Introduction {#s1}
============

Small regulatory RNA pathways are highly conserved mechanisms present in most eukaryotic organisms and play an important role in post-transcriptional gene regulation. The gene-regulatory function of microRNAs (miRNAs) and short interfering RNAs (siRNAs) is mainly through translational repression or degradation of cytoplasmic mRNAs by an RNA-induced silencing complex (RISC). miRNA and siRNA pathways share a common RNase III processing enzyme, Dicer, and together with other proteins it constitutes RISC for gene transcriptional repression [@pone.0095350-Lamontagne1]. Dicer is responsible for recognizing a hairpin (in pre-miRNA) or long double-strand RNA (dsRNA), and processing them into miRNA-miRNA\* duplexes or siRNA duplexes [@pone.0095350-He1]. These small RNA duplexes are converted to a single-stranded form and bound to Argonaute (AGO), a key component of RISC, through a process coordinated by Dicer and other RNA-binding proteins [@pone.0095350-Hammond1]. Then small RNAs target specific mRNA sequences, leading to cleavage or translational repression of these [@pone.0095350-Wang1].

Dicer proteins are present in many eukaryotic organisms including plants, fungi, and metazoans [@pone.0095350-Cerutti1], [@pone.0095350-Shabalina1]. Vertebrates and nematodes have only one Dicer gene (Dicer1), whereas insects and flatworms possess two, (Dicer1 and Dicer2). Dicers normally contain a number of functional domains: an N-terminal DEAD box, an RNA helicase domain, a Dicer dimer domain, a Piwi-Argonaute-Zwille (PAZ) domain, two RNase III domains and a dsRNA binding domain [@pone.0095350-Bernstein1], [@pone.0095350-Margis1]. The crystal structure of Dicer from *Giardia intestinalis* revealed that the PAZ domain was responsible for binding of the 3′ terminus of dsRNA [@pone.0095350-Blaszczyk1]. After the 3′ end bound to the PAZ domain, pre-miRNAs or dsRNAs are cleaved by the two RNase III domains which form a single dsRNA processing center through intramolecular dimerization [@pone.0095350-Zhang1]. In Dicer1, binding of the PAZ domain to the 3′ terminus of pre-miRNA is crucial for orienting the RNase III domains for cleavage, however, recent publications have revealed that 5′ terminus recognition of pre-miRNAs is also important for mature miRNAs synthesis [@pone.0095350-MacRae1], [@pone.0095350-Park1].

Previous studies have focused on Dicers of plants and model organisms, little is known about Dicers of invertebrates. The recent availability of genome sequences of over 34 invertebrate species from 10 phyla, including 1 choanoflagellate, 2 cnidarians, 1 placozoan, 2 annelids, 1 mollusc, 7 platyhelminths, 7 nematodes, 10 arthropods, 1 echinoderm and 3 chordates, have allowed us to perform an extensive phylogenetic analysis of Dicers.

Materials and Methods {#s2}
=====================

Acquisition of sequence {#s2a}
-----------------------

For some well-annotated genomes, Dicer sequences were directly retrieved from the databases. In addition, BLASTP and TBLASTN were performed to search against their databases using *Drosophila melanogaster* Dicers (NP_524453 and NP_523778), *Caenorhabditis elegans* Dicer (NP_498761) or *Schistosoma mansoni* Dicers (Smp_169750.1 and Smp_033600) as query sequences. An E-value of 1×e-10 was used as a cutoff in BLAST searches and the hits were filtered to keep only those with at least 25% identity to the query sequence. Protein functional domains were identified using Pfam database and SMART database [@pone.0095350-Punta1], [@pone.0095350-Letunic1]. The species names, abbreviations and accession numbers are provided in [Table 1](#pone-0095350-t001){ref-type="table"}.

10.1371/journal.pone.0095350.t001

###### Distribution of endoribonuclease Dicer genes in invertebrates.

![](pone.0095350.t001){#pone-0095350-t001-1}

  Species                              Species Abbreviation          Gene Symbol           Accession Number    Length[a](#nt101){ref-type="table-fn"}   Data origin[b](#nt102){ref-type="table-fn"}
  ----------------------------------- ---------------------- --------------------------- -------------------- ---------------------------------------- ---------------------------------------------
  **Choanoflagellatea**                                                                                                                                
  * Monosiga brevicollis*                     Mbrev                       /                       /                              /                                           /
  **Cnidaria**                                                                                                                                         
  * Nematostella vectensis*                   NvecA                    Sca_186               ABZ10549^c^                        1099                                     NCBI JGI
                                              NvecB                   167046039              ABZ10551^c^                        1469                   
  * Hydra magnipapillata*                     HmagA                 HYDRA_212274             Hma2.212274                        835                                      Metazome
                                              HmagB                 HYDRA_205202             Hma2.205202                        769                    
                                              HmagC                 HYDRA_222700             Hma2.222700                        565                    
  **Placozoa**                                                                                                                                         
  * Trichoplax adhaerens*                     TadhA               TRIADDRAFT_18215           XP_002108754                       1018                                     NCBI JGI
                                              TadhB               TRIADDRAFT_51673           XP_002107798                       1418                   
                                              TadhC               TRIADDRAFT_51985           XP_002107959                       685                    
                                              TadhD               TRIADDRAFT_52058           XP_002108012                       915                    
                                              TadhE                     DCL-E                ABZ10547^c^                        707                    
  **Annelida**                                                                                                                                         
  * Capitella teleta*                          Ctel               CAPTEDRAFT_223153            ELU12939                         1651                                       NCBI
  * Helobdella robusta*                        Hrob           estExt_Genewise1.C_710069         103772                          1316                                        JGI
  **Mollusca**                                                                                                                                         
  * Lottia gigantea*                           Lgig                  gw1.12.81.1                61365                           1800                                        JGI
  **Platyhelminthes**                                                                                                                                  
  * Schmidtea mediterranea*                   Smed1                    Dicer-1               ASA.00018.01                       1328                                      SmedGD
                                              Smed2                    Dicer-2             mk4.000125.07.01                     1589                   
  * Schistosoma mansoni*                      Sman1                    Dicer-1               Smp_169750.1                       2319                                      Sanger
                                              Sman2                    Dicer-2                Smp_033600                        928                    
  * Schistosoma japonicum*                    Sjap1                    Dicer-1               Sjp_0069770                        2480                                      Sanger
                                              Sjap2                    Dicer-2               Sjp_0043700                        923                    
  * Echinococcus granulosus*                  Egra1                    Dicer-1              EgrG_000085200                      1904                                      Sanger
                                              Egra2                    Dicer-2              EgrG_000181800                      1924                   
  * Echinococcus multilocularis*              Emul1                    Dicer-1              EmuJ_000085200                      1906                                      Sanger
                                              Emul2                    RNC3.1               EmuJ_000180900                      1812                   
                                              Emul2                    RNC3.2               EmuJ_000181800                      1929                   
  * Hymenolepis microstoma*                   Hmic1                    Dicer-1              HmN_000252400                       1897                                      Sanger
                                              Hmic2            Endoribonuclease Dicer       HmN_000200100                       1183                   
  * Taenia solium*                            Tsol1                    Dicer-1              TsM_000872800                       1890                                      Sanger
                                              Tsol2            Endoribonuclease Dicer       TsM_000756400                       829                    
  **Nematoda**                                                                                                                                         
  * Caenorhabditis elegans*                    Cele                    Dicer-1                NP_498761                         1910                                       NCBI
  * Bursaphelenchus xylophilus*                Bxyl                    Dicer-1              BUX_s00116.153                      1040                                      Sanger
  * Pristionchus pacificus*                    Ppac                    Dicer-1              WBGene00096444                      2769                                     WormBase
  * Strongyloides ratti*                       Srat                    Dicer-1                  g5271                           1824                                     WormBase
  * Brugia malayi*                             Bmal                Dicer-1/Bm5026           WBGene00225287                      1930                                     WormBase
  * Trichinella spiralis*                     TspiA                   Tsp_01223              XP_003377020                       2029                                       NCBI
                                              TspiB                   Tsp_00125              XP_003375890                       1903                                       NCBI
  * Loa loa filariasis*                        Lloa                  LOAG_02227              XP_003137813                       1928                                       NCBI
  **Arthropoda**                                                                                                                                       
  * Daphnia pulex*                            DpulA               DAPPUDRAFT_308316            EFX72380                         1979                                     NCBI JGI
                                              DpulB               DAPPUDRAFT_329028            EFX69538                         1459                   
                                              DpulC               DAPPUDRAFT_309030            EFX86072                         1604                   
  * Pediculus humanus corporis*                Phum                    Dcier-1               XP_002429494                       2179                                       NCBI
  * Tribolium castaneum*                      Tcas1                    Dicer-1                XP_968993                         1865                                       NCBI
                                              Tcas2                    Dicer-2               NP_001107840                       1623                   
  * Nasonia vitripennis*                      Nvit1                    Dicer-1               XP_001605287                       1917                                       NCBI
                                              Nvit2                    Dicer-2               XP_001602524                       1450                   
  * Acyrthosiphon pisum*                      ApisA                 LOC100159500             XP_001943370                       1626                                       NCBI
                                              ApisB                 LOC100166428             XP_001945890                       1691                   
  * Drosophila melanogaster*                  Dmel1                    Dicer-1                NP_524453                         2249                                   NCBI FlyBase
                                              Dmel2                    Dicer-2                NP_523778                         1772                   
  * Anopheles gambiae*                        Agam1                AgaP_AGAP002836           XP_003436256                       2336                                       NCBI
                                              Agam2                AgaP_AGAP012289            XP_320248                         1672                   
  * Aedes aegypti*                            Aaeg1                AaeL_AAEL006794           XP_001652212                       1658                                       NCBI
                                              Aaeg2                AaeL_AAEL001612           XP_001659747                       2193                   
  * Culex pipiens quinquefasciatus*           Cpip1               CpipJ_CPIJ003169           XP_001844757                       2270                                       NCBI
                                              Cpip2               CpipJ_CPIJ010534           XP_001855187                       1165                   
  **Echinodermata**                                                                                                                                    
  * Strongylocentrotus purpuratus*             Spur                   LOC586001               XP_790894                         1850                                     Metazome
  **Chordata**                                                                                                                                         
  * Branchiostoma floridae*                    Bflo               BRAFLDRAFT_202604          XP_002610617                       1868                                       NCBI
  * Ciona intestinalis*                       Ciona            Endoribonuclease Dicer     ENSCINP00000017117                    1872                                     Ensemble
  * Saccoglossus kowalevskii*                  Skow               Sakowv30031161m.g        Sakowv30031161m                      1905                                     Metazome

Amino acid length;

JGI: Joint Genome Institute; NCBI: National Center for Biotechnology Information; SmedGD: *Schmidatea mediterranea* Genome Database; Sanger: Wellcome Trust Sanger Insitute; FlyBase: Drosophila database; SilkDB: silkworm database; Ensemble: Ensemble Genome Browser.

Sequence alignment and phylogenetic analysis {#s2b}
--------------------------------------------

The data sets contained a total of 58 sequences from 34 species (in a size from 565aa to 2769aa, [Text S1](#pone.0095350.s001){ref-type="supplementary-material"}). The amino acid sequences of Dicer were aligned by MUSCLE [@pone.0095350-Edgar1] with default parameters and manually optimized by Jalview 2.8 [@pone.0095350-Waterhouse1]. The alignments were subsequently processed using Gblocks v0.91b [@pone.0095350-Castresana1] for phylogenetic reconstruction, allowing gaps in 1/2 of the sequences. ProtTest 3.2 was applied to find an appropriate model of amino acid substitution for tree building analysis [@pone.0095350-Darriba1]. A maximum likelihood tree was constructed using PhyML 3.0 program [@pone.0095350-Guindon1]. Clade support was calculated using SH-like approximate likelihood ratio test, Bayes likelihood test and bootstrap proportions (500 replicates).

Results {#s3}
=======

Identification and distribution of Dicer genes across invertebrates {#s3a}
-------------------------------------------------------------------

The final data sets contained 58 Dicer gene sequences from two cnidarians, one placozoa, two annelids, one mollusc, seven platyhelminths, seven nematodes, eleven arthropods, one echinoderm and three chordates ([Table 1](#pone-0095350-t001){ref-type="table"}). No Dicer homologues were identified in the choanoflagellate *Monosiga brevicollis*. Our results of genomic database searches revealed that one placozoan, two annelids, one mollusc, one echinoderm and three of the chordates investigated possessed only one Dicer1 gene. Each of nematodes had only one Dicer1 gene, except *Trichinella spiralis*, which expressed both Dicer1 and Dicer2 genes. Platyhelminths and arthropods possessed two Dicer genes in their genomes, with the exceptions of *Daphnia pulex* (three genes), *Pediculus humanus corporis* (one gene) and *Echinococcus multilocularis* (three genes).

Phylogenetic analysis of Dicers {#s3b}
-------------------------------

As shown in the Maximum likelihood tree ([Fig. 1](#pone-0095350-g001){ref-type="fig"}), Dicers of invertebrates were grouped into two lineages: Dicer1 subfamily and Dicer2 subfamily. Almost all of the arthopods and platyhelminths surveyed possessed one member of each of these subfamilies, and annelids, molluscs, nematodes, echinoderms and chordates investigated had only one Dicer gene that belongs to Dicer1 subfamily. The placozoan *Trichoplax adhaerens* had the most copies of Dicer genes in our investigated species; however, all of the five Dicer genes were classed into the Dicer2 subfamily. The two cnidarians *N. vectensis* and *Hydra magnipapillata* each had only one Dicer2 gene, but possessed other Dicer genes that fell outside the two subfamilies.

![A maximum likelihood tree of invertebrate Dicers.\
The tree was constructed using maximum likelihood method. Two number sets, 1.00/1.00/0.99 and 0.87/0.96/0.28, at the nodes were SH-like approximate likelihood ratio, Bayes likelihood and bootstrap values, respectively.](pone.0095350.g001){#pone-0095350-g001}

Organization of functional domains of Dicer family {#s3c}
--------------------------------------------------

We identified the functional domains using the Pfam database and confirmed each inferred domain using the SMART database. As shown in [Fig. 1](#pone-0095350-g001){ref-type="fig"}, Dicers had significant variability in domain organization. For instance, Dicers initially characterized in humans are multidomain proteins, consisting of an N-terminal DEAD box, an RNA helicase domain, a Dicer dimer domain, a PAZ domain, two RNase III domains and a dsRNA binding domain [@pone.0095350-Zhang1]. However, *Taenia solium* Dicer2 processed only one RNase III domain. We also observed the loss of the DEAD domain, which contains two RecA-like domains as a catalytic core and can regulate various processes involving RNA [@pone.0095350-Kurimoto1], in Dicer1 of mollusks, annelids, platyhelminths and most arthropods.

A PAZ domain is an RNA-binding module found in PPD proteins (PAZ and Piwi domain proteins) and Dicer orthologs, and anchors the 2-nucleotide 3′ overhang of dsRNA with its highly conserved binding pocket [@pone.0095350-Zhang1]. After searching annotated domains using Pfam and SMART databases, we did not find the PAZ domain in Dicer2 of the platyhelminths *Schmidtea mediterranea, Hymenolepis microstoma*, *T. solium*, *Echinococcus granulosus*, *E. multilocularis*, the placozoan *T. adhaerens* and the nematode *T. spiralis*. There are two possibilities: the sequences are too divergent to be clearly recognized or they may have lost the PAZ domain during evolution. We therefore aligned the key amino acid residues in the PAZ domain of Dicers in the above species. As shown in [Fig. 2a](#pone-0095350-g002){ref-type="fig"}, most of the key residues in the PAZ domain were conserved in Dicer2 sequences with the exceptions of *S. mediterranea* and *T. adhaerens*, indicating the absence of the PAZ domain in those two species. Recently studies have revealed that human Dicer anchors not only the 3′ end but also the 5′ end, and the 5′ end recognition by Dicer is important for the precise and effective biogenesis of miRNAs [@pone.0095350-Park1]. A previous study revealed that the 5′ binding residues (Arg778, Arg780 and Arg811 within the N-terminal extension of PAZ domain, and Arg996 and Arg1003 within PAZ domain) were conserved across invertebrate Dicer1 and absent in other Dicers [@pone.0095350-Park1]. However, we found that these five key residues were present in *N. vectensis* DicerB, which was classed into the Dicer2 family ([Fig. 2b](#pone-0095350-g002){ref-type="fig"}).

![The key residues critical for recognition of 3′ and 5′ pockets.\
The key residues involved in 3′ pocket (a) and 5′ pocket recognition (b) were indicated using asterisks in red. The Dicers in which a PAZ domain was not identified using Pfam and SMART are highlighted in grey (a). Gaps are filled using question marks (?).](pone.0095350.g002){#pone-0095350-g002}

After dsRNA binding to the 5′ and 3′ pockets, two RNase III domains of Dicer cleave targeted molecules. Based on the alignment of Dicer RNase III domain, we found that the catalytic core in most invertebrates was highly conserved ([Fig. 3](#pone-0095350-g003){ref-type="fig"}). However, *Schistosoma mansoni* Dicer1, *S. mediterranea Dicer1*, *T. solium* Dicer2 and *E. multilocularis* Dicer RNC3.1 showed variations in this key region ([Fig. 3](#pone-0095350-g003){ref-type="fig"}). Compared to platyhelminths, the RNase III domain seemed to be divergent in cnidarians, and most of the key residues were altered in *H. magnipapillata* DicerC ([Fig. 3](#pone-0095350-g003){ref-type="fig"}), indicating the possibility of loss of dsRNA cleavage ability. During the cleavage of targeted molecules by sRNAs, two RNase III domains of Dicer form a tight dimer of which the subunit interface is hydrophobic [@pone.0095350-Blaszczyk1]. The crystal structure showed that a tight dimer was formed by two *Aquifex aeolicus* RNase III proteins, each of which possessed only one RNase III domain. A total of 128 hydrophobic interactions (\<4.0 Å) were found between the two molecules, whereas only 20 hydrogen bonds/salt bridges existed at the dimer interface. In the dimer, two identical "ball-and-socket" junctions were formed at each end of the interface. The 'ball' was the hydrophobic side chain of Phe41 and the 'socket' was a cavity formed by side chains of Val52, Val56, Leu67, Ser68, and Lys71 [@pone.0095350-Blaszczyk1]. Subsequent studies showed that a Met1317 within the human RNase III a domain was located in the position of the 'ball' residue and the corresponding socket residues Thr1717, Tyr1721, Leu1732, Thr1732 and Arg1736 were located in RNaseIII b domain [@pone.0095350-Ji1]. Interestingly, we found that the 'ball' residue in the RNase III a domain of platyhelminths Dicer1 was replaced by a hydrophilic amino acid-threonine, whereas the corresponding 'socket' residues were still conserved ([Fig. 4](#pone-0095350-g004){ref-type="fig"}).

![The key residues of RNase III domains.\
Catalytic residues are marked by red asterisks and gaps filled using question marks (?).](pone.0095350.g003){#pone-0095350-g003}

![The residues of "ball and socket" junction.\
The 'Ball' residue within RNaseIII a domain is marked by a black asterisk, and the 'socket' residues within RNase III b domain are marked by red asterisks. Gaps are filled using question marks (?).](pone.0095350.g004){#pone-0095350-g004}

Discussion {#s4}
==========

In our study, the number of the Dicer genomic loci was variable, from one in several invertebrates to five in *T. adhaerens*. Dicers of invertebrates were clearly classed into two subfamilies, Dicer1 and Dicer2, except for several Dicers from cnidarians. Our results support the model of Dicer evolution in which a eukaryote Dicer may have duplicated independently. Interestingly, Dicer2 of *E. multilocularis* may have undergone duplication after species formation. Mature miRNAs have been identified in all the invertebrates investigated in our research with the exceptions of the choanoflagellate *M. brevicollis* and the placozoan *T. adhaerens* [@pone.0095350-Grimson1]. Similarly, we failed to find Dicer genes and other RISC proteins genes in the genome of *M. brevicollis*, a close known relative of metazoans. However, *T. adhaerens*, a simple known metazoan, possessed five Dicer proteins and all of them belonged to Dicer2 subfamily. These Dicers may constitute an immune defense mechanism against viral infection as placozoans are exposed to a high viral load [@pone.0095350-deJong1].

Both cnidarians possessed multiple Dicers, and only *N. vectensis* DicerB and *H. magnipapillata* DicerB were classed into Dicer2 group, while the others fell outside the two recognized subfamilies. Recent analysis has revealed that cnidarians express species-specific miRNAs and share few miRNA families with bilateria [@pone.0095350-Krishna1], [@pone.0095350-Moran1]. These distinct cnidarian Dicers may provide some clues to understanding of the biogenesis of species-specific miRNAs.

The recognition and cleavage of dsRNA by Dicer is a core step in miRNA and siRNA pathways. The 3′ pocket of Dicer is involved in 3′ end binding of dsRNA. The absence of key sites in the pocket in *S. mediterranea* Dicer2 and *T. adhaerens* DicerA could lead to loss of the binding ability. But these two Dicer2 genes may function with the help of other RNA binding protein, such as Drosha [@pone.0095350-Han1]. The 5′ pocket is positioned in close proximity to the 3′ pocket on the same surface of Dicer1, and the binding residues of the 5′ pocket are conserved [@pone.0095350-Park1]. Interestingly, we found most of the key binding residues of the 5′ pocket, which were previously found only in Dicer1, in *N. vectensis* DicerB that belonged to Dicer2 lineage. It suggests that *N. vectensis* DicerB may retain the bioactivities of Dicer1 as well.

After dsRNA recognition by the conserved pockets, the cleavage of dsRNA is conducted by two RNase III domains [@pone.0095350-Ji2]. The loss of the catalytic residue-aspartate in *E. multilocularis* Dicer RNC3.1 could reduce its catalytic activity. However, Dicer RNC3.2, a paralogue of Dicer RNC3.1, possessed all the key residues, and therefore it may compensate for the reduced activity of Dicer RNC3.1. The dimerization of RNase III domains creates a catalytic valley which can accommodate a dsRNA substrate. The two "ball and socket" junctions may be responsible in part for the accurate positioning of the catalytic residues in the valley. A Previous study suggested that the 'ball' consisted of the hydrophobic side chain of amino acids, and the interaction between 'ball' and 'socket' was precluded by charged side chain or the absence of any side chain in the position of the 'ball' [@pone.0095350-Blaszczyk1]. Interestingly, we found that the 'ball' within the RNase III a domain. In platyhelminths Dicer1 proteins contained a hydrophilic side chain of threonine, but the residues of the hydrophobic 'socket' were conserved across different species. This substitution may results in reduced affinity between the 'ball' and the hydrophobic 'socket' and altered conformation.

Supporting Information {#s5}
======================

###### 

**Amino acid sequences of Dicers.**

(TXT)

###### 

Click here for additional data file.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: YD ZG YZ. Analyzed the data: ZG MW DB. Contributed reagents/materials/analysis tools: ZG. Wrote the paper: ZG YZ DB.
