MASPs Knockout Mice 167

**2.2. The alternative pathway**

(Brouwer, et al., 2006).

et al., 2010).

**3. MBL-associated serine proteases**

**3.1. Three MASP proteins were associated with MBL and ficolins**

MASP is homologue of C1r and C1s of the classical pathway, sharing the well-described domains structure in the order from N-terminus, CUB-I, EGF, CUB-II, CCP-I, CCP-II and SP (Sato, et al., 1994). The CUB (C1r/C1s, embryonic sea Urchin protein [Uefg], and Bonemorphogenetic protein 1 [Bmp1]) domain is approximately 110 aa, predicting a molecular structure of an antiparallel beta-barrel similar to those in immunoglobulins (Bork and Beckmann, 1993). The EGF (epidermal growth factor-like) domain of approximately 50 aa is also found in many proteins and is known to mediate protein-protein interactions via calcium ion. The N-terminal three domains consisting of CUB-I, EGF and CUB-II of the MASP are responsible for dimerization and for the calcium-dependent binding to MBL and ficolins(Feinberg, et al., 2003). The two contiguous CCPs (complement control protein) of

**2.3. The lectin pathway**

In the alternative pathway, spontaneous hydrolysis of C3, designated C3(H2O) results in triggering complement activation with complement factor B, making another C3 convertase, C3(H2O)Bb on foreign cells (Muller-Eberhard and Gotze, 1972, Pangburn, et al., 1981). This leads to the cleavage of factor B by factor D, giving rise to an active enzyme complex with the fragment Bb as the enzyme. The alternative pathway does not involve specific recognition molecules and also functions to amplify C3 activation (amplification loop)

Activation of the lectin pathway is similar with that of the classical pathway (Degn, et al., 2010). The lectin pathway is initiated by some serum lectins binding to pathogen-associated molecular patterns, mainly carbohydrate structures present on bacterial, fungal, or viral pathogens. In 1978, a serum lectin, designated mannose-binding lectin (MBL), which recognizes carbohydrates such as mannose and N-acetylglucosamine was first isolated from rabbit liver (Kawasaki, et al., 1978). MBL acts as the pattern recognition molecule, which recognizes sugar chains on some foreign pathogens. MBL is also found to have an avidity of complement activation (Ikeda, et al., 1987) (Holmskov, et al., 2003) (Turner, 1996). It has been thought that MBL activates complement by C1r2C1s2 protease complex that consists of classical pathway (Ohta, et al., 1990). However, in 1992, Matsushita and Fujita found a new plasma serum protease designated MBL-associated serine protease (MASP) that binds MBL (Matsushita and Fujita, 1992) (Matsushita, et al., 1998). Recent studies identified ficolins that are also plasma proteins with binding activity for carbohydrates to associate with MASP and to activate complement (Matsushita, et al., 2000, Matsushita, et al., 2001) (Cseh, et al., 2002). Ficolins has a collagen-like domain and a fibrinogen-like domain. Furthermore, CL-K1 (Keshi, et al., 2006) was also identified as a collectin that associates with MASP(Hansen,

**Figure 1.** Activation pathways for complement system.

In the mammalian complement system, the pivotal molecule circulating C3 is cleaved into C3a and C3b by two different C3 convertases, C4b2a and C3bBb. C4b2a is generated by the classical and lectin pathway and C3bBb is generated by the alternative pathway.

### **2.1. The classical pathway**

The classic pathway is initiated by recognition of the first C1 binding to a variety of targets, most prominently immune complexes (Walport, 2001a) (Walport, 2001b). C1 consists of a single C1q molecule associated with dimers of C1r and C1s(Lepow, et al., 1963). C1r and C1s are plasma serine proteases, normally existing in an inactive proenzyme form. The conformational exchange of C1q by binding to immune complexes results in the activation of C1r. C1r is thought to be cleaved in some autocatalytic manner and once C1r molecule is activated, it activate C1s, which in turn cleaves C4 and then C2(Arlaud, et al., 2002). The C4 cleavage products are C4a and C4b. The latter molecule may be bound to non-self surfaces on pathogens and is bound to C2 to form the classical pathway C3 convertase.

## **2.2. The alternative pathway**

166 Binding Protein

(Walport, 2001a)(Fig. 1).

**Figure 1.** Activation pathways for complement system.

**2.1. The classical pathway**

pathway C3 convertase.

up to C3 cleavage is called the activation pathway. There are three distinct activation pathways of the complement cascade; the classical, alternative, and lectin pathways, that all converge on factor C3 and lead to activation of complement effector functions as above

In the mammalian complement system, the pivotal molecule circulating C3 is cleaved into C3a and C3b by two different C3 convertases, C4b2a and C3bBb. C4b2a is generated by the

The classic pathway is initiated by recognition of the first C1 binding to a variety of targets, most prominently immune complexes (Walport, 2001a) (Walport, 2001b). C1 consists of a single C1q molecule associated with dimers of C1r and C1s(Lepow, et al., 1963). C1r and C1s are plasma serine proteases, normally existing in an inactive proenzyme form. The conformational exchange of C1q by binding to immune complexes results in the activation of C1r. C1r is thought to be cleaved in some autocatalytic manner and once C1r molecule is activated, it activate C1s, which in turn cleaves C4 and then C2(Arlaud, et al., 2002). The C4 cleavage products are C4a and C4b. The latter molecule may be bound to non-self surfaces on pathogens and is bound to C2 to form the classical

classical and lectin pathway and C3bBb is generated by the alternative pathway.

In the alternative pathway, spontaneous hydrolysis of C3, designated C3(H2O) results in triggering complement activation with complement factor B, making another C3 convertase, C3(H2O)Bb on foreign cells (Muller-Eberhard and Gotze, 1972, Pangburn, et al., 1981). This leads to the cleavage of factor B by factor D, giving rise to an active enzyme complex with the fragment Bb as the enzyme. The alternative pathway does not involve specific recognition molecules and also functions to amplify C3 activation (amplification loop) (Brouwer, et al., 2006).

## **2.3. The lectin pathway**

Activation of the lectin pathway is similar with that of the classical pathway (Degn, et al., 2010). The lectin pathway is initiated by some serum lectins binding to pathogen-associated molecular patterns, mainly carbohydrate structures present on bacterial, fungal, or viral pathogens. In 1978, a serum lectin, designated mannose-binding lectin (MBL), which recognizes carbohydrates such as mannose and N-acetylglucosamine was first isolated from rabbit liver (Kawasaki, et al., 1978). MBL acts as the pattern recognition molecule, which recognizes sugar chains on some foreign pathogens. MBL is also found to have an avidity of complement activation (Ikeda, et al., 1987) (Holmskov, et al., 2003) (Turner, 1996). It has been thought that MBL activates complement by C1r2C1s2 protease complex that consists of classical pathway (Ohta, et al., 1990). However, in 1992, Matsushita and Fujita found a new plasma serum protease designated MBL-associated serine protease (MASP) that binds MBL (Matsushita and Fujita, 1992) (Matsushita, et al., 1998). Recent studies identified ficolins that are also plasma proteins with binding activity for carbohydrates to associate with MASP and to activate complement (Matsushita, et al., 2000, Matsushita, et al., 2001) (Cseh, et al., 2002). Ficolins has a collagen-like domain and a fibrinogen-like domain. Furthermore, CL-K1 (Keshi, et al., 2006) was also identified as a collectin that associates with MASP(Hansen, et al., 2010).

## **3. MBL-associated serine proteases**

### **3.1. Three MASP proteins were associated with MBL and ficolins**

MASP is homologue of C1r and C1s of the classical pathway, sharing the well-described domains structure in the order from N-terminus, CUB-I, EGF, CUB-II, CCP-I, CCP-II and SP (Sato, et al., 1994). The CUB (C1r/C1s, embryonic sea Urchin protein [Uefg], and Bonemorphogenetic protein 1 [Bmp1]) domain is approximately 110 aa, predicting a molecular structure of an antiparallel beta-barrel similar to those in immunoglobulins (Bork and Beckmann, 1993). The EGF (epidermal growth factor-like) domain of approximately 50 aa is also found in many proteins and is known to mediate protein-protein interactions via calcium ion. The N-terminal three domains consisting of CUB-I, EGF and CUB-II of the MASP are responsible for dimerization and for the calcium-dependent binding to MBL and ficolins(Feinberg, et al., 2003). The two contiguous CCPs (complement control protein) of MASP, especially the second CCP domain, have been implicated in the binding of macromolecular substrates. The CCP domains of around 60 aa are found in a number of complement factors and other proteins(Chou and Heinrikson, 1997). The SP (serine protease) domain is the catalytically active unit of the proteases and defines them as part of the S1A family of chymotrypsin-like proteases (Yousef, et al., 2004). MASP is able to cleave C4 and C2 to generate a C3 convertase, C4b2a. Recent studies isolated two additional MASPs in human MBL complex (Thiel, et al., 1997) (Dahl, et al., 2001). These newly identified MASPs are called as MASP-2 and MASP-3 and the former one is MASP-1(Schwaeble, et al., 2002).

The Study of MASPs Knockout Mice 169

**Figure 2.** Schematic representation of *MASP1* gene

MAP44 by alternative splicing.

MASP-2, lacking catalytic domain.

*3.3.2. MASP2*

MASP1 gene consists of 18 exons, encoding three gene products, MASP-1, MASP-3 and

MASP2 gene is located on human chromosome 1p36.3-p36.2(Stover, et al., 1999a). And mouse Masp2 gene is located on chromosome 4(Lawson and Reid, 2000). It was shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed sMAP/MAp19 by alternative splicing (Takahashi, et al., 1999) (Stover, et al., 1999b). sMAP/MAp19 consist of only CUB-I and EGF-like domain of

## **3.2. Substrates for MASP**

It is apparently defined that MASP-2 cleaves C4 that is similar with C1s in the classical pathway (Vorup-Jensen, et al., 1998) (Ambrus, et al., 2003). However, substrates for MASP-1 and MASP-3 are still obscure. Several candidates were demonstrated by recent studies as shown in Table 1.

**Table 1.** Substrates for MASPs

#### **3.3.** *MASP* **genes**

#### *3.3.1. MASP1*

*MASP1* is located on chromosome 3q27-q28 in human and chromosome 16 (B2-B3) in mouse (Takada, et al., 1995). Three gene products, MASP-1, MASP-3 and MAP44 are encoded from this gene by alternative splicing. MAP44 is a truncated protein of MASP-1/3 and lacks serine protease domain (Degn, et al., 2009) (Skjoedt, et al., 2010). MAP44 is thought to be a regulatory factor, attenuating activation of the lectin pathway. *MASP1* gene has a unique structure. A single exon, encoding whole MASP-3 light-chain and the six sprit exons, encoding MASP-1 are tandem located (Dahl, et al., 2001). Therefore, MASP-1 and MASP-3 consist of a common heavy-chain and the distinct light-chain.

**Figure 2.** Schematic representation of *MASP1* gene

MASP1 gene consists of 18 exons, encoding three gene products, MASP-1, MASP-3 and MAP44 by alternative splicing.

#### *3.3.2. MASP2*

168 Binding Protein

1(Schwaeble, et al., 2002).

shown in Table 1.

C1r C1s

C1s C4, C2

**3.3.** *MASP* **genes**

*3.3.1. MASP1*

**Table 1.** Substrates for MASPs

**3.2. Substrates for MASP**

MASP Substrates (reference)

MASP, especially the second CCP domain, have been implicated in the binding of macromolecular substrates. The CCP domains of around 60 aa are found in a number of complement factors and other proteins(Chou and Heinrikson, 1997). The SP (serine protease) domain is the catalytically active unit of the proteases and defines them as part of the S1A family of chymotrypsin-like proteases (Yousef, et al., 2004). MASP is able to cleave C4 and C2 to generate a C3 convertase, C4b2a. Recent studies isolated two additional MASPs in human MBL complex (Thiel, et al., 1997) (Dahl, et al., 2001). These newly identified MASPs are called as MASP-2 and MASP-3 and the former one is MASP-

It is apparently defined that MASP-2 cleaves C4 that is similar with C1s in the classical pathway (Vorup-Jensen, et al., 1998) (Ambrus, et al., 2003). However, substrates for MASP-1 and MASP-3 are still obscure. Several candidates were demonstrated by recent studies as

MASP-1 C3 (Matsushita and Fujita, 1995), C2, fibrinogen, Factor XIII(Hajela, et al., 2002), PAR4

*MASP1* is located on chromosome 3q27-q28 in human and chromosome 16 (B2-B3) in mouse (Takada, et al., 1995). Three gene products, MASP-1, MASP-3 and MAP44 are encoded from this gene by alternative splicing. MAP44 is a truncated protein of MASP-1/3 and lacks serine protease domain (Degn, et al., 2009) (Skjoedt, et al., 2010). MAP44 is thought to be a regulatory factor, attenuating activation of the lectin pathway. *MASP1* gene has a unique structure. A single exon, encoding whole MASP-3 light-chain and the six sprit exons, encoding MASP-1 are tandem located (Dahl, et al., 2001). Therefore, MASP-1 and MASP-3

(Megyeri, et al., 2009), Df(Takahashi, et al., 2010)

MASP-3 IGFBP-5 (Cortesio and Jiang, 2006), Df(Iwaki, et al., 2011)

consist of a common heavy-chain and the distinct light-chain.

MASP-2 C4, C2(Ambrus, et al., 2003), prothrombin

MASP2 gene is located on human chromosome 1p36.3-p36.2(Stover, et al., 1999a). And mouse Masp2 gene is located on chromosome 4(Lawson and Reid, 2000). It was shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed sMAP/MAp19 by alternative splicing (Takahashi, et al., 1999) (Stover, et al., 1999b). sMAP/MAp19 consist of only CUB-I and EGF-like domain of MASP-2, lacking catalytic domain.

The Study of MASPs Knockout Mice 171

*4.1.1. Masp1/3-/- shows the abnormality of the lectin pathway activation*

*4.1.2. Masp1/3-/- shows the abnormality of the alternative pathway activation*

arthritis and ischemia/reperfusion injury (Thurman and Holers, 2006).

**Figure 4.** MASP-1 and/or MASP-3 involve in activating a zymogen of complement factor D

observed.

Complement factor D (Df) is synthesized as a zymogen (Pro-Df) from adipocytes. In serum of *Masp1/3*-/-, Pro-Df that has an activation peptide (QPRGR) at N-terminal of Df was

MASP-2 activation (Takahashi, et al., 2008).

Serum from *Masp1/3*-/- shows the abnormality of both C4 and C3 activation on mannan and it is restored by adding recombinant MASP-1. This result supported that MASP-1 contributes the lectin pathway through C4 activation. Furthermore, MASP-2 activation is delayed in *Masp1/3*-/- to be compared with that of wild type. This result reveals that MASP-1 and/or MASP-3 may involve in the lectin pathway activation through the acceleration of

Further study noticed us that not only lectin pathway but also alternative pathway is abnormal in *Masp1/3*-/-. We found that complement factor D (Df) circulates as a zymogen in *Masp1/3*-/- (Takahashi, et al., 2010). Df was known to be active-form, but not a zymogen in circulation (Lesavre and Muller-Eberhard, 1978). However, it has become evident that most proteases in blood are secreted as zymogen. Df was thought to be an exception. We also found that Df is synthesized as zymogen from adipocytes (Takahashi, et al., 2010) (Fig. 4). This result supports the general consensus for Df. Interestingly, increasing evidence suggests that the alternative pathway is involved in human disease, such as inflammatory

**Figure 3.** Schematic representation of *MASP2* gene

*MASP2* gene consists of 12 exons, encoding two gene products, MASP-2 and sMAP/Map19 by alternative splicing.

## **4. Studies for the Masp-knockout mice**

**Table 2.** Masps knockout mice

## **4.1. MASP-1 and MASP-3-deficient mice (***Masp1/3***-/-)**

To investigate the role of MASP-1 in complement activation, we planned to disrupt the second exon of Masp1 gene by a conventional gene targeting (Takahashi, et al., 2008). When this project was proceeding, MASP-3 was identified (Dahl, et al., 2001). Surprisingly, both gene products were produced from *MASP1* gene. Since the targeted second exon is at upstream of both transcripts, it was predicted that MASP-3 is also absent in this knockout mice. It was confirmed that not only MASP-1, but also MASP-3, is absent in *MASP1/3*–/– mice (Takahashi, et al., 2008).

## *4.1.1. Masp1/3-/- shows the abnormality of the lectin pathway activation*

170 Binding Protein

**Figure 3.** Schematic representation of *MASP2* gene

**4. Studies for the Masp-knockout mice**

by alternative splicing.

**Table 2.** Masps knockout mice

(Takahashi, et al., 2008).

*MASP2* gene consists of 12 exons, encoding two gene products, MASP-2 and sMAP/Map19

To investigate the role of MASP-1 in complement activation, we planned to disrupt the second exon of Masp1 gene by a conventional gene targeting (Takahashi, et al., 2008). When this project was proceeding, MASP-3 was identified (Dahl, et al., 2001). Surprisingly, both gene products were produced from *MASP1* gene. Since the targeted second exon is at upstream of both transcripts, it was predicted that MASP-3 is also absent in this knockout mice. It was confirmed that not only MASP-1, but also MASP-3, is absent in *MASP1/3*–/– mice

Knockout mice Mutant allele chromosome Targeted exon

Masp1/3-/- Masp1tm1Tefu 16 2

sMAP/Masp2-/- Masp2tm1Tefu 4 5

**4.1. MASP-1 and MASP-3-deficient mice (***Masp1/3***-/-)**

Masp2-/- Masp2tm1Wjsc 4 11 &12

Serum from *Masp1/3*-/- shows the abnormality of both C4 and C3 activation on mannan and it is restored by adding recombinant MASP-1. This result supported that MASP-1 contributes the lectin pathway through C4 activation. Furthermore, MASP-2 activation is delayed in *Masp1/3*-/- to be compared with that of wild type. This result reveals that MASP-1 and/or MASP-3 may involve in the lectin pathway activation through the acceleration of MASP-2 activation (Takahashi, et al., 2008).

## *4.1.2. Masp1/3-/- shows the abnormality of the alternative pathway activation*

Further study noticed us that not only lectin pathway but also alternative pathway is abnormal in *Masp1/3*-/-. We found that complement factor D (Df) circulates as a zymogen in *Masp1/3*-/- (Takahashi, et al., 2010). Df was known to be active-form, but not a zymogen in circulation (Lesavre and Muller-Eberhard, 1978). However, it has become evident that most proteases in blood are secreted as zymogen. Df was thought to be an exception. We also found that Df is synthesized as zymogen from adipocytes (Takahashi, et al., 2010) (Fig. 4). This result supports the general consensus for Df. Interestingly, increasing evidence suggests that the alternative pathway is involved in human disease, such as inflammatory arthritis and ischemia/reperfusion injury (Thurman and Holers, 2006).

**Figure 4.** MASP-1 and/or MASP-3 involve in activating a zymogen of complement factor D

Complement factor D (Df) is synthesized as a zymogen (Pro-Df) from adipocytes. In serum of *Masp1/3*-/-, Pro-Df that has an activation peptide (QPRGR) at N-terminal of Df was observed.

#### *4.1.3. MASP-1 and/or MASP-3 involve the fat metabolism through Df activation*

It was also reported that the alternative pathway is involved in fat metabolism in adipose tissue (Paglialunga, et al., 2008). Recent studies have indicated that acylation-stimulating protein (ASP), which is identical to C3adesArg, stimulates fat storage in adipocytes (Yasruel, et al., 1991) (Maslowska, et al., 1997). ASP is a derivative of complement C3; thus, *C3*-/- mice are lean owing to ASP deficiency. Furthermore, plasma ASP levels are decreased in Bfdeficient and Df-deficient mice, indicating that the alternative pathway stimulates production of ASP. We found that *Masp1/3*-/- mice are also apparently lean (Takahashi, et al., 2008), strongly indicating a contribution of MASP-1 to fat metabolism via alternative pathway. We measured the plasma concentration of leptin and TNF-alpha (Fig. 5). Leptin plays a critical role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Leptin appears to be a hormone secreted by adipocyte (Zhang, et al., 1994). It was shown that level of leptin in *Masp1/3*-/- significantly decrease. Furthermore, we determined that one of inflammatory factor, TNF-alpha increases in *Masp1/3*-/-. As shown in Fig. 4, adipose tissues in Masp1/3-/- apparently show atrophy. Therefore, fat metabolisms in Masp1/3-/- adipose tissue might be reduced.

The Study of MASPs Knockout Mice 173

**Figure 6.** H&E staining of mouse adipose tissue

*4.1.5. MASP3 mutation causes 3MC syndrome*

the alternative pathway.

*4.1.4. Masp1/3-/- is resistant to AP-mediated joint damage*

Banda et al. demonstrated that *Masp1/3*−/− mice are highly resistant to CAIA as evidenced by a significant decrease in the histological scores as compared with WT mice (Banda, et al., 2010). Recent studies supported that the alternative pathway is both necessary and sufficient to induce disease in murine collagen Ab-induced arthritis (CAIA) (Banda, et al., 2006) (Banda, et al., 2007). This model mouse confirmed that *Masp1/3*-/- shows the abnormality of

3MC syndrome (Malpuech-Michels-Mingarelli-Carnevale syndrome) are four rare autosomal recessive disorders (Carnevale, et al., 1989) (Mingarelli, et al., 1996) (Malpuech, et al., 1983) (Michels, et al., 1978). This syndrome shows facial dysmorphic traits. Recent

**Figure 5.** Serum leptin and TNF-alpha level in *Masp1/3*-/-

**Figure 6.** H&E staining of mouse adipose tissue

*4.1.3. MASP-1 and/or MASP-3 involve the fat metabolism through Df activation*

Therefore, fat metabolisms in Masp1/3-/- adipose tissue might be reduced.

**Figure 5.** Serum leptin and TNF-alpha level in *Masp1/3*-/-

It was also reported that the alternative pathway is involved in fat metabolism in adipose tissue (Paglialunga, et al., 2008). Recent studies have indicated that acylation-stimulating protein (ASP), which is identical to C3adesArg, stimulates fat storage in adipocytes (Yasruel, et al., 1991) (Maslowska, et al., 1997). ASP is a derivative of complement C3; thus, *C3*-/- mice are lean owing to ASP deficiency. Furthermore, plasma ASP levels are decreased in Bfdeficient and Df-deficient mice, indicating that the alternative pathway stimulates production of ASP. We found that *Masp1/3*-/- mice are also apparently lean (Takahashi, et al., 2008), strongly indicating a contribution of MASP-1 to fat metabolism via alternative pathway. We measured the plasma concentration of leptin and TNF-alpha (Fig. 5). Leptin plays a critical role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Leptin appears to be a hormone secreted by adipocyte (Zhang, et al., 1994). It was shown that level of leptin in *Masp1/3*-/- significantly decrease. Furthermore, we determined that one of inflammatory factor, TNF-alpha increases in *Masp1/3*-/-. As shown in Fig. 4, adipose tissues in Masp1/3-/- apparently show atrophy.

### *4.1.4. Masp1/3-/- is resistant to AP-mediated joint damage*

Banda et al. demonstrated that *Masp1/3*−/− mice are highly resistant to CAIA as evidenced by a significant decrease in the histological scores as compared with WT mice (Banda, et al., 2010). Recent studies supported that the alternative pathway is both necessary and sufficient to induce disease in murine collagen Ab-induced arthritis (CAIA) (Banda, et al., 2006) (Banda, et al., 2007). This model mouse confirmed that *Masp1/3*-/- shows the abnormality of the alternative pathway.

#### *4.1.5.  This syndrome shows facial dysmorphic traits. Recent

observations for families, including patients who suffer from 3MC syndrome found the genetic mutations in *CL-K1* and *MASP1* genes (Rooryck, et al., 2011). This result was very interesting, since a possibility was raised that MASP-3 may be responsible to not only complement system, but also development system with a recognition molecule, CL-K1. In 2010, Sirmaci, et al. also found the mutations of *MASP1* gene in two Turkish familes (Sirmaci, et al., 2010). Preliminary results was obtained that *Masp1/3* knockout mice have some developmental disorders (publication preparing).

The Study of MASPs Knockout Mice 175

Recently MASP1 mutants were identified in human patients, suffering from 3MC syndrome. However, the mechanisms how MASP-1 and/or MASP-3 contribute the facial development are still unclear. Further study using Masp1/3-/- would provide a powerful tool to resolve

Ambrus, G., Gal, P., Kojima, M., Szilagyi, K., Balczer, J., Antal, J., Graf, L., Laich, A., Moffatt, B. E., Schwaeble, W., Sim, R. B. and Zavodszky, P. (2003). Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on

Arlaud, G. J., Gaboriaud, C., Thielens, N. M., Budayova-Spano, M., Rossi, V. and Fontecilla-Camps, J. C. (2002). Structural biology of the C1 complex of complement unveils the mechanisms of its activation and proteolytic activity. *Molecular Immunology,* Vol. 39, No.

Banda, N. K., Takahashi, K., Wood, A. K., Holers, V. M. and Arend, W. P. (2007). Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway. *J Immunol,* Vol. 179, No. 6, pp. 4101-4109 Banda, N. K., Takahashi, M., Levitt, B., Glogowska, M., Nicholas, J., Takahashi, K., Stahl, G. L., Fujita, T., Arend, W. P. and Holers, V. M. (2010). Essential role of complement mannose-binding lectin-associated serine proteases-1/3 in the murine collagen antibody-induced model of inflammatory arthritis. *J Immunol,* Vol. 185, No. 9, pp. 5598-

Banda, Nirmal K., Thurman, Joshua M., Kraus, Damian, Wood, Allyson, Carroll, Michael C., Arend, William P. and Holers, V. Michael. (2006). Alternative Complement Pathway Activation Is Essential for Inflammation and Joint Destruction in the Passive Transfer Model of Collagen-Induced Arthritis. *The Journal of Immunology,* Vol. 177, No. 3, pp.

Bork, P. and Beckmann, G. (1993). The CUB domain. A widespread module in developmentally regulated proteins. *Journal of molecular biology,* Vol. 231, No. 2, pp. 539-

Brouwer, N., Dolman, K. M., van Zwieten, R., Nieuwenhuys, E., Hart, M., Aarden, L. A., Roos, D. and Kuijpers, T. W. (2006). Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop. *Mol Immunol,*

Carnevale, F., Krajewska, G., Fischetto, R., Greco, M. G. and Bonvino, A. (1989). Ptosis of eyelids, strabismus, diastasis recti, hip defect, cryptorchidism, and developmental delay

Minoru Takahashi, Daisuke Iwaki, Yuichi Endo and Teizo Fujita

*Department of Immunology, Fukushima Medical University School of Medicine, Japan*

recombinant catalytic fragments. *J Immunol,* Vol. 170, No. 3, pp. 1374-1382

this problem.

**Author details**

**6. References**

7-8, pp. 383-394

5606

1904-1912

Vol. 43, No. 13, pp. 2051-2060

in two sibs. *Am J Med Genet,* Vol. 33, No. 2, pp. 186-189

545

## **4.2. sMAP and MASP-2-deficient mice (***sMAP/Masp2***-/-)**

To clarify the role of sMAP/Map19, we also generated another mutant mice, disrupting the fifth exon of *MASP2* gene by replacement with *neor* -gene (Iwaki, et al., 2006). Since this targeted region is the sMAP/Map19-specific exon, it was predicted that MASP-2 might be intact in this knockout mice. However, MASP-2 was not detected in their serum. Therefore, these mutant mice were named as *sMAP*/*Masp2*-/-.

When recombinant sMAP and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2. On the other hand, the addition of recombinant sMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP plays a regulatory role in the activation of the lectin pathway(Iwaki, et al., 2006).

## **4.3. MASP-2-deficient mice (***Masp2***-/-)**

An England group generated MASP-2-deficient mice(Schwaeble, et al., 2011). This strain lacks exon 11 and 12 of *Masp2* gene, encoding the C-terminal part of the CCPII and the SP domains. In their knockout mice, sMAP/Map19 is predicted to be intact. In vitro analysis of *MASP2*−/− plasma showed a total absence of lectin pathway-dependent C4 cleavage on mannan- and zymosan-coated surfaces. They investigated whether MASP-2 affect the inflammatory process using a model of myocardial ischemia reperfusion injury (MIRI). It was observed that *MASP2*-/- was protected from MIRI.

## **5. Conclusion**

Here, we focus on analyses of three strains for *Masps* knockout mice, *Masp1/3*-/-, *sMAP/Masp2*-/ and *Masp2*-/-. All strains show that activation of lectin pathway is deficient. We also detected the abnormality of the alternative pathway in *Masp1/3*-/-. But *Masp2*-deficient phenotype does not affect the activity. MASPs are associated with MBL, ficolins and CL-K1. MBL-deficient mice were generated and analysed (Takahashi, et al., 2002, Shi, et al., 2004). Surprisingly, MBLnull mice show the comparable level of the alternative pathway with that of wild type. If MASP-1 and/or MASP-3 involve the activation of alternative pathway with MBL, MBL-null mice must be affected. Other recognition molecules, ficolin or CL-K1 might be involved in this phenomenon. This problem should be resolved in future study.

Recently MASP1 mutants were identified in human patients, suffering from 3MC syndrome. However, the mechanisms how MASP-1 and/or MASP-3 contribute the facial development are still unclear. Further study using Masp1/3-/- would provide a powerful tool to resolve this problem.

## **Author details**

174 Binding Protein

observations for families, including patients who suffer from 3MC syndrome found the genetic mutations in *CL-K1* and *MASP1* genes (Rooryck, et al., 2011). This result was very interesting, since a possibility was raised that MASP-3 may be responsible to not only complement system, but also development system with a recognition molecule, CL-K1. In 2010, Sirmaci, et al. also found the mutations of *MASP1* gene in two Turkish familes (Sirmaci, et al., 2010). Preliminary results was obtained that *Masp1/3* knockout mice have

To clarify the role of sMAP/Map19, we also generated another mutant mice, disrupting the

targeted region is the sMAP/Map19-specific exon, it was predicted that MASP-2 might be intact in this knockout mice. However, MASP-2 was not detected in their serum. Therefore,

When recombinant sMAP and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2. On the other hand, the addition of recombinant sMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP

An England group generated MASP-2-deficient mice(Schwaeble, et al., 2011). This strain lacks exon 11 and 12 of *Masp2* gene, encoding the C-terminal part of the CCPII and the SP domains. In their knockout mice, sMAP/Map19 is predicted to be intact. In vitro analysis of *MASP2*−/− plasma showed a total absence of lectin pathway-dependent C4 cleavage on mannan- and zymosan-coated surfaces. They investigated whether MASP-2 affect the inflammatory process using a model of myocardial ischemia reperfusion injury (MIRI). It

Here, we focus on analyses of three strains for *Masps* knockout mice, *Masp1/3*-/-, *sMAP/Masp2*-/ and *Masp2*-/-. All strains show that activation of lectin pathway is deficient. We also detected the abnormality of the alternative pathway in *Masp1/3*-/-. But *Masp2*-deficient phenotype does not affect the activity. MASPs are associated with MBL, ficolins and CL-K1. MBL-deficient mice were generated and analysed (Takahashi, et al., 2002, Shi, et al., 2004). Surprisingly, MBLnull mice show the comparable level of the alternative pathway with that of wild type. If MASP-1 and/or MASP-3 involve the activation of alternative pathway with MBL, MBL-null mice must be affected. Other recognition molecules, ficolin or CL-K1 might be involved in this

plays a regulatory role in the activation of the lectin pathway(Iwaki, et al., 2006).

some developmental disorders (publication preparing).

fifth exon of *MASP2* gene by replacement with *neor*

these mutant mice were named as *sMAP*/*Masp2*-/-.

**4.3. MASP-2-deficient mice (***Masp2***-/-)**

**5. Conclusion**

was observed that *MASP2*-/- was protected from MIRI.

phenomenon. This problem should be resolved in future study.

**4.2. sMAP and MASP-2-deficient mice (***sMAP/Masp2***-/-)**

Minoru Takahashi, Daisuke Iwaki, Yuichi Endo and Teizo Fujita *Department of Immunology, Fukushima Medical University School of Medicine, Japan*

#### **6. References**

Carroll, M. C. (2004). The complement system in regulation of adaptive immunity. *Nature immunology,* Vol. 5, No. 10, pp. 981-986

The Study of MASPs Knockout Mice 177

Iwaki, D., Kanno, K., Takahashi, M., Endo, Y., Lynch, N. J., Schwaeble, W. J., Matsushita, M., Okabe, M. and Fujita, T. (2006). Small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway. *J Immunol,* Vol. 177, No. 12, pp.

Iwaki, Daisuke, Kanno, Kazuko, Takahashi, Minoru, Endo, Yuichi, Matsushita, Misao and Fujita, Teizo. (2011). The Role of Mannose-Binding Lectin-Associated Serine Protease-3 in Activation of the Alternative Complement Pathway. *The Journal of Immunology,* Vol.

Kawasaki, T., Etoh, R. and Yamashina, I. (1978). Isolation and characterization of a mannanbinding protein from rabbit liver. *Biochem Biophys Res Commun,* Vol. 81, No. 3, pp. 1018-

Keshi, H., Sakamoto, T., Kawai, T., Ohtani, K., Katoh, T., Jang, S. J., Motomura, W., Yoshizaki, T., Fukuda, M., Koyama, S., Fukuzawa, J., Fukuoh, A., Yoshida, I., Suzuki, Y. and Wakamiya, N. (2006). Identification and characterization of a novel human collectin

Lawson, P. R. and Reid, K. B. (2000). A novel PCR-based technique using expressed sequence tags and gene homology for murine genetic mapping: localization of the

Lepow, I. H., Naff, G. B., Todd, E. W., Pensky, J. and Hinz, C. F. (1963). Chromatographic resolution of the first component of human complement into three activities. *The Journal*

Lesavre, P. H. and Muller-Eberhard, H. J. (1978). Mechanism of action of factor D of the

Malpuech, G., Demeocq, F., Palcoux, J. B. and Vanlieferinghen, P. (1983). A previously undescribed autosomal recessive multiple congenital anomalies/mental retardation (MCA/MR) syndrome with growth failure, lip/palate cleft(s), and urogenital anomalies.

Maslowska, M., Sniderman, A. D., Germinario, R. and Cianflone, K. (1997). ASP stimulates glucose transport in cultured human adipocytes. *Int J Obes Relat Metab Disord,* Vol. 21,

Matsushita, M., Endo, Y. and Fujita, T. (1998). MASP1 (MBL-associated serine protease 1).

Matsushita, M., Endo, Y. and Fujita, T. (2000). Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease. *J Immunol,* Vol. 164,

Matsushita, M., Endo, Y., Hamasaki, N. and Fujita, T. (2001). Activation of the lectin complement pathway by ficolins. *Int Immunopharmacol,* Vol. 1, No. 3, pp. 359-363 Matsushita, M. and Fujita, T. (1992). Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. *J Exp*

Matsushita, M. and Fujita, T. (1995). Cleavage of the third component of complement (C3) by mannose-binding protein-associated serine protease (MASP) with subsequent

complement activation. *Immunobiology,* Vol. 194, No. 4-5, pp. 443-448

complement genes. *International immunology,* Vol. 12, No. 3, pp. 231-240

alternative complement pathway. *J Exp Med,* Vol. 148, No. 6, pp. 1498-1509

CL-K1. *Microbiol Immunol,* Vol. 50, No. 12, pp. 1001-1013

*of experimental medicine,* Vol. 117, No. pp. 983-1008

*Am J Med Genet,* Vol. 16, No. 4, pp. 475-480

*Immunobiology,* Vol. 199, No. 2, pp. 340-347

*Med,* Vol. 176, No. 6, pp. 1497-1502

No. 4, pp. 261-266

No. 5, pp. 2281-2284

8626-8632

1024

187, No. 7, pp. 3751-3758

Iwaki, D., Kanno, K., Takahashi, M., Endo, Y., Lynch, N. J., Schwaeble, W. J., Matsushita, M., Okabe, M. and Fujita, T. (2006). Small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway. *J Immunol,* Vol. 177, No. 12, pp. 8626-8632

176 Binding Protein

773

127-135

No. pp.

6104

*immunology,* Vol. 5, No. 10, pp. 981-986

*Biochem Biophys,* Vol. 449, No. 1-2, pp. 164-170

*Immunol,* Vol. 183, No. 11, pp. 7371-7378

*Immunol,* Vol. 107, No. 3, pp. 140-151

Vol. 262, No. 16, pp. 7451-7454

Carroll, M. C. (2004). The complement system in regulation of adaptive immunity. *Nature*

Chou, K. C. and Heinrikson, R. L. (1997). Prediction of the tertiary structure of the complement control protein module. *Journal of protein chemistry,* Vol. 16, No. 8, pp. 765-

Cortesio, C. L. and Jiang, W. (2006). Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5. *Arch*

Cseh, S., Vera, L., Matsushita, M., Fujita, T., Arlaud, G. J. and Thielens, N. M. (2002). Characterization of the interaction between L-ficolin/p35 and mannan-binding lectin-

Degn, S. E., Hansen, A. G., Steffensen, R., Jacobsen, C., Jensenius, J. C. and Thiel, S. (2009). MAp44, a human protein associated with pattern recognition molecules of the complement system and regulating the lectin pathway of complement activation. *J*

Degn, S. E., Jensenius, J. C. and Bjerre, M. (2010). The lectin pathway and its implications in coagulation, infections and auto-immunity. *Current opinion in organ transplantation,* Vol.

Feinberg, H., Uitdehaag, J. C., Davies, J. M., Wallis, R., Drickamer, K. and Weis, W. I. (2003). Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated

Fujita, T., Matsushita, M. and Endo, Y. (2004). The lectin-complement pathway--its role in

Hajela, K., Kojima, M., Ambrus, G., Wong, K. H., Moffatt, B. E., Ferluga, J., Hajela, S., Gal, P. and Sim, R. B. (2002). The biological functions of MBL-associated serine proteases

Hansen, S., Selman, L., Palaniyar, N., Ziegler, K., Brandt, J., Kliem, A., Jonasson, M., Skjoedt, M. O., Nielsen, O., Hartshorn, K., Jorgensen, T. J., Skjodt, K. and Holmskov, U. (2010). Collectin 11 (CL-11, CL-K1) is a MASP-1/3-associated plasma collectin with microbialbinding activity. *Journal of immunology (Baltimore, Md. : 1950),* Vol. 185, No. 10, pp. 6096-

Holers, V. M. (2003). The complement system as a therapeutic target in autoimmunity. *Clin*

Holmskov, U., Thiel, S. and Jensenius, J. C. (2003). Collections and ficolins: humoral lectins

Ikeda, K., Sannoh, T., Kawasaki, N., Kawasaki, T. and Yamashina, I. (1987). Serum lectin with known structure activates complement through the classical pathway. *J Biol Chem,*

of the innate immune defense. *Annu Rev Immunol,* Vol. 21, No. pp. 547-578

serine protease-2. *The EMBO journal,* Vol. 22, No. 10, pp. 2348-2359

(MASPs). *Immunobiology,* Vol. 205, No. 4-5, pp. 467-475

innate immunity and evolution. *Immunol Rev,* Vol. 198, No. pp. 185-202

associated serine proteases-1 and -2. *J Immunol,* Vol. 169, No. 10, pp. 5735-5743 Dahl, M. R., Thiel, S., Matsushita, M., Fujita, T., Willis, A. C., Christensen, T., Vorup-Jensen, T. and Jensenius, J. C. (2001). MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. *Immunity,* Vol. 15, No. 1, pp.

Megyeri, M., Mako, V., Beinrohr, L., Doleschall, Z., Prohaszka, Z., Cervenak, L., Zavodszky, P. and Gal, P. (2009). Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function. *J Immunol,* Vol. 183, No. 5, pp. 3409-3416

The Study of MASPs Knockout Mice 179

gastrointestinal ischemia/reperfusion injury. *Proc Natl Acad Sci U S A,* Vol. 108, No. 18,

Shi, L., Takahashi, K., Dundee, J., Shahroor-Karni, S., Thiel, S., Jensenius, J. C., Gad, F., Hamblin, M. R., Sastry, K. N. and Ezekowitz, R. A. (2004). Mannose-binding lectindeficient mice are susceptible to infection with Staphylococcus aureus. *J Exp Med,* Vol.

Sirmaci, A., Walsh, T., Akay, H., Spiliopoulos, M., Sakalar, Y. B., Hasanefendioglu-Bayrak, A., Duman, D., Farooq, A., King, M. C. and Tekin, M. (2010). MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory findings of Carnevale, Malpuech, OSA, and Michels syndromes. *Am J Hum Genet,* Vol. 87, No. 5, pp. 679-686 Skjoedt, M. O., Hummelshoj, T., Palarasah, Y., Honore, C., Koch, C., Skjodt, K. and Garred, P. (2010). A novel mannose-binding lectin/ficolin-associated protein is highly expressed in heart and skeletal muscle tissues and inhibits complement activation. *The Journal of*

Stover, C. M., Schwaeble, W. J., Lynch, N. J., Thiel, S. and Speicher, M. R. (1999a). Assignment of the gene encoding mannan-binding lectin-associated serine protease 2 (MASP2) to human chromosome 1p36.3-->p36.2 by in situ hybridization and somatic

Stover, C. M., Thiel, S., Thelen, M., Lynch, N. J., Vorup-Jensen, T., Jensenius, J. C. and Schwaeble, W. J. (1999b). Two constituents of the initiation complex of the mannanbinding lectin activation pathway of complement are encoded by a single structural

Takada, F., Seki, N., Matsuda, Y., Takayama, Y. and Kawakami, M. (1995). Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27-q28 and mouse 16B2-B3. *Genomics,* Vol. 25, No. 3,

Takahashi, K., Gordon, J., Liu, H., Sastry, K. N., Epstein, J. E., Motwani, M., Laursen, I., Thiel, S., Jensenius, J. C., Carroll, M. and Ezekowitz, R. A. (2002). Lack of mannosebinding lectin-A enhances survival in a mouse model of acute septic peritonitis.

Takahashi, M., Endo, Y., Fujita, T. and Matsushita, M. (1999). A truncated form of mannosebinding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. *Int Immunol,* Vol.

Takahashi, M., Ishida, Y., Iwaki, D., Kanno, K., Suzuki, T., Endo, Y., Homma, Y. and Fujita, T. (2010). Essential role of mannose-binding lectin-associated serine protease-1 in

Takahashi, M., Iwaki, D., Kanno, K., Ishida, Y., Xiong, J., Matsushita, M., Endo, Y., Miura, S., Ishii, N., Sugamura, K. and Fujita, T. (2008). Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway. *J*

Thiel, S., Vorup-Jensen, T., Stover, C. M., Schwaeble, W., Laursen, S. B., Poulsen, K., Willis, A. C., Eggleton, P., Hansen, S., Holmskov, U., Reid, K. B. and Jensenius, J. C. (1997). A

activation of the complement factor D. *J Exp Med,* Vol. 207, No. 1, pp. 29-37

cell hybrid analysis. *Cytogenet Cell Genet,* Vol. 84, No. 3-4, pp. 148-149

pp. 7523-7528

pp. 757-759

11, No. 5, pp. 859-863

199, No. 10, pp. 1379-1390

*biological chemistry,* Vol. 285, No. 11, pp. 8234-8243

gene. *J Immunol,* Vol. 162, No. 6, pp. 3481-3490

*Microbes Infect,* Vol. 4, No. 8, pp. 773-784

*Immunol,* Vol. 180, No. 9, pp. 6132-6138

gastrointestinal ischemia/reperfusion injury. *Proc Natl Acad Sci U S A,* Vol. 108, No. 18, pp. 7523-7528

Shi, L., Takahashi, K., Dundee, J., Shahroor-Karni, S., Thiel, S., Jensenius, J. C., Gad, F., Hamblin, M. R., Sastry, K. N. and Ezekowitz, R. A. (2004). Mannose-binding lectindeficient mice are susceptible to infection with Staphylococcus aureus. *J Exp Med,* Vol. 199, No. 10, pp. 1379-1390

178 Binding Protein

183, No. 5, pp. 3409-3416

294, No. 3, pp. E521-529

No. 3, pp. 856-867

797

pp. 197-203

4-5, pp. 455-466

*Genet,* Vol. 33, No. 10, pp. 884-886

action. *J Exp Med,* Vol. 135, No. 4, pp. 1003-1008

protein. *Int Immunol,* Vol. 6, No. 4, pp. 665-669

*Biol Chem,* Vol. 265, No. 4, pp. 1980-1984

Megyeri, M., Mako, V., Beinrohr, L., Doleschall, Z., Prohaszka, Z., Cervenak, L., Zavodszky, P. and Gal, P. (2009). Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function. *J Immunol,* Vol.

Michels, V. V., Hittner, H. M. and Beaudet, A. L. (1978). A clefting syndrome with ocular anterior chamber defect and lid anomalies. *J Pediatr,* Vol. 93, No. 3, pp. 444-446 Mingarelli, R., Castriota Scanderbeg, A. and Dallapiccola, B. (1996). Two sisters with a syndrome of ocular, skeletal, and abdominal abnormalities (OSA syndrome). *J Med*

Muller-Eberhard, H. J. and Gotze, O. (1972). C3 proactivator convertase and its mode of

Ohta, M., Okada, M., Yamashina, I. and Kawasaki, T. (1990). The mechanism of carbohydrate-mediated complement activation by the serum mannan-binding protein. *J*

Paglialunga, S., Fisette, A., Yan, Y., Deshaies, Y., Brouillette, J. F., Pekna, M. and Cianflone, K. (2008). Acylation-stimulating protein deficiency and altered adipose tissue in alternative complement pathway knockout mice. *Am J Physiol Endocrinol Metab,* Vol.

Pangburn, M. K., Schreiber, R. D. and Muller-Eberhard, H. J. (1981). Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3. *J Exp Med,* Vol. 154,

Ricklin, D., Hajishengallis, G., Yang, K. and Lambris, J. D. (2010). Complement: a key system for immune surveillance and homeostasis. *Nature immunology,* Vol. 11, No. 9, pp. 785-

Rooryck, C., Diaz-Font, A., Osborn, D. P., Chabchoub, E., Hernandez-Hernandez, V., Shamseldin, H., Kenny, J., Waters, A., Jenkins, D., Kaissi, A. A., Leal, G. F., Dallapiccola, B., Carnevale, F., Bitner-Glindzicz, M., Lees, M., Hennekam, R., Stanier, P., Burns, A. J., Peeters, H., Alkuraya, F. S. and Beales, P. L. (2011). Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome. *Nat Genet,* Vol. 43, No. 3,

Sato, T., Endo, Y., Matsushita, M. and Fujita, T. (1994). Molecular characterization of a novel serine protease involved in activation of the complement system by mannose-binding

Schwaeble, W., Dahl, M. R., Thiel, S., Stover, C. and Jensenius, J. C. (2002). The mannanbinding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes. *Immunobiology,* Vol. 205, No.

Schwaeble, W. J., Lynch, N. J., Clark, J. E., Marber, M., Samani, N. J., Ali, Y. M., Dudler, T., Parent, B., Lhotta, K., Wallis, R., Farrar, C. A., Sacks, S., Lee, H., Zhang, M., Iwaki, D., Takahashi, M., Fujita, T., Tedford, C. E. and Stover, C. M. (2011). Targeting of mannanbinding lectin-associated serine protease-2 confers protection from myocardial and

second serine protease associated with mannan-binding lectin that activates complement. *Nature,* Vol. 386, No. 6624, pp. 506-510

**Chapter 8**

© 2012 Vlasova-St. Louis et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Various experimental approaches have been developed to understand the interaction between RBPs and the network of transcripts that they regulate. One of the most widely used techniques involves immunopurification of specific RNA-binding proteins from

and reproduction in any medium, provided the original work is properly cited.

**CELF1, a Multifunctional Regulator**

Daniel Beisang, Paul R. Bohjanen and Irina A. Vlasova-St. Louis

In order to assure the precise utilization of genetic information, gene expression is regulated at the level of transcription as well as multiple post-transcriptional levels including splicing, transport, localization, mRNA stability, and translation [1],[2],[3],[4],[5],[6],[7]. During evolution, cells developed precise mechanisms to ensure that each transcript is appropriately stored, modified, translated or degraded, depending on the need for the mRNA or encoded protein by the cell. Steady-state protein levels within a cell correlate poorly with steady-state levels of mRNA, leading scientists to hypothesize that the gene expression is regulated at post-transcriptional levels [8]. Work over the past quarter century has resulted in the identification of unifying concepts in post-transcriptional regulation. One unifying concept states is that post-transcriptional regulation is mediated by two major molecular components: *cis*-acting regulatory sequence elements and *trans*-acting factors. *Cis*acting regulatory sequence elements are sub-sequences contained in the 5' untranslated region (UTR), coding region, and 3'UTR of mRNA that are selectively recognized by a complementary set of one or more *trans*-acting factors to regulate post-transcriptional gene expression. *Trans*-acting factors include RNA-binding proteins (RBPs) and microRNAs (miRNAs) which are able to influence the fate of mRNA by controlling processes such as translation and mRNA degradation (reviewed in references [9],[10],[11],[12]). The combinatorial interplay between various miRNAs and RBPs binding to a given mRNA allows for the transcript specific regulation critical to many cellular decisions during development [13],[14],[15],[16] and in response to environmental stimuli (reviewed in

**of Posttranscriptional Networks**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48780

references [17],[18],[19],[20],[21],[22])*.*

**1. Introduction**

## **CELF1, a Multifunctional Regulator of Posttranscriptional Networks**

Daniel Beisang, Paul R. Bohjanen and Irina A. Vlasova-St. Louis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48780

## **1. Introduction**

180 Binding Protein

2, pp. 348-357

1058-1066

1140-1144

No. 6505, pp. 425-432

second serine protease associated with mannan-binding lectin that activates

Thurman, J. M. and Holers, V. M. (2006). The central role of the alternative complement

Turner, M. W. (1996). Mannose-binding lectin: the pluripotent molecule of the innate

Vorup-Jensen, T., Jensenius, J. C. and Thiel, S. (1998). MASP-2, the C3 convertase generating protease of the MBLectin complement activating pathway. *Immunobiology,* Vol. 199, No.

Walport, M. J. (2001a). Complement. First of two parts. *N Engl J Med,* Vol. 344, No. 14, pp.

Walport, M. J. (2001b). Complement. Second of two parts. *N Engl J Med,* Vol. 344, No. 15, pp.

Yasruel, Z., Cianflone, K., Sniderman, A. D., Rosenbloom, M., Walsh, M. and Rodriguez, M. A. (1991). Effect of acylation stimulating protein on the triacylglycerol synthetic

Yousef, G. M., Elliott, M. B., Kopolovic, A. D., Serry, E. and Diamandis, E. P. (2004). Sequence and evolutionary analysis of the human trypsin subfamily of serine

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L. and Friedman, J. M. (1994). Positional cloning of the mouse obese gene and its human homologue. *Nature,* Vol. 372,

pathway in human disease. *J Immunol,* Vol. 176, No. 3, pp. 1305-1310

pathway of human adipose tissue. *Lipids,* Vol. 26, No. 7, pp. 495-499

peptidases. *Biochimica et biophysica acta,* Vol. 1698, No. 1, pp. 77-86

immune system. *Immunology today,* Vol. 17, No. 11, pp. 532-540

complement. *Nature,* Vol. 386, No. 6624, pp. 506-510

In order to assure the precise utilization of genetic information, gene expression is regulated at the level of transcription as well as multiple post-transcriptional levels including splicing, transport, localization, mRNA stability, and translation [1],[2],[3],[4],[5],[6],[7]. During evolution, cells developed precise mechanisms to ensure that each transcript is appropriately stored, modified, translated or degraded, depending on the need for the mRNA or encoded protein by the cell. Steady-state protein levels within a cell correlate poorly with steady-state levels of mRNA, leading scientists to hypothesize that the gene expression is regulated at post-transcriptional levels [8]. Work over the past quarter century has resulted in the identification of unifying concepts in post-transcriptional regulation. One unifying concept states is that post-transcriptional regulation is mediated by two major molecular components: *cis*-acting regulatory sequence elements and *trans*-acting factors. *Cis*acting regulatory sequence elements are sub-sequences contained in the 5' untranslated region (UTR), coding region, and 3'UTR of mRNA that are selectively recognized by a complementary set of one or more *trans*-acting factors to regulate post-transcriptional gene expression. *Trans*-acting factors include RNA-binding proteins (RBPs) and microRNAs (miRNAs) which are able to influence the fate of mRNA by controlling processes such as translation and mRNA degradation (reviewed in references [9],[10],[11],[12]). The combinatorial interplay between various miRNAs and RBPs binding to a given mRNA allows for the transcript specific regulation critical to many cellular decisions during development [13],[14],[15],[16] and in response to environmental stimuli (reviewed in references [17],[18],[19],[20],[21],[22])*.*

Various experimental approaches have been developed to understand the interaction between RBPs and the network of transcripts that they regulate. One of the most widely used techniques involves immunopurification of specific RNA-binding proteins from

© 2012 Vlasova-St. Louis et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cellular extracts followed by high-throughput analysis of the co-purified RNA species [23]. The coupling of this technique to powerful bioinformatic analysis methods has lead researchers to understand the binding specificity of a wide-variety of RBPs. The advent of new technology such as next generation sequencing and chemical cross-linking procedures have improved these methodologies and allowed for the fine-scale mapping of RBP binding sites, as well as the refinement of RBP binding motifs. Microarray-based studies that evaluated mRNA decay rates on a global basis have also provided valuable information about the role of post-transcriptional regulation of a wide variety of transcripts that have important physiological functions [24],[25],[26],[27],[28],[29].

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 183

Embryo Deadenylation Element Binding Protein (EDEN-BP), which is 88% identical to CELF1, regulates transcript deadenylation, and human CELF1 was able to functionally replace the deadenylation function of EDEN-BP in *Xenopus* extracts [51]. In HeLa cell extracts CELF1 also promotes transcript deadenylation and was shown to recruit PolyA Ribonuclease (PARN) [52]. In addition to the evolutionary conservation of the biochemical function of CELF1, the developmental programs regulated by CELF1 may also be conserved. For example, CELF1 appears to be an important factor in muscle development in diverse species. Studies investigating the function of CELF1 in *Drosophila*, *Xenopus* and mice have shown that CELF1 is critical for regulating the muscle developmental program [45], [53], [54]. More recent work suggests that CELF1 is a crucial factor in the regulation of mRNA degradation in mouse myoblasts [55]. Thus, in addition to the conservation of its biochemical function as a regulator of deadenylation, the role of CELF1 in muscle

As described below, CELF proteins from diverse species bind to RNA preferentially at GUrich sequences and thereby regulate post-transcriptional processes such as mRNA splicing, translation, deadenylation and mRNA degradation. The structure and biochemical properties of CELF family members suggest functional redundancy [56], yet each CELF protein targets specific sub-populations of RNA transcripts and appears to have distinct functions [57]. We are starting to understand the mechanisms by which an individual CELF protein can serve multiple biochemical functions to coordinately regulate gene expression at

CELF 1 and 2 proteins were first isolated and characterized as novel heterogeneous nuclear ribonucleoproteins (hnRNPs). Timchenko et.al. demonstrated that these proteins bound to RNA containing the sequence (CUG)8 within the 3'UTR of myotonin protein kinase mRNA *in vitro* [58],[59]. Subsequent searches for the RNA-binding specificities of CELF1 and CELF2 used systemic evolution of ligands exponential enrichment (SELEX), revealing that CELF1 and CELF2 both bound preferentially to GU-rich RNA sequences [60]. Binding by CELF1 to GU-rich sequences *in vitro* and *in vivo* was abrogated by mutation of G nucleotides to C [30],[61]. Takanashi et. al. used a yeast three hybrid system for evaluating RNA-protein interactions, and found CELF1 bound preferentially to UG repeats rather than to CUG repeats [32]. CELF1 bound with high specificity to (UG)15 based on a surface plasmon resonance (SPR) quantitative binding assay [62]. Orthologues of CELF1 in other species also appear to have preferences for binding to GU-rich sequences. In *Xenopus*, the CELF1 orthologue EDEN-BP (embryo deadenylation element binding protein) binds to the GU-rich EDEN element, which contains the sequence (UGUA)12, and functions as a deadenylation signal in *Xenopus* embryos after fertilization [31],[51]. In *Drosophila*, the CELF1 orthologue Bru-3 was found to bind specifically to (UG)15 repeats and also was able to bind to the *Xenopus* EDEN element [46]. The *Zebrafish* protein Brul, a homologue of EDEN-BP with 81% identity, was also shown to preferentially bind to GU-rich RNAs [63]. EDEN-BP and Bru-3 can bind to GRE-RNA as dimers [64],[65] and may require GU-rich sequences of sufficient

**3. Biochemistry of binding by CELF proteins to target mRNA**

development may also be conserved.

posttranscriptional levels [30].

This chapter focuses on the role of CELF1 (CUGBP and embryonically lethal abnormal vision-type RNA binding protein 3-like factor 1) in the regulation of posttranscriptional gene expression. CELF1 functions to regulate posttranscriptional gene expression by binding to RNA sequences known as GU-rich elements (GREs). Genome-wide measurements of mRNA decay and bioinformatic sequence motif discovery methods were used to identify the GRE as a highly conserved sequence that was enriched in the 3'UTR of mRNA transcripts with short half lives in primary human T lymphocytes [30]. This sequence resembled previously characterized binding sites for CELF1 [31],[32], and CELF1 was found to bind with high affinity to GRE sequences and mediate mRNA degradation [30]. This chapter reviews how CELF1 and its target transcripts function as an evolutionarily conserved posttranscriptional regulatory network which plays important roles in health and disease.

## **2. Evolutionary conservation of CELF proteins**

The CELF protein family is an evolutionarily conserved family of RNA-binding proteins that play essential roles in post-transcriptional gene regulation [28],[33]. These proteins contain three highly conserved RNA-Recognition Motifs (RRM) with the 2 N-terminal RRMs and the C-terminal RRM being separated by a highly divergent linker domain [34]. The RRMs confer RNA binding activity, and it is postulated that the divergent linker domain is an important site for functional regulation. Six members of the CELF family have been identified in humans and mice: CELF1 (CUGBP1) and CELF2 (CUGBP2) proteins are expressed ubiquitously and play vital role in embryogenesis [35],[36],[37],[38],[39], whereas CELF proteins 3-6 are restricted to adult tissues and found almost exclusively in the nervous system [40],[41]. CELF proteins often serve multiple functions in both the cytoplasm and the nucleus [42],[43]. Human CELF1 and its orthologs in *Gallus gallus*, *Zebrafish, Xenopus, Drosophila* and *C. elegans* have been known for many years to regulate gene expression at posttranscriptional levels and to control important developmental processes [31],[44],[45],[46],[47],[48],[49].

CELF1 function is conserved across evolution at the level of biochemical mechanism as well as its function in regulating development. Transcript deadenylation is often the first step in the mRNA degradation process, and CELF1 has been shown to promote transcript deadenylation in diverse species [28],[50]. In *Xenopus* embryos the CELF1 homologue Embryo Deadenylation Element Binding Protein (EDEN-BP), which is 88% identical to CELF1, regulates transcript deadenylation, and human CELF1 was able to functionally replace the deadenylation function of EDEN-BP in *Xenopus* extracts [51]. In HeLa cell extracts CELF1 also promotes transcript deadenylation and was shown to recruit PolyA Ribonuclease (PARN) [52]. In addition to the evolutionary conservation of the biochemical function of CELF1, the developmental programs regulated by CELF1 may also be conserved. For example, CELF1 appears to be an important factor in muscle development in diverse species. Studies investigating the function of CELF1 in *Drosophila*, *Xenopus* and mice have shown that CELF1 is critical for regulating the muscle developmental program [45], [53], [54]. More recent work suggests that CELF1 is a crucial factor in the regulation of mRNA degradation in mouse myoblasts [55]. Thus, in addition to the conservation of its biochemical function as a regulator of deadenylation, the role of CELF1 in muscle development may also be conserved.

182 Binding Protein

roles in health and disease.

[31],[44],[45],[46],[47],[48],[49].

cellular extracts followed by high-throughput analysis of the co-purified RNA species [23]. The coupling of this technique to powerful bioinformatic analysis methods has lead researchers to understand the binding specificity of a wide-variety of RBPs. The advent of new technology such as next generation sequencing and chemical cross-linking procedures have improved these methodologies and allowed for the fine-scale mapping of RBP binding sites, as well as the refinement of RBP binding motifs. Microarray-based studies that evaluated mRNA decay rates on a global basis have also provided valuable information about the role of post-transcriptional regulation of a wide variety of transcripts that have

This chapter focuses on the role of CELF1 (CUGBP and embryonically lethal abnormal vision-type RNA binding protein 3-like factor 1) in the regulation of posttranscriptional gene expression. CELF1 functions to regulate posttranscriptional gene expression by binding to RNA sequences known as GU-rich elements (GREs). Genome-wide measurements of mRNA decay and bioinformatic sequence motif discovery methods were used to identify the GRE as a highly conserved sequence that was enriched in the 3'UTR of mRNA transcripts with short half lives in primary human T lymphocytes [30]. This sequence resembled previously characterized binding sites for CELF1 [31],[32], and CELF1 was found to bind with high affinity to GRE sequences and mediate mRNA degradation [30]. This chapter reviews how CELF1 and its target transcripts function as an evolutionarily conserved posttranscriptional regulatory network which plays important

The CELF protein family is an evolutionarily conserved family of RNA-binding proteins that play essential roles in post-transcriptional gene regulation [28],[33]. These proteins contain three highly conserved RNA-Recognition Motifs (RRM) with the 2 N-terminal RRMs and the C-terminal RRM being separated by a highly divergent linker domain [34]. The RRMs confer RNA binding activity, and it is postulated that the divergent linker domain is an important site for functional regulation. Six members of the CELF family have been identified in humans and mice: CELF1 (CUGBP1) and CELF2 (CUGBP2) proteins are expressed ubiquitously and play vital role in embryogenesis [35],[36],[37],[38],[39], whereas CELF proteins 3-6 are restricted to adult tissues and found almost exclusively in the nervous system [40],[41]. CELF proteins often serve multiple functions in both the cytoplasm and the nucleus [42],[43]. Human CELF1 and its orthologs in *Gallus gallus*, *Zebrafish, Xenopus, Drosophila* and *C. elegans* have been known for many years to regulate gene expression at posttranscriptional levels and to control important developmental processes

CELF1 function is conserved across evolution at the level of biochemical mechanism as well as its function in regulating development. Transcript deadenylation is often the first step in the mRNA degradation process, and CELF1 has been shown to promote transcript deadenylation in diverse species [28],[50]. In *Xenopus* embryos the CELF1 homologue

important physiological functions [24],[25],[26],[27],[28],[29].

**2. Evolutionary conservation of CELF proteins**

As described below, CELF proteins from diverse species bind to RNA preferentially at GUrich sequences and thereby regulate post-transcriptional processes such as mRNA splicing, translation, deadenylation and mRNA degradation. The structure and biochemical properties of CELF family members suggest functional redundancy [56], yet each CELF protein targets specific sub-populations of RNA transcripts and appears to have distinct functions [57]. We are starting to understand the mechanisms by which an individual CELF protein can serve multiple biochemical functions to coordinately regulate gene expression at posttranscriptional levels [30].

## **3. Biochemistry of binding by CELF proteins to target mRNA**

CELF 1 and 2 proteins were first isolated and characterized as novel heterogeneous nuclear ribonucleoproteins (hnRNPs). Timchenko et.al. demonstrated that these proteins bound to RNA containing the sequence (CUG)8 within the 3'UTR of myotonin protein kinase mRNA *in vitro* [58],[59]. Subsequent searches for the RNA-binding specificities of CELF1 and CELF2 used systemic evolution of ligands exponential enrichment (SELEX), revealing that CELF1 and CELF2 both bound preferentially to GU-rich RNA sequences [60]. Binding by CELF1 to GU-rich sequences *in vitro* and *in vivo* was abrogated by mutation of G nucleotides to C [30],[61]. Takanashi et. al. used a yeast three hybrid system for evaluating RNA-protein interactions, and found CELF1 bound preferentially to UG repeats rather than to CUG repeats [32]. CELF1 bound with high specificity to (UG)15 based on a surface plasmon resonance (SPR) quantitative binding assay [62]. Orthologues of CELF1 in other species also appear to have preferences for binding to GU-rich sequences. In *Xenopus*, the CELF1 orthologue EDEN-BP (embryo deadenylation element binding protein) binds to the GU-rich EDEN element, which contains the sequence (UGUA)12, and functions as a deadenylation signal in *Xenopus* embryos after fertilization [31],[51]. In *Drosophila*, the CELF1 orthologue Bru-3 was found to bind specifically to (UG)15 repeats and also was able to bind to the *Xenopus* EDEN element [46]. The *Zebrafish* protein Brul, a homologue of EDEN-BP with 81% identity, was also shown to preferentially bind to GU-rich RNAs [63]. EDEN-BP and Bru-3 can bind to GRE-RNA as dimers [64],[65] and may require GU-rich sequences of sufficient

length to allow dimer formation [66]. In addition to the primary GU-rich sequence, adjacent sequence elements may also be important for assembly of CELF proteins on RNA by allowing optimal secondary structure to facilitate the formation of RNA-protein complexes [67],[68].

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 185

abundance as well as increased splicing activity [78]. Additionally, in transgenic mouse models of DM1, mice treated with specific inhibitors of the PKC pathway showed amelioration of cardiac abnormalities associated with the disease phenotype [79]. Phosphorylation of CELF1 also influences its ability to regulate muscle development (reviewed in [80]). CELF1 phosphorylation by Akt kinase at Ser 28 in normal muscle myoblasts influences its ability to affect the translation of its target transcripts during differentiation [65]. Phosphorylation of CELF1 also directly influences its RNA-binding activity. For example, cyclin D3-Cdk4/6 phosphorylates CELF1 at Ser 302, altering the binding specificity of CELF1 to RNA and translation initiation proteins, such as eIF2α [81]. During the process of T cell activation, phosphorylation of CELF1 alters binding by CELF1 to target transcripts. Shortly following T cell activation, CELF1 becomes phosphorylated, dramatically decreasing its affinity for mRNA and leading to stabilization of CELF1 target transcripts [77]. Overall, these studies show that phosphorylation regulates the many

Insight into the biological significance of CELF1 function as a coordinate regulator of posttranscriptional network was revealed through the experimental determination of CELF1 target transcripts. A technique involving RNA-immunoprecipitation followed by microarray analysis of associate transcripts (RIP-Chip) has allowed for the unbiased, genome-wide experimental identification of RNA-binding protein target transcripts. This technique involves immunoprecipitating an RNA-binding protein of interest from cell lysates under conditions that preserve RNA:Protein interactions. The co-purified RNA found associated with the immunoprecipitated RNA-binding protein is then isolated and interrogated using high throughput methods such as microarrays. Using this methodology, CELF1 targets have been identified in HeLa cells, resting and activated human T cells, and mouse myoblasts [55],[77],[82]. CELF1 targets, identified in cytoplasmic extracts from HeLa cells using an anti-CELF1 antibody, were analyzed to identify the CELF1 target sequence, which is known as the GRE. The sequence profile of CELF1 target transcripts was analyzed for enriched sequences using a Markov Chain Monte Carlo based gibbs sampler algorithm (BioProspector) as well as an overrepresentation algorithm, and the previously described GRE sequence, UGUUUGUUUGU, and a GU-repeat sequence, UGUGUGUGUGU, were found to be highly overrepresented in the 3'UTRs of the CELF1 target transcripts [83]. Both sequences were validated as CELF1-binding targets and were shown to function as mRNA decay elements by accelerating the decay of reporter transcripts. While GU-repeat sequences had previously been identified as a CELF1 recognition motif through *in vitro* SELEX protocol [60]. These and other experiments identified putative binding targets of CELF1 in cells [32],[62]. Because (as described later in this chapter) the UGUUUGUUUGU sequence and the GU-repeat sequence both bound to CELF1 and functioned as decay elements, the GRE was redefined to contain both of these sequences [83],[84]. The RIP-Chip approach was also used to immunoprecipitate endogenous RNA binding complexes from mouse myoblasts,

functions of CELF1 in posttranscriptional gene regulation.

**3.2. Identification of CELF1 target transcripts**

Structural studies have provided valuable insight into the mechanisms underlying the RNAbinding activity of CELF1. CELF proteins all contain two N-terminal and one C-terminal RNA recognition motifs (RRMs), separated by a 160-230 residue divergent domain [69],[70]. The highly conserved RRMs bind to RNA in a sequence-specific manner [69],[71]. Nuclear Magnetic Resonance spectroscopic (NMR)-based solution studies demonstrated that both RRM1 and RRM2 each contribute to binding to a 12-nt target RNA containing two UUGUU motifs. The tandem RRM1/2 domains together show increased affinity compared to the binding by each domain separately to an RNA sequence with two sequential UUGU(U) motifs, thus indicating binding cooperativity between the two RRMs [34],[72]. Crystallographic studies showed that both RRM2 and RRM1 bind to GRE-RNA, and RRM1 is important for crystal-packing interactions [73].

In addition to RRM1 and RRM2, RRM3 also has RNA-binding activity. According to NMR analysis, RRM3 specifically recognizes the UGU trinucleotide segment of bound (UG)3 RNA through extensive stacking and hydrogen-bonding interactions within the pocket formed by the beta-sheet and the conserved N-terminal extension [71]. Experiments investigating CELF1 function through a yeast three hybrid system suggested that deletion/mutation of RRM1 or RRM2 does not abrogate binding to GU-rich RNA, suggesting that RRM3 may recognize GU-repeats more avidly than RRM1 or RRM2 [62]. Additionally, it has been reported that RRM3 is able to recognize a poorly defined G/C-rich sequence from the 5'UTR of Cyclin D1 when combined with the divergent domain [65]. The divergent domain also appears to be important for RNA-binding since the presence of divergent domain within recombinant CELF1/CELF4 chimeric proteins increased RNA-binding affinity, perhaps by conveying important conformational changes necessary for RNA-binding [32],[62],[70]. The divergent domain may also facilitate CELF:CELF homotypic interactions [64] which may influence its activity. For example, CELF:CELF interactions appear to activate RNA deadenylation in *Xenopus* extracts [66].

### **3.1. Regulation of CELF1 function through phosphorylation**

CELF1 is a known phosphoprotein with multiple predicted phosphorylation sites, and CELF1 phosphorylation appears to regulate its function as a mediator of alternative splicing, mRNA decay, and translational regulation [74],[75],[76],[77]. One of the pathologic events which occurs in the disease Myotonic Dystrophy type 1 (DM1) is an increase in the protein abundance of CELF1 and an associated increase in CELF1 mediated alternative splicing activity. This increase in CELF1 protein abundance is a result of increased CELF1 protein stability secondary to hyperphosphorylation [75]. In DM1, the (CUG)n expansion of the DMPK 3'UTR leads to protein kinase C (PKC) activation through an unknown mechanism. PKC, in turn, hyperphosphorylates CELF1, resulting in increased protein stability and abundance as well as increased splicing activity [78]. Additionally, in transgenic mouse models of DM1, mice treated with specific inhibitors of the PKC pathway showed amelioration of cardiac abnormalities associated with the disease phenotype [79]. Phosphorylation of CELF1 also influences its ability to regulate muscle development (reviewed in [80]). CELF1 phosphorylation by Akt kinase at Ser 28 in normal muscle myoblasts influences its ability to affect the translation of its target transcripts during differentiation [65]. Phosphorylation of CELF1 also directly influences its RNA-binding activity. For example, cyclin D3-Cdk4/6 phosphorylates CELF1 at Ser 302, altering the binding specificity of CELF1 to RNA and translation initiation proteins, such as eIF2α [81]. During the process of T cell activation, phosphorylation of CELF1 alters binding by CELF1 to target transcripts. Shortly following T cell activation, CELF1 becomes phosphorylated, dramatically decreasing its affinity for mRNA and leading to stabilization of CELF1 target transcripts [77]. Overall, these studies show that phosphorylation regulates the many functions of CELF1 in posttranscriptional gene regulation.

#### **3.2. Identification of CELF1 target transcripts**

184 Binding Protein

[67],[68].

is important for crystal-packing interactions [73].

deadenylation in *Xenopus* extracts [66].

**3.1. Regulation of CELF1 function through phosphorylation**

length to allow dimer formation [66]. In addition to the primary GU-rich sequence, adjacent sequence elements may also be important for assembly of CELF proteins on RNA by allowing optimal secondary structure to facilitate the formation of RNA-protein complexes

Structural studies have provided valuable insight into the mechanisms underlying the RNAbinding activity of CELF1. CELF proteins all contain two N-terminal and one C-terminal RNA recognition motifs (RRMs), separated by a 160-230 residue divergent domain [69],[70]. The highly conserved RRMs bind to RNA in a sequence-specific manner [69],[71]. Nuclear Magnetic Resonance spectroscopic (NMR)-based solution studies demonstrated that both RRM1 and RRM2 each contribute to binding to a 12-nt target RNA containing two UUGUU motifs. The tandem RRM1/2 domains together show increased affinity compared to the binding by each domain separately to an RNA sequence with two sequential UUGU(U) motifs, thus indicating binding cooperativity between the two RRMs [34],[72]. Crystallographic studies showed that both RRM2 and RRM1 bind to GRE-RNA, and RRM1

In addition to RRM1 and RRM2, RRM3 also has RNA-binding activity. According to NMR analysis, RRM3 specifically recognizes the UGU trinucleotide segment of bound (UG)3 RNA through extensive stacking and hydrogen-bonding interactions within the pocket formed by the beta-sheet and the conserved N-terminal extension [71]. Experiments investigating CELF1 function through a yeast three hybrid system suggested that deletion/mutation of RRM1 or RRM2 does not abrogate binding to GU-rich RNA, suggesting that RRM3 may recognize GU-repeats more avidly than RRM1 or RRM2 [62]. Additionally, it has been reported that RRM3 is able to recognize a poorly defined G/C-rich sequence from the 5'UTR of Cyclin D1 when combined with the divergent domain [65]. The divergent domain also appears to be important for RNA-binding since the presence of divergent domain within recombinant CELF1/CELF4 chimeric proteins increased RNA-binding affinity, perhaps by conveying important conformational changes necessary for RNA-binding [32],[62],[70]. The divergent domain may also facilitate CELF:CELF homotypic interactions [64] which may influence its activity. For example, CELF:CELF interactions appear to activate RNA

CELF1 is a known phosphoprotein with multiple predicted phosphorylation sites, and CELF1 phosphorylation appears to regulate its function as a mediator of alternative splicing, mRNA decay, and translational regulation [74],[75],[76],[77]. One of the pathologic events which occurs in the disease Myotonic Dystrophy type 1 (DM1) is an increase in the protein abundance of CELF1 and an associated increase in CELF1 mediated alternative splicing activity. This increase in CELF1 protein abundance is a result of increased CELF1 protein stability secondary to hyperphosphorylation [75]. In DM1, the (CUG)n expansion of the DMPK 3'UTR leads to protein kinase C (PKC) activation through an unknown mechanism. PKC, in turn, hyperphosphorylates CELF1, resulting in increased protein stability and Insight into the biological significance of CELF1 function as a coordinate regulator of posttranscriptional network was revealed through the experimental determination of CELF1 target transcripts. A technique involving RNA-immunoprecipitation followed by microarray analysis of associate transcripts (RIP-Chip) has allowed for the unbiased, genome-wide experimental identification of RNA-binding protein target transcripts. This technique involves immunoprecipitating an RNA-binding protein of interest from cell lysates under conditions that preserve RNA:Protein interactions. The co-purified RNA found associated with the immunoprecipitated RNA-binding protein is then isolated and interrogated using high throughput methods such as microarrays. Using this methodology, CELF1 targets have been identified in HeLa cells, resting and activated human T cells, and mouse myoblasts [55],[77],[82]. CELF1 targets, identified in cytoplasmic extracts from HeLa cells using an anti-CELF1 antibody, were analyzed to identify the CELF1 target sequence, which is known as the GRE. The sequence profile of CELF1 target transcripts was analyzed for enriched sequences using a Markov Chain Monte Carlo based gibbs sampler algorithm (BioProspector) as well as an overrepresentation algorithm, and the previously described GRE sequence, UGUUUGUUUGU, and a GU-repeat sequence, UGUGUGUGUGU, were found to be highly overrepresented in the 3'UTRs of the CELF1 target transcripts [83]. Both sequences were validated as CELF1-binding targets and were shown to function as mRNA decay elements by accelerating the decay of reporter transcripts. While GU-repeat sequences had previously been identified as a CELF1 recognition motif through *in vitro* SELEX protocol [60]. These and other experiments identified putative binding targets of CELF1 in cells [32],[62]. Because (as described later in this chapter) the UGUUUGUUUGU sequence and the GU-repeat sequence both bound to CELF1 and functioned as decay elements, the GRE was redefined to contain both of these sequences [83],[84]. The RIP-Chip approach was also used to immunoprecipitate endogenous RNA binding complexes from mouse myoblasts, using an anti-CELF1 antibody and similar G and U rich target sequences were identified [55]. In *Xenopus* extracts, target transcripts identified by RIP-Chip using an antibody against the CELF1 sequence homolog, EDEN-BP, were enriched in GU-rich sequences very similar to GREs [85]. These GU-rich containing target transcripts represented approximately 5% of the tested transcripts on the microarray [85]. In this work, the authors proposed a 15-nucleotide consensus motif (UGU/UG)3 to be the target motif of EDEN-BP [85],[86]. The RIP-Chip approach was also used to investigate the cytoplasmic target transcripts of CELF1 in resting and activated primary human T cells, and target transcripts were highly enriched for the presence of the GRE in their 3'UTRs, but the number of CELF1 target transcripts decreased dramatically following T cell activation [77]. Overall, numerous CELF1 target transcripts have been identified in several different systems indicating the CELF1 functions to regulate an important posttranscriptional network of gene expression.

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 187

**Figure 1.** Alternative pre-mRNA splicing by CELF1 protein modulates the temporal and spatial diversity of genes during development. CELF1 binds to GU-rich intronic sequences in precursor mRNA and regulates exon inclusion or exon skipping during stage-specific alternative splicing transitions.

CELF1-mediated regulation of alternative splicing is critical for maintenance of normal muscle structure and function [43],[96],[97]. Much of what we know about the role of CELF1 in alternative splicing comes from studies investigating the role of CELF1 in the pathogenesis of the neuromuscular disease myotonic dystrophy type 1 (DM1). In this disease, aberrant gain of CELF1 function is combined with a corresponding loss of function of the splicing factor MBNL1, resulting in the mis-splicing of a number of crucial genes (reviewed in [11]). Minigene reporter systems that contain alternative splice sites proved to be a useful tools for the identification of pre-mRNA targets for CELF1, including genes for cardiac troponin T (TNNT2) [98], insulin receptor (INSR) [99], and chloride channel1 (CLCN1) [100],[101]. Interestingly, these genes were all shown to be mis-regulated in tissues from patients who suffered from DM1. Minigene systems have been particularly useful in demonstrating that individual pre-mRNA splicing events are affected by loss or gain of activities of specific regulatory proteins. Studies performed in cultured cells with transiently transfected minigenes have identified a number of alternative gene regions regulated by CELF1 and other family members [43], [69], [98], [99], [100], [101], [102], [103], [104], [105], [106],[107],[108]. However, as in other chimeric systems, the results of minigene overexpression experiments may not necessarily reflect the full-length pre-mRNA splicing patterns observed *in vivo*, especially during certain stages of organism development [109]. CELF proteins have been found to regulate the switch from fetal to adult splicing patterns of several skeletal muscle transcripts through the use of transgenic mouse models [101],[100],[110]*.* In mice, splicing microarray studies found that nearly half of transcripts that undergo fetal-to-adult alternative splicing transitions in heart respond to overexpression of CELF1, suggesting that the level of CELF1 activity directly regulates the alternative splicing pattern of endogenous transcripts [111]. The development of dominant

Another approach to identify targets of RNA-binding proteins utilizes a cross-linking step prior to immunoprecipitation (CLIP) and subsequent high throughput methods to identify protein binding sequences. Using this method, 315 CELF1 RNA targets were identified in whole cell extracts from mouse hindbrain [87]. These RNA-binding targets for CELF1 were enriched in UG repeat sequences, with 64% of target sequences found in introns and 25% found in 3' UTR sequences [87]. Similar analysis of CELF1 in the C2C12 mouse myoblast cell line [88] extensively characterized RNA-binding sites of CELF1 and found that CELF1 bound predominantly in 3'UTRs and caused mRNA decay. The authors found significant enrichment of CELF1 binding sites in intronic regions flanking exons, supporting a role for CELF1 in alternative splicing [88]. Overall, these studies suggest that GU-rich sequences serve as genuine binding sites for CELF1 in a manner that has been conserved through evolution. In the next sections, we review the data supporting the model that CELF1 recognizes GU-rich sequences and thereby regulates pre-mRNA splicing, translation, and/or mRNA deadenylation/decay depending on the cellular and environmental context.

## **4. CELF1 as a regulator of splicing**

Pre-mRNA alternative splicing is a common mechanism for generating transcript and protein diversity. An estimated 90% of human genes produce alternatively spliced transcripts [89],[90]. Alignment of the genomic regions adjacent to mammalian intron-exon splice sites, identified TG-rich motifs (TTCTG and TGTT) as conserved *cis*-elements found at splicing acceptor sites associated with alternative splicing [91],[92]. These C/UG-rich sequences serve as binding sites for CELF proteins which activate or repress the splicing of pre-mRNA targets, depending on the context [93]. Recent evidence has re-confirmed the position-dependence of CELF1-binding sites in regulating exon inclusion or skipping (Figure 1) [88]. Although alternative splicing regulation was initially considered the primary function of CELF1 proteins in the nucleus, CELF members have also been implicated in nuclear C to U RNA editing in mammalian cells [94],[95].

gene expression.

environmental context.

**4. CELF1 as a regulator of splicing**

nuclear C to U RNA editing in mammalian cells [94],[95].

using an anti-CELF1 antibody and similar G and U rich target sequences were identified [55]. In *Xenopus* extracts, target transcripts identified by RIP-Chip using an antibody against the CELF1 sequence homolog, EDEN-BP, were enriched in GU-rich sequences very similar to GREs [85]. These GU-rich containing target transcripts represented approximately 5% of the tested transcripts on the microarray [85]. In this work, the authors proposed a 15-nucleotide consensus motif (UGU/UG)3 to be the target motif of EDEN-BP [85],[86]. The RIP-Chip approach was also used to investigate the cytoplasmic target transcripts of CELF1 in resting and activated primary human T cells, and target transcripts were highly enriched for the presence of the GRE in their 3'UTRs, but the number of CELF1 target transcripts decreased dramatically following T cell activation [77]. Overall, numerous CELF1 target transcripts have been identified in several different systems indicating the CELF1 functions to regulate an important posttranscriptional network of

Another approach to identify targets of RNA-binding proteins utilizes a cross-linking step prior to immunoprecipitation (CLIP) and subsequent high throughput methods to identify protein binding sequences. Using this method, 315 CELF1 RNA targets were identified in whole cell extracts from mouse hindbrain [87]. These RNA-binding targets for CELF1 were enriched in UG repeat sequences, with 64% of target sequences found in introns and 25% found in 3' UTR sequences [87]. Similar analysis of CELF1 in the C2C12 mouse myoblast cell line [88] extensively characterized RNA-binding sites of CELF1 and found that CELF1 bound predominantly in 3'UTRs and caused mRNA decay. The authors found significant enrichment of CELF1 binding sites in intronic regions flanking exons, supporting a role for CELF1 in alternative splicing [88]. Overall, these studies suggest that GU-rich sequences serve as genuine binding sites for CELF1 in a manner that has been conserved through evolution. In the next sections, we review the data supporting the model that CELF1 recognizes GU-rich sequences and thereby regulates pre-mRNA splicing, translation, and/or mRNA deadenylation/decay depending on the cellular and

Pre-mRNA alternative splicing is a common mechanism for generating transcript and protein diversity. An estimated 90% of human genes produce alternatively spliced transcripts [89],[90]. Alignment of the genomic regions adjacent to mammalian intron-exon splice sites, identified TG-rich motifs (TTCTG and TGTT) as conserved *cis*-elements found at splicing acceptor sites associated with alternative splicing [91],[92]. These C/UG-rich sequences serve as binding sites for CELF proteins which activate or repress the splicing of pre-mRNA targets, depending on the context [93]. Recent evidence has re-confirmed the position-dependence of CELF1-binding sites in regulating exon inclusion or skipping (Figure 1) [88]. Although alternative splicing regulation was initially considered the primary function of CELF1 proteins in the nucleus, CELF members have also been implicated in

**Figure 1.** Alternative pre-mRNA splicing by CELF1 protein modulates the temporal and spatial diversity of genes during development. CELF1 binds to GU-rich intronic sequences in precursor mRNA and regulates exon inclusion or exon skipping during stage-specific alternative splicing transitions.

CELF1-mediated regulation of alternative splicing is critical for maintenance of normal muscle structure and function [43],[96],[97]. Much of what we know about the role of CELF1 in alternative splicing comes from studies investigating the role of CELF1 in the pathogenesis of the neuromuscular disease myotonic dystrophy type 1 (DM1). In this disease, aberrant gain of CELF1 function is combined with a corresponding loss of function of the splicing factor MBNL1, resulting in the mis-splicing of a number of crucial genes (reviewed in [11]). Minigene reporter systems that contain alternative splice sites proved to be a useful tools for the identification of pre-mRNA targets for CELF1, including genes for cardiac troponin T (TNNT2) [98], insulin receptor (INSR) [99], and chloride channel1 (CLCN1) [100],[101]. Interestingly, these genes were all shown to be mis-regulated in tissues from patients who suffered from DM1. Minigene systems have been particularly useful in demonstrating that individual pre-mRNA splicing events are affected by loss or gain of activities of specific regulatory proteins. Studies performed in cultured cells with transiently transfected minigenes have identified a number of alternative gene regions regulated by CELF1 and other family members [43], [69], [98], [99], [100], [101], [102], [103], [104], [105], [106],[107],[108]. However, as in other chimeric systems, the results of minigene overexpression experiments may not necessarily reflect the full-length pre-mRNA splicing patterns observed *in vivo*, especially during certain stages of organism development [109]. CELF proteins have been found to regulate the switch from fetal to adult splicing patterns of several skeletal muscle transcripts through the use of transgenic mouse models [101],[100],[110]*.* In mice, splicing microarray studies found that nearly half of transcripts that undergo fetal-to-adult alternative splicing transitions in heart respond to overexpression of CELF1, suggesting that the level of CELF1 activity directly regulates the alternative splicing pattern of endogenous transcripts [111]. The development of dominant negative (DN) and tissue specific transgenic mice was advantageous for studying CELFspecific alternative splicing *in vivo* [96],[104],[109],[110]. For example, dominant negative CELF (DNΔCELF) expressed under the control of a cardiac muscle-specific promoter, promoted the development of dilatated cardiomyopathy and cardiac dysfunction over time [96]. In contrast, when DNΔCELF was expressed under the control of a skeletal musclespecific promoter, mice exhibit reduction in muscle intersticia and an increase in slow twitch fibers [110]. In near future, we will see more phenotypic studies using a nucleus-restricted form of the dominant negative CELF protein which would specifically block only the CELF1 nuclear function, leaving the cytoplasmic function intact [112], [113], [114].

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 189

[122]. Since EDEN-BP in *Xenopus* is a regulator of deadenylation, it is likely that CELF1 also regulates deadenylation in mammalian cells, leading to transcript degradation through

Translation is a critical layer of post-transcriptional control of gene expression that is regulated in response to environmental and developmental changes. CELF proteins have been shown to be involved in the activation of translation of several mRNA species at various stages of development [124]. Additionally, CELF proteins have been shown to function as inhibitors of translation under conditions of stress, where they act as translational silencers in conjunction with other protein binding partners. The involvement of CELF1 in translational regulation is evolutionarily conserved, with several CELF1 homologues having been shown to regulate translation. For example, in the *Drosophila*  oocyte, translational repression is mediated by the protein Bruno (CELF1 orthologue), that binds specifically to bruno response elements (BREs) within the oskar mRNA 3'UTR. Binding by the CELF1 orthologue Bru-3 to GU-rich sequences in 3'UTR of gurken, cyclin A and oskar mRNA leads to their translational repression [44]. The suggested mechanism underlying Bru-3 mediated translational regulation is through the formation of a Bru-3/eIF4E/5'-cap translational silencing complex during specific stages of embryo development [125]. CELF1 has also been shown to play a role in translational regulation in mammalian cells. In human cell lines, tethering of CELF1 to the 3'UTR of mRNA through an interaction with the MS2 coat protein led to decreased steady state levels of reporter transcripts that contained a MS2 RNA-binding site, while reporter protein levels increased [126]. CELF1 increases the translation of p21 (CDKN1A28) protein [127], and Mef2a29 [128] during normal muscle cell differentiation via direct interaction with (GC)n repeats located within the 5'UTR of those mRNAs. The data suggests that CELF1 mediates translational regulation through interaction with a G- and C-rich motif in the 5'UTR, whereas CELF1 mediates its splicing and degradation effects through interaction with a G- and U-rich motif in introns and 3'UTRs, respectively. Our experiments determining CELF1 binding targets through high-throughput means have failed to identify enrichment of GC-rich motifs or 5'UTR binding by CELF1 [82]. It may be that CELF1 mediated translational regulation is rare, and only occurs in the context of very specific mRNA species and cellular contexts. Recently, an additional mechanism for CELF1 mediated translational regulation through interaction with the 3'UTR was discovered. Binding of CELF1 to the 3'UTR of Serine hydroxymethyltransferase (SHMT) RNA [129],[130] and cyclin dependent kinase inhibitor p27 (Kip1) RNA [131], was found to regulate internal ribosome entry site (IRES) mediated translation activation. This implicated CELF1 in participating in an IRES mode of initiation of mRNA translation. In addition, IRES translation is achieved through CELF1/hnRNPH complex formation, which promotes circularization of RNA transcripts by mediating 5'/3' ends interactions [129]. Whether CELF1 recruits the translation machinery to the 5'UTR via additional interaction with eIF2 (Eukaryotic Initiation Factor 2) or another initiation factor

unknown mechanisms.

remains to be determined (Figure 2).

**5.2. Translation**

## **5. CELF proteins as regulators of deadenylation, translation, and mRNA decay**

## **5.1. Deadenylation**

CELF1 plays important roles in mRNA stability and translation in diverse species. In eukaryotic organisms, the length of a transcript's polyA tail influences the translational state of a transcript, and deadenylation is regulated by GU-rich sequences and CELF1 proteins across evolution. Regulation of translation through deadenylation in *Xenopus* embryos is the best characterized model of posttranscriptional gene regulation by CELF proteins. In this model, the shortening or lengthening of the polyA tail causes activation or repression of translation of a number of transcripts [115]. However, as we describe below, deadenylation can also be the first step leading to mRNA degradation in mammalian cells, and the deadenylation machinery seems to be conserved although the consequences of deadenylation (translation or degradation) is regulated differently in diverse species.

In *Xenopus,* maternal transcripts are stored in the cytoplasm of oocytes in a translationally silent form (reviewed in [116]). After fertilization of *Xenopus* oocytes, the CELF1 homologue EDEN-BP binds to the EDEN element which activates transcript deadenylation and leads to the translation of EDEN-containing mRNA transcripts, including transcripts that encode important cell cycle regulators [31],[33],[50],[117],[118],[119]. Furthermore, human CELF1, which has 88% identity with EDEN-BP, was able to functionally substitute for EDEN-BP to mediate transcript deadenylation in *Xenopus* extracts [51], suggesting that the deadenylation function of GU-rich sequences and CELF proteins were conserved in diverse species.

Removal of the polyA tail is the rate-limiting step in the degradation of the majority of mammalian mRNAs [120],[121].In human cell lines, CELF1 has been shown to associate with the deadenylase enzyme polyA ribonuclease (PARN) and to stimulate polyA tail shortening in a cell-free assay using S100 extracts from human cells [52]. It is not known if CELF1 activates other deadenylases in mammalian cells or how deadenylated transcripts are subsequently degraded. PARN, EDEN-BP and cytoplasmic polyadenylation elementbinding proteins (CPEB) are present in *Xenopus* oocyte extracts [122], [123]. Theoretically, the balance between the rate of deadenylation versus polyadenylation depends upon the ability of EDEN-BP to recruit PARN and bind to polyA tail with higher affinity than CPEB [122]. Since EDEN-BP in *Xenopus* is a regulator of deadenylation, it is likely that CELF1 also regulates deadenylation in mammalian cells, leading to transcript degradation through unknown mechanisms.

#### **5.2. Translation**

188 Binding Protein

**decay**

**5.1. Deadenylation**

negative (DN) and tissue specific transgenic mice was advantageous for studying CELFspecific alternative splicing *in vivo* [96],[104],[109],[110]. For example, dominant negative CELF (DNΔCELF) expressed under the control of a cardiac muscle-specific promoter, promoted the development of dilatated cardiomyopathy and cardiac dysfunction over time [96]. In contrast, when DNΔCELF was expressed under the control of a skeletal musclespecific promoter, mice exhibit reduction in muscle intersticia and an increase in slow twitch fibers [110]. In near future, we will see more phenotypic studies using a nucleus-restricted form of the dominant negative CELF protein which would specifically block only the CELF1

**5. CELF proteins as regulators of deadenylation, translation, and mRNA**

CELF1 plays important roles in mRNA stability and translation in diverse species. In eukaryotic organisms, the length of a transcript's polyA tail influences the translational state of a transcript, and deadenylation is regulated by GU-rich sequences and CELF1 proteins across evolution. Regulation of translation through deadenylation in *Xenopus* embryos is the best characterized model of posttranscriptional gene regulation by CELF proteins. In this model, the shortening or lengthening of the polyA tail causes activation or repression of translation of a number of transcripts [115]. However, as we describe below, deadenylation can also be the first step leading to mRNA degradation in mammalian cells, and the deadenylation machinery seems to be conserved although the consequences of

deadenylation (translation or degradation) is regulated differently in diverse species.

function of GU-rich sequences and CELF proteins were conserved in diverse species.

Removal of the polyA tail is the rate-limiting step in the degradation of the majority of mammalian mRNAs [120],[121].In human cell lines, CELF1 has been shown to associate with the deadenylase enzyme polyA ribonuclease (PARN) and to stimulate polyA tail shortening in a cell-free assay using S100 extracts from human cells [52]. It is not known if CELF1 activates other deadenylases in mammalian cells or how deadenylated transcripts are subsequently degraded. PARN, EDEN-BP and cytoplasmic polyadenylation elementbinding proteins (CPEB) are present in *Xenopus* oocyte extracts [122], [123]. Theoretically, the balance between the rate of deadenylation versus polyadenylation depends upon the ability of EDEN-BP to recruit PARN and bind to polyA tail with higher affinity than CPEB

In *Xenopus,* maternal transcripts are stored in the cytoplasm of oocytes in a translationally silent form (reviewed in [116]). After fertilization of *Xenopus* oocytes, the CELF1 homologue EDEN-BP binds to the EDEN element which activates transcript deadenylation and leads to the translation of EDEN-containing mRNA transcripts, including transcripts that encode important cell cycle regulators [31],[33],[50],[117],[118],[119]. Furthermore, human CELF1, which has 88% identity with EDEN-BP, was able to functionally substitute for EDEN-BP to mediate transcript deadenylation in *Xenopus* extracts [51], suggesting that the deadenylation

nuclear function, leaving the cytoplasmic function intact [112], [113], [114].

Translation is a critical layer of post-transcriptional control of gene expression that is regulated in response to environmental and developmental changes. CELF proteins have been shown to be involved in the activation of translation of several mRNA species at various stages of development [124]. Additionally, CELF proteins have been shown to function as inhibitors of translation under conditions of stress, where they act as translational silencers in conjunction with other protein binding partners. The involvement of CELF1 in translational regulation is evolutionarily conserved, with several CELF1 homologues having been shown to regulate translation. For example, in the *Drosophila*  oocyte, translational repression is mediated by the protein Bruno (CELF1 orthologue), that binds specifically to bruno response elements (BREs) within the oskar mRNA 3'UTR. Binding by the CELF1 orthologue Bru-3 to GU-rich sequences in 3'UTR of gurken, cyclin A and oskar mRNA leads to their translational repression [44]. The suggested mechanism underlying Bru-3 mediated translational regulation is through the formation of a Bru-3/eIF4E/5'-cap translational silencing complex during specific stages of embryo development [125]. CELF1 has also been shown to play a role in translational regulation in mammalian cells. In human cell lines, tethering of CELF1 to the 3'UTR of mRNA through an interaction with the MS2 coat protein led to decreased steady state levels of reporter transcripts that contained a MS2 RNA-binding site, while reporter protein levels increased [126]. CELF1 increases the translation of p21 (CDKN1A28) protein [127], and Mef2a29 [128] during normal muscle cell differentiation via direct interaction with (GC)n repeats located within the 5'UTR of those mRNAs. The data suggests that CELF1 mediates translational regulation through interaction with a G- and C-rich motif in the 5'UTR, whereas CELF1 mediates its splicing and degradation effects through interaction with a G- and U-rich motif in introns and 3'UTRs, respectively. Our experiments determining CELF1 binding targets through high-throughput means have failed to identify enrichment of GC-rich motifs or 5'UTR binding by CELF1 [82]. It may be that CELF1 mediated translational regulation is rare, and only occurs in the context of very specific mRNA species and cellular contexts. Recently, an additional mechanism for CELF1 mediated translational regulation through interaction with the 3'UTR was discovered. Binding of CELF1 to the 3'UTR of Serine hydroxymethyltransferase (SHMT) RNA [129],[130] and cyclin dependent kinase inhibitor p27 (Kip1) RNA [131], was found to regulate internal ribosome entry site (IRES) mediated translation activation. This implicated CELF1 in participating in an IRES mode of initiation of mRNA translation. In addition, IRES translation is achieved through CELF1/hnRNPH complex formation, which promotes circularization of RNA transcripts by mediating 5'/3' ends interactions [129]. Whether CELF1 recruits the translation machinery to the 5'UTR via additional interaction with eIF2 (Eukaryotic Initiation Factor 2) or another initiation factor remains to be determined (Figure 2).

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 191

involved in the age-associated induction of the CELF1-eIF2 complex [136]. In the rat aging model, the CELF1-eIF2 complex binds to the 5'UTR of HDAC1 mRNA and increases histone deacetylase 1 protein levels in aging liver [136],[137]. It was further shown that during rat aging, CELF1 phosphorylation promotes its interaction with a GC-rich sequence in 5'UTR of p21 mRNA causing p21 translational arrest and senescence in fibroblasts [138]. In myocytes, p21 mRNA is stabilized in discrete cytoplasmic structures called stress granules, which serve as reversible storage sites for mRNA under conditions of stress. Interestingly, only during late senescence did p21s localization in stress granules interfere with its translation [138],[139]. One important component of stress granules is the RNA-binding protein T cell internal antigen 1 (TIA1). Consistent with CELF1's recruitment to stress granules, CELF1 has been shown to function as a translational silencer through interaction with the TIA1 protein [140]. Further support for this model comes from experiments utilizing DM1 cell harboring CUG repeat RNA. The presence of a CUG repeat expansion was found to cause stress and activation of the PKR-phospho-eIF2α–CELF1 pathway leading to stress granule formation and inhibition of mRNA translation [81]. This disruption to physiologic mRNA translation pathways by cellular stress signals might contribute to the progressive muscle loss in DM1 patients. Taken together, this data suggests that CELF proteins may function as

Bioinformatic analysis of short lived-transcripts in primary human T cells led to the identification of the conserved, GU-rich element (GRE) enriched in transcript's 3'UTRs. CELF1 was subsequently identified as a protein that specifically bound to the GRE *in vitro* and then to regulate the decay of exogenously expressed GRE-containing transcripts within cells [30],[141]. Further verification of the role of CELF1 in GRE-mediated mRNA decay came from the observation that in HeLa cells, siRNA-mediated knockdown of CELF1 led to stabilization of GRE-containing beta-globin reporter transcripts, as well as endogenous GRE-containing transcripts [30],[83],[142]. These results implicated CELF1 as a mediator of GRE-dependent mRNA decay. In primary human T cells, GREs and CELF1 appear to be involved in the rapid changes in gene expression patterns observed following T cell receptor-mediated activation. Identification the cytoplasmic binding targets of CELF1 before and after T cell activation led to the discovery that CELF1 dissociated from GREcontaining transcripts following T cell activation in a manner correlated with a transient upregulation of CELF1 target mRNAs [77]. The dissociation of CELF1 from its target transcripts upon T cell activation was the result of an activation-dependent phosphorylation of CELF1 and a resultant decrease in the ability of CELF1 to bind to GRE-containing RNAs [77]. Many of the transiently up-regulated CELF1 target transcripts encoded proteins necessary for the transition from a quiescent state to a state of cellular activation and proliferation. This supported a model whereby CELF1 suppresses a network of transcripts involved in activation and proliferation in resting T cells, and subsequent activationinduced phosphorylation of CELF1 allows for de-repression and accumulation of these

activators or repressors of translation, depending on the context.

**5.3. mRNA Decay**

transcripts within activated cells.

**Figure 2.** Simplified representation of the role of CELF1 in mRNA translation. Several mechanisms have been proposed for CELF1 mediated regulation of translation. Many of these mechanisms involve CELF1 interacting with mRNA through sequences in the 5'UTR and subsequent translation via an internal ribosomal entry site. If (hyper)/phosphorylated, CELF1 interacts with eIF2 and other translation initiation factors, this serves to promote the production of truncated protein products.

One well studied instance of CELF1 mediated translational control involves the translation of alternative isoforms of the transcription factor CCAAT/enhancer-binding protein (CEBPbeta) [132],[133],[134]. In a rat model, CELF1 phosphorylation was activated by partial hepatectomy, which promoted the formation of a complex between CELF1 and eIF2a. This subsequently led to selective translation of the liver enriched inhibitory protein (LIP) isoform of CCAAT/enhancer-binding protein [76]. It was later shown that in liver, CELF1 undergoes hyper-phosphorylation through a GSK3beta-cyclin D3-cdk4 kinase pathway, and the activity of this pathway seemed to increase with age [135]. Similar to the partial hepatectomy model, the cdk4-mediated hyper-phosphorylation of CELF1 was involved in the age-associated induction of the CELF1-eIF2 complex [136]. In the rat aging model, the CELF1-eIF2 complex binds to the 5'UTR of HDAC1 mRNA and increases histone deacetylase 1 protein levels in aging liver [136],[137]. It was further shown that during rat aging, CELF1 phosphorylation promotes its interaction with a GC-rich sequence in 5'UTR of p21 mRNA causing p21 translational arrest and senescence in fibroblasts [138]. In myocytes, p21 mRNA is stabilized in discrete cytoplasmic structures called stress granules, which serve as reversible storage sites for mRNA under conditions of stress. Interestingly, only during late senescence did p21s localization in stress granules interfere with its translation [138],[139]. One important component of stress granules is the RNA-binding protein T cell internal antigen 1 (TIA1). Consistent with CELF1's recruitment to stress granules, CELF1 has been shown to function as a translational silencer through interaction with the TIA1 protein [140]. Further support for this model comes from experiments utilizing DM1 cell harboring CUG repeat RNA. The presence of a CUG repeat expansion was found to cause stress and activation of the PKR-phospho-eIF2α–CELF1 pathway leading to stress granule formation and inhibition of mRNA translation [81]. This disruption to physiologic mRNA translation pathways by cellular stress signals might contribute to the progressive muscle loss in DM1 patients. Taken together, this data suggests that CELF proteins may function as activators or repressors of translation, depending on the context.

#### **5.3. mRNA Decay**

190 Binding Protein

**Figure 2.** Simplified representation of the role of CELF1 in mRNA translation. Several mechanisms have been proposed for CELF1 mediated regulation of translation. Many of these mechanisms involve CELF1 interacting with mRNA through sequences in the 5'UTR and subsequent translation via an internal ribosomal entry site. If (hyper)/phosphorylated, CELF1 interacts with eIF2 and other translation

One well studied instance of CELF1 mediated translational control involves the translation of alternative isoforms of the transcription factor CCAAT/enhancer-binding protein (CEBPbeta) [132],[133],[134]. In a rat model, CELF1 phosphorylation was activated by partial hepatectomy, which promoted the formation of a complex between CELF1 and eIF2a. This subsequently led to selective translation of the liver enriched inhibitory protein (LIP) isoform of CCAAT/enhancer-binding protein [76]. It was later shown that in liver, CELF1 undergoes hyper-phosphorylation through a GSK3beta-cyclin D3-cdk4 kinase pathway, and the activity of this pathway seemed to increase with age [135]. Similar to the partial hepatectomy model, the cdk4-mediated hyper-phosphorylation of CELF1 was

initiation factors, this serves to promote the production of truncated protein products.

Bioinformatic analysis of short lived-transcripts in primary human T cells led to the identification of the conserved, GU-rich element (GRE) enriched in transcript's 3'UTRs. CELF1 was subsequently identified as a protein that specifically bound to the GRE *in vitro* and then to regulate the decay of exogenously expressed GRE-containing transcripts within cells [30],[141]. Further verification of the role of CELF1 in GRE-mediated mRNA decay came from the observation that in HeLa cells, siRNA-mediated knockdown of CELF1 led to stabilization of GRE-containing beta-globin reporter transcripts, as well as endogenous GRE-containing transcripts [30],[83],[142]. These results implicated CELF1 as a mediator of GRE-dependent mRNA decay. In primary human T cells, GREs and CELF1 appear to be involved in the rapid changes in gene expression patterns observed following T cell receptor-mediated activation. Identification the cytoplasmic binding targets of CELF1 before and after T cell activation led to the discovery that CELF1 dissociated from GREcontaining transcripts following T cell activation in a manner correlated with a transient upregulation of CELF1 target mRNAs [77]. The dissociation of CELF1 from its target transcripts upon T cell activation was the result of an activation-dependent phosphorylation of CELF1 and a resultant decrease in the ability of CELF1 to bind to GRE-containing RNAs [77]. Many of the transiently up-regulated CELF1 target transcripts encoded proteins necessary for the transition from a quiescent state to a state of cellular activation and proliferation. This supported a model whereby CELF1 suppresses a network of transcripts involved in activation and proliferation in resting T cells, and subsequent activationinduced phosphorylation of CELF1 allows for de-repression and accumulation of these transcripts within activated cells.

In mouse myoblasts, cytoplasmic CELF1 bound hundreds of target transcripts that contained GU-rich sequences, including networks of transcripts that regulated cell cycle, intracellular transport and cell survival [55]. Knockdown of CELF1 in this myoblast cell line led to the stabilization of many endogenous GRE-containing targets, as well as luciferase reporter RNAs [88]. Many CELF1 target transcripts were found to be significantly stabilized in CELF1 knockout myoblasts, suggesting that CELF1 mediates the decay of a network of transcripts during myoblast growth and differentiation [55]. In the DM1 disease model, there is aberrant activation of the protein kinase C pathway as a result of the CTG expansion, and this results in CELF1 phosphorylation. Mouse myoblasts (C2C12 cells) made to express CTG expanded RNA were shown to experience stabilization of tumor necrosis factor alpha (TNF-alpha) mRNA [143]. This result suggested that the overexpression of TNF-alpha observed in DM1 could be coming from muscle, and this TNFalpha overexpression may contribute to the muscle wasting and insulin resistance that are characteristic of this disease [143]. In summary, CELF1 and its GRE-containing target transcripts define posttranscriptional regulatory networks that function to control cellular growth, activation, and differentiation (Figure 3).

CELF1, a Multifunctional Regulator of Posttranscriptional Networks 193