# RNA DISEASES IN HUMANS – FROM FUNDAMENTAL RESEARCH TO THERAPEUTIC APPLICATIONS

EDITED BY : Naoyuki Kataoka, Akila Mayeda and Kinji Ohno PUBLISHED IN : Frontiers in Molecular Biosciences and Frontiers in Genetics

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# RNA DISEASES IN HUMANS – FROM FUNDAMENTAL RESEARCH TO THERAPEUTIC APPLICATIONS

Topic Editors: Naoyuki Kataoka, The University of Tokyo, Japan Akila Mayeda, Fujita Health University, Japan Kinji Ohno, Nagoya University, Japan

This Research Topic addresses the human diseases caused by a malfunction of the RNA metabolism. We aim at strengthening the link between fundamental research and therapeutic applications.

In eukaryotes, RNA is transcribed from genomic DNA. RNA molecules undergo multiple post-transcriptional processes such as splicing, editing, modification, translation, and degradation. A defect, mis-regulation, or malfunction of these processes often results in diseases in humans, referred to as 'RNA diseases'. There is an increasing number of studies focused on RNA diseases, which are aimed at uncovering the fundamental molecular mechanisms at play in order to develop therapeutic approaches.

Citation: Kataoka, N., Mayeda, A., Ohno, K., eds. (2019). RNA Diseases in Humans – From Fundamental Research to Therapeutic Applications. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-097-4

# Table of Contents

*05 Editorial: RNA Diseases in Humans—From Fundamental Research to Therapeutic Applications*

Naoyuki Kataoka, Akila Mayeda and Kinji Ohno

### CHAPTER 1

### RNA SPLICING

So Masaki, Shun Ikeda, Asuka Hata, Yusuke Shiozawa, Ayana Kon, Seishi Ogawa, Kenji Suzuki, Fumihiko Hakuno, Shin-Ichiro Takahashi and Naoyuki Kataoka

*55 HMGA1a Induces Alternative Splicing of the* Estrogen Receptor-αlpha *Gene by Trapping U1 snRNP to an Upstream Pseudo-5*′ *Splice Site* Kenji Ohe, Shinsuke Miyajima, Tomoko Tanaka, Yuriko Hamaguchi, Yoshihiro Harada, Yuta Horita, Yuki Beppu, Fumiaki Ito, Takafumi Yamasaki, Hiroki Terai, Masayoshi Mori, Yusuke Murata, Makito Tanabe, Ichiro Abe, Kenji Ashida, Kunihisa Kobayashi, Munechika Enjoji, Takashi Nomiyama, Toshihiko Yanase, Nobuhiro Harada, Toshiaki Utsumi and Akila Mayeda

### CHAPTER 2

### TRANSLATION

*63 Translation of Hepatitis a Virus IRES is Upregulated by a Hepatic Cell-Specific Factor*

Akitoshi Sadahiro, Akira Fukao, Mio Kosaka, Yoshinori Funakami, Naoki Takizawa, Osamu Takeuchi, Kent E. Duncan and Toshinobu Fujiwara

*73 Micropeptides Encoded in Transcripts Previously Identified as Long Noncoding RNAs: A New Chapter in Transcriptomics and Proteomics* Fouzia Yeasmin, Tetsushi Yada and Nobuyoshi Akimitsu

### CHAPTER 3

### LONG NON-CODING RNA

*83 Identification of Minimal* p53 *Promoter Region Regulated by MALAT1 in Human Lung Adenocarcinoma Cells*

Keiko Tano, Rena Onoguchi-Mizutani, Fouzia Yeasmin, Fumiaki Uchiumi, Yutaka Suzuki, Tetsushi Yada and Nobuyoshi Akimitsu

*93 Wnt/*β*-catenin Signaling Pathway Regulates Specific lncRNAs That Impact Dermal Fibroblasts and Skin Fibrosis* Nathaniel K. Mullin, Nikhil V. Mallipeddi, Emily Hamburg-Shields,

Beatriz Ibarra, Ahmad M. Khalil and Radhika P. Atit

*106 Distinct and Modular Organization of Protein Interacting Sites in Long Non-coding RNAs*

Saakshi Jalali, Shrey Gandhi and Vinod Scaria

# Editorial: RNA Diseases in Humans—From Fundamental Research to Therapeutic Applications

Naoyuki Kataoka1 \*, Akila Mayeda2 and Kinji Ohno3

*1 Laboratory of Cell Regulation, Departments of Applied Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan, 2 Division of Gene Expression Mechanism, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan, 3 Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan*

Keywords: RNA, RNA disease, splicing, non-coding RNA (ncRNA), translation, virus

**Editorial on the Research Topic**

#### Edited by:

*William Cho, Queen Elizabeth Hospital (QEH), Hong Kong*

#### Reviewed by:

*Venugopal Thayanithy, Medical School, University of Minnesota, United States Mohammadreza Hajjari, Shahid Chamran University of Ahvaz, Iran*

\*Correspondence: *Naoyuki Kataoka<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Molecular Biosciences*

> Received: *15 May 2019* Accepted: *26 June 2019* Published: *16 July 2019*

#### Citation:

*Kataoka N, Mayeda A and Ohno K (2019) Editorial: RNA Diseases in Humans—From Fundamental Research to Therapeutic Applications. Front. Mol. Biosci. 6:53. doi: 10.3389/fmolb.2019.00053* **RNA Diseases in Humans—From Fundamental Research to Therapeutic Applications**

In higher eukaryotes, many different RNAs are encoded in and transcribed from genomic DNAs. Transcribed RNA molecules undergo multiple post-transcriptional processes such as splicing, editing, modification, translation, and degradation. A defect, mis-regulation, or malfunction of these processes often results in diseases in humans, and recently they have been referred to as "RNA diseases" (Scotti and Swanson, 2016; Kataoka, 2017; Ohno et al., 2018). There is an increasing number of studies focused on RNA diseases, which are aimed at unraveling the fundamental molecular mechanisms and developing therapeutic approaches. The goal of this special issue is to introduce RNA diseases to not only RNA scientists but also clinical scientists who are interested in seeking for cures of the diseases.

Pre-mRNA splicing is one of the major processes for post-transcriptional regulatory steps in eukaryotes. Defects in this step result in many diseases, such as neuromuscular diseases and cancers. Splicing takes place in a large multi-RNA-protein complex, called spliceosome. Over one hundred proteins are involved in this step, and many splicing regulatory proteins have been reported. It has been demonstrated that cancer cells undergo cancer-specific splicing patterns. In this special issue on RNA diseases, El Marabti and Younis describe distinct cases of alternative splicing in cancer. They report several categories of splicing aberrations causing alterations in cancer-related genes, mutations in genes encoding splicing factors or core spliceosomal subunits and the disruptions of the balance of RNA-binding proteins. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by ineffective hematopoiesis, peripheral blood cytopenia and a high risk of progression to acute myeloid leukemia (AML). Recently it has been shown that mutations on splicing factors are causes of MDS. SRSF2 is one of the major responsible genes for MDS. Masaki et al. found that MDS associated mutations in SRSF2 alter binding affinity to CCWG sequence in RNA and cause aberrant splicing of a subset of genes including EZH2. Estrogen Receptor (ER) α has a critical role in majority of breast cancer. Ohe et al. analyzed the alternative splicing of ERα by using in vitro splicing assays and found that HMGA1a induced exon skipping of a shortened exon 1 of ERα. Regulation of ERα alternative splicing by an HMGA1a gives us a novel insight on 5′ splice site regulation by U1 snRNP, as well as a possible target in breast cancer therapy.

**5**

Pre-mRNA splicing and associated proteins are also involved in tissue development and diseases. Su et al. describe the importance of alternative splicing during neuronal differentiation. Alternative splicing modulates signaling activity, centriolar dynamics, and metabolic pathways, and it also contributes to neurogenesis and brain development. This review shed light on how splicing defects cause brain disorders and diseases. Among those diseases, one of the most famous disease genes is Fused in sarcoma (FUS). FUS is an RNA binding protein that regulates RNA metabolism including alternative splicing, transcription, and RNA transportation. This protein is genetically and pathologically involved in frontotemporal lobar degeneration (FTLD)/amyotrophic lateral sclerosis (ALS). Ishigaki and Sobue summarize the functions of FUS protein in alternative splicing, transcription, mRNA destabilization, axonal transport, and morphological maintenance of neurons. They conclude that a biological link between loss of FUS function, Tau isoform alteration, aberrant post-synaptic function, and phenotypic expression might lead to the sequential cascade culminating in FTLD. RNA splicing factor is also involved in muscle differentiation and cardiomyopathy. RBM20 is a vertebrate-specific RNA-binding protein, and it has initially been identified as one of dilated cardiomyopathy (DCM)-linked genes. RBM20 is a regulator of heart-specific alternative splicing, and one of the major targets for RBM20 is the titin (TTN) gene. As titin is the most important factor for passive tension of cardiomyocytes, extensive heart-specific and developmentally regulated alternative splicing of the TTN pre-mRNA by RBM20 plays a critical role in passive stiffness and diastolic function of the heart. Manipulation of the Ttn pre-mRNA splicing raises RBM20 as a potential therapeutic target (Watanabe et al.).

Translation of mRNA is another critical step for gene expression. Translation of most mRNAs in cells are mediated by the cap structure at the 5′ terminus. However, translation of some cellular mRNAs and viral mRNAs are cap-independent. Internal ribosome entry site (IRES) can recruit eukaryotic initiation factor (eIF) complex and ribosome subunit on mRNA in a cap-independent manner. Hepatitis A virus (HAV) utilizes IRES-dependent translation, but unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3′ end of HAV RNA is polyadenylated. Sadahiro et al. analyzed HAV-IRESmediated translation in a cell-free system derived from either non-hepatic cells or hepatoma cells and revealed that HAV IRESmediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Their results strongly suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Recently, it has been well-accepted that long non-coding RNAs (lncRNAs) also play important roles in gene expression in cells. Some of them can be good markers for cancer progression (Yoshimoto et al., 2016). In the manuscript by Tano et al. demonstrated that MALAT1 has a function in repressing the promoter of p53 (TP53) tumor suppressor gene. The p53 targets were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells, which were mediated by transcriptional activation of p53 through MALAT1 depletion. They also identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity.

On the other hand, the expression of lncRNAs is also regulated by signal cascade. Mullin et al. identified lncRNAs and protein-coding RNAs that are induced by β-catenin activity in mouse dermal fibroblasts. They identified 111 lncRNAs that are differentially expressed in response to activation of Wnt/βcatenin signaling. Among them, two novel Wnt signaling-Induced Non-Coding RNA (Wincr) transcripts, Wincr1 and Wincr2, were validated. These two lncRNAs are highly expressed in mouse embryonic skin and perinatal dermal fibroblasts, and Wincr1 expression levels in perinatal dermal fibroblasts affects the expression of key markers of fibrosis. These results suggest that β-catenin signaling-responsive lncRNAs modulate dermal fibroblast behavior and collagen accumulation in dermal fibrosis, providing nodes for therapeutic intervention.

The lncRNAs are thought to exhibit their functions as RNA-Protein complex. Jalali et al. utilized the available genomescale experimental datasets of RNA binding proteins (RBP) to understand the role of lncRNAs in terms of its interactions with RBPs. Their analysis suggests that density of interaction sites for the RBPs was significantly higher for specific sub-classes of lncRNAs when compared to protein-coding transcripts. The significant enrichment of RBP sites across some lncRNA classes strongly suggests that these interactions might be important in the functional roles of lncRNA such as silencing, mRNA processing, and transport.

Interestingly, lncRNAs also serve as templates of micropeptides. In the review article by Yeasmin et al. they summarize the current progress and view of micropeptides encoded in putative short open reading frames (sORFs) within transcripts previously identified as lncRNAs or transcripts of unknown function (TUFs). There are several lines of evidence for significant divergent roles of micropeptides in many fundamental biological processes and even important relationships with pathogenesis.

In conclusion, RNA and RNA binding proteins are involved in most of gene expression steps in higher eukaryotes. The mutations in them cause RNA diseases in human. We sincerely hope the increasing numbers of scientific findings including papers in this special issue will shed light on the disease mechanisms and lead to the development of novel therapeutic strategies for them.

### AUTHOR CONTRIBUTIONS

NK, AM, and KO wrote the paper. NK took the primary responsibility for the final content. NK, AM, and KO read and approved the final manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kataoka, Mayeda and Ohno. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Cancer Spliceome: Reprograming of Alternative Splicing in Cancer

Ettaib El Marabti and Ihab Younis\*

*Biological Sciences Program, Carnegie Mellon University in Qatar, Doha, Qatar*

Alternative splicing allows for the expression of multiple RNA and protein isoforms from one gene, making it a major contributor to transcriptome and proteome diversification in eukaryotes. Advances in next generation sequencing technologies and genome-wide analyses have recently underscored the fact that the vast majority of multi-exon genes under normal physiology engage in alternative splicing in tissue-specific and developmental-specific manner. On the other hand, cancer cells exhibit remarkable transcriptome alterations partly by adopting cancer-specific splicing isoforms. These isoforms and their encoded proteins are not insignificant byproducts of the abnormal physiology of cancer cells, but either drivers of cancer progression or small but significant contributors to specific cancer hallmarks. Thus, it is paramount that the pathways that regulate alternative splicing in cancer, including the splicing factors that bind to pre-mRNAs and modulate spliceosome recruitment. In this review, we present a few distinct cases of alternative splicing in cancer, with an emphasis on their regulation as well as their contribution to cancer cell phenotype. Several categories of splicing aberrations are highlighted, including alterations in cancer-related genes that directly affect their pre-mRNA splicing, mutations in genes encoding splicing factors or core spliceosomal subunits, and the seemingly mutation-free disruptions in the balance of the expression of RNA-binding proteins, including components of both the major (U2 dependent) and minor (U12-dependent) spliceosomes. Given that the latter two classes cause global alterations in splicing that affect a wide range of genes, it remains a challenge to identify the ones that contribute to cancer progression. These challenges necessitate a systematic approach to decipher these aberrations and their impact on cancer. Ultimately, a sufficient understanding of splicing deregulation in cancer is predicted to pave the way for novel and innovative RNA-based therapies.

Keywords: splicing, cancer spliceome, alternative splicing, exons, introns

### SPLICING IS AN EVOLUTIONARY CONSERVED AND ESSENTIAL STEP IN GENE EXPRESSION IN EUKARYOTES

Most genes in eukaryotes contain intervening sequences (introns) that disrupt the expressed sequences (exons). Introns in eukaryotes are much longer (median size ∼1,000 bp but can be >100,000 bp) compared to exons (median size ∼120 bp), making introns the major contributors to the sequence of genes. After transcription, in order for the expressed transcript

#### Edited by:

*Naoyuki Kataoka, The University of Tokyo, Japan*

#### Reviewed by:

*Daisuke Kaida, University of Toyama, Japan Rahul N. Kanadia, University of Connecticut, United States Claudia Ghigna, Istituto di Genetica Molecolare (IGM), Italy*

> \*Correspondence: *Ihab Younis<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Molecular Biosciences*

Received: *30 April 2018* Accepted: *09 August 2018* Published: *07 September 2018*

#### Citation:

*El Marabti E and Younis I (2018) The Cancer Spliceome: Reprograming of Alternative Splicing in Cancer. Front. Mol. Biosci. 5:80. doi: 10.3389/fmolb.2018.00080* (pre-mRNA) to become a suitable message for downstream processes such as translation of the encoded protein, the premRNA from any multi-exon gene has to undergo extensive processing to remove the introns by an extraordinary molecular machine, the spliceosome. Given their large size, introns add a long time, sometimes hours, to the transcription process of genes in eukaryotes. Thus, introns present a conundrum as their transcription, just to be spliced out and degraded, seems to be a wasteful process both in terms of the time it takes to transcribe them and the energy consumed in their transcription as well as their removal and degradation. In addition, the splicing process needs to be extremely efficient as well as be executed with high fidelity. Efficiency is required to make sure all introns are removed from the pre-mRNA on time and in a coordinated manner. Fidelity is of paramount importance because joining exons with any mistake of even one base could have catastrophic effects on the reading frame. Furthermore, the cis-sequences or splice sites at the boundaries of each intron are too simple, sometimes degenerate and highly redundant outside the actual splice sites to serve alone as efficient landmarks for spliceosome assembly. Taken together, these features of introns and splicing in general make the presence of introns in eukaryotes counterintuitive. However, introns are not simply extra sequences that are removed by splicing, but rather have several advantages such as coupling multiple RNA processing events for higher gene expression efficiency as well as regulation and providing a checkpoint for quality control of the mRNA. They also allow any gene that harbor them to have a tremendous capacity for diversification through the process of alternative splicing. Thus, it is likely that the advantages of harboring introns outweigh the disadvantages as their presence in eukaryotic genomes and to some extent their position in the genes are highly conserved (Fedorov et al., 2002; Rogozin et al., 2003), in some cases between humans and the plant Arabidopsis thaliana.

### DIVERSIFICATION OF TRANSCRIPTOMES BY ALTERNATIVE SPLICING

The advent of high-throughput sequencing has uncovered the fact that most multi-exon genes in eukaryotes undergo at least one event of alternative splicing (Pan et al., 2008), generating two or more distinct mRNAs from the same gene, with the number of alternatively spliced transcripts potentially staggering for some genes. Interestingly, many such transcripts are expressed in a tissue-specific manner, at specific developmental stage, or in a disease-specific manner (Castle et al., 2008; Wang et al., 2008). While the function of some of these alternative transcripts is not always immediately interpretable or even recognized, a plethora of work indicates that alternatively spliced exons are translated and tend to encode important domains in the encoded polypeptide (Kalsotra and Cooper, 2011; Ellis et al., 2012; Weatheritt et al., 2016; Tapial et al., 2017). This suggests an evolutionary conserved molecular design for transcriptome diversification without the need to expand the genome that would require creating genes that are homologous to existing ones that serve similar yet distinct functions (Nilsen and Graveley, 2010).

Alternative splicing is a term used to collectively refer to several splicing events. As shown in **Figure 1**, there are various distinct forms of alternative splicing, including alternative exons (cassette exons: skipped/included whole exons), retained introns, and alternative 5′ and 3′ splice sites (5′ ss and 3′ ss). There are also several less obvious alternative splicing events that are tightly coupled to and could be a consequence of transcription regulation such as alternative first and last exons. Nevertheless, all these events are well documented in eukaryotes with remarkable impacts on transcriptome diversification. One class of alternative splicing, intron retention, is often overlooked because it is interpreted as a splicing mistake that lead to an intron not being spliced out. While this might be true in several cases, a lot of evidence points to intron retention being regulated to control the expression of genes post-transcriptionally. In fact, cancer cells of all types are characterized by high levels of retained intron, leading to a higher diversity of their transcriptomes compared to normal cells (Dvinge and Bradley, 2015).

Intron retention and its regulation are obvious in a class of introns referred to as minor or U12 introns, which are conserved in almost all eukaryotes. Unlike the vast majority of introns in cells, which rely on the canonical spliceosome that is composed of U1, U2, U4, U5, and U6 snRNPs for their splicing, minor introns utilize a less abundant and seemingly less efficient spliceosome that is made of U11, U12, U4atac, U5, and U6atac snRNPs. Around 800 minor introns in the human genome are embedded in genes that function in signal transduction and information relay, cell cycle control and DNA damage repair (Turunen et al., 2013). We previously showed that hundreds of U12 introns are extremely conserved and are used as molecular switches that provide rapid control of gene expression that does not depend on transcription of new pre-mRNA especially when the gene product is needed instantaneously such as when cells are under stress (Younis et al., 2013). Given the functions of the genes that host minor introns, it is likely that they are regulated in a similar fashion in cancer.

### MECHANISMS OF SPLICING REGULATION

It is of note that some documented alternative splicing events constitute only a small fraction of the processed mRNA that are expressed at any given time. While this suggests that such alternative splicing events represent the expected biological noise of a process that is extremely active in cells, we argue that these events are tightly regulated and serve significant roles in various cell types and tissues. More specifically, the low abundance of these events in one cell type could have evolved to be so because the encoded protein from these specific splicing isoforms have a cell type- or condition-specific function. In addition, some of these events are only expressed at a high level when cells are faced with certain environmental conditions, such as stress, in when the specific splicing isoform becomes absolutely required (Younis et al., 2013). Thus, exhaustive searches are now needed to identify these conditions in which these isoforms become abundant and their function more significant. Finally, some disease tissues show enrichment of these events, suggesting specific functions.

This infers that the abundance of specific alternatively spliced transcripts as well as the choice of the specific alternative splicing events for a given pre-mRNA are under tight regulation. This regulation is dictated by both cis-elements in the pre-mRNA itself and trans-factors such as RNA binding proteins (RBPs). The fact that the human genome encodes for thousands of RBPs, of which a big fraction functions in RNA splicing and its regulation strongly supports the notion that alternative splicing is not random but rather a highly regulated process and a key step in gene expression regulation.

Splicing factors historically have been classified into hnRNPs, which typically suppress splicing, and SR proteins, which tend to have a positive role in splicing regulation. However, a more thorough analysis of the function of any given hnRNP or SR protein quickly reveals that they do not always conform to these classifications. The ultimate role of an RBP in splicing regulation depends on multiple factors. These include the strength and context of its binding sites on the pre-mRNA in addition to either competitive or cooperative binding of multiple RBPs on or around the regulated exon or intron. This combinatorial regulation makes it very hard to predict the splicing outcome of reduced or increased binding of a single splicing factor in normal or diseased cells. Another complication is that a splicing factor is likely to regulated pre-mRNA splicing of other splicing factors in endless feedback loops and complex networks. A better approach to understand the regulation of alternative splicing in a given condition requires a systems biology approach in which the expression status and targets of multiple if not all RBPs be assessed to start building these networks of co-regulated pathways.

### DEREGULATION OF ALTERNATIVE SPLICING IN CANCER

All data to date indicate that alternative splicing is a welldesigned process that is tightly regulated in order to produce a network of alternatively spliced transcripts, which we refer to in this review as the splice-ome (spliceome). Work in the last two decades have moreover showed that the spliceome is significantly altered in disease states, such as cancer (Reviewed in, David and Manley, 2010; Chabot and Shkreta, 2016; Scotti and Swanson, 2016). In fact, every hallmark of cancer can be represented by several examples of proto-oncogenes, tumor suppressor genes, or other genes whose splicing is altered to produce isoforms that are needed for the transformation process (Oltean and Bates, 2014). In this review, we do not aim to provide a comprehensive list of all the cancer-related abnormal alternative splicing events, but rather highlight a few TABLE 1 | Selected examples of genes with cancer-related alternatively spliced isoforms.

*(Continued)*

TABLE 1 | Continued

that exemplify a deregulated splicing program in cancer that is not a byproduct of the cancer phenotype but a driving force in cancer development and maintenance (**Table 1**). We thus discuss four main categories of splicing aberrations: (1) Cancerspecific splicing alterations in oncogenes and tumor suppressor genes. (2) Cancer-specific mutations in splicing factors. (3) Changes in upstream signaling pathways that deregulate splicing factors. And (4) aberrations in spliceosomal components that are linked to cancer. The involvement of alternative splicing in the 10 hallmarks of cancer has been reviewed elsewhere (Sveen et al., 2016). Here we summarize some of these changes to point to the fact that the cancer phenotype in several cancer types is heavily reliant on altering one or several splicing choices.

### CANCER-SPECIFIC SPLICING ALTERATIONS IN ONCOGENES AND TUMOR SUPPRESSOR GENES

Some of the earliest and most studied examples of alternative splicing events that lead to isoforms amiable for cancer are in genes involved in apoptosis such as the members of the Bcl2 family and several caspases. For example, intron 2 of the Bcl2L1 gene, which encodes the Bcl-X protein, is alternatively spliced. More specifically, the spliceosome has a choice between two 5 ′ splice sites (5′ ss) for intron 2. Depending on which 5 ′ ss is chosen, the mRNA produced could be large (Bcl-XL), which encodes a Bcl-X protein with anti-apoptotic function, or small (Bcl-XS), encoding a Bcl-X protein that is missing an essential BH domain and is pro-apoptotic. Another example is Caspase 2 pre-mRNA splicing, whereas the spliceosome faces a choice of including exon 9, generating a caspase 2L mRNA or skipping exon 9, leading to the caspase 2S isoform. The large isoform encodes the pro-apoptotic Casp2L protein, whereas the anti-apoptotic Casp2S protein is encoded by the short isoform. Given that cancer cells are resistant to cell death by apoptosis, they need to ensure the production of Bcl-XS and/or Casp2L. In the absence of mutations in Bcl2L1 and Caspase 2 genes that would affect splice sites or other cis-elements leading to Bcl-XS and Casp2L production, cancer cells reprogram the splicing machinery and/or splicing factors that bind to these pre-mRNAs to ensure that the cancer-specific isoforms are enriched.

Several tumor suppressor genes undergo alternative splicing in cancer that leads to either complete or partial loss of function. For example, complex alternative splicing of TP53, which encodes the p53 protein, generates several isoforms with significant impact on the protein function (Surget et al., 2013). Once activated, by DNA damage for example, p53 can induce cell-cycle arrest in either the G1 or G2 phase of the cell cycle. p53 can also activate Growth Arrest and DNA Damage 45 (GADD45), which regulates cell-cycle arrest in the G2/M phases. Thus, the presence of a functional p53 is essential for the multiple cell cycle checkpoints that allow cells to repair DNA damage or commit to apoptosis. Some of the protein products from the TP53 splicing isoforms are dominant negative, and since p53 acts as a tetramer, the production of these dominant negative subunits, even at low level, can have dramatic effects as they act as poison subunits. Four isoforms of these p53 transcripts are depicted in **Figure 2**.

Interestingly, even tumor viruses take advantage of alternative splicing to produce oncoproteins that cause host cell transformation. For example, the production of the two Human Papilloma Virus (HPV) oncoproteins E6 and E7 in patient tissues, which are encoded by one pre-mRNA, depends on alternative splicing. Briefly, unspliced transcripts (that is, the intron is retained) produce the E6 mRNA (and ORF) whereas complete splicing of the pre-mRNA produces the E7 mRNA and ORF. Other transcripts including E6∧E7 or E6∗ III are generated due to alternative 3′ -splice usage (Graham and Faizo, 2017).

These few examples underscore the capacity of alternative splicing to produce two or more proteins from a single gene that could have completely opposite functions with major consequences on cell fate and the transformation process.

### CANCER-SPECIFIC MUTATIONS OR ALTERATIONS IN SPLICING FACTORS

Given that cancer cells do reprogram the spliceome, it is not surprising that splicing factors are common targets for deregulation in this disease (Dvinge et al., 2016).

The SR protein SRSF2, for example, is a splicing factor that is commonly mutated in a collection of neoplastic diseases or cancers of immature blood cells known as Myelodisplastic Syndromes (MDS). Interestingly, mutations in SRSF2 that alter its sequence specificity on its target pre-mRNAs are more likely to be linked to MDS than nonsense mutations, indicating that a gain-of-function (binding to differential pre-mRNA targets) rather than loss-of-function of SRSF2 produces a new set of alternatively spliced mRNAs that are relevant to MDS development (Kim et al., 2015).

Of note, not all changes in splicing factors are due to mutations in their encoding genes as mutation-free disruptions in the repertoire of RNA-binding proteins (splicing factors) due to the imbalance in their expression is emerging as a common feature in many diseases including cancer. For example, frequent upregulation of mutation-free SRSF2 is a driver in the development of Hepatocellular Carcinoma (HCC) (Luo et al., 2017).  These alterations in splicing factors, whether due to mutations or altered expression, tend to have large effects on cell phenotype as these splicing factors bind to and regulate the splicing of hundreds of pre-mRNAs. Thus, cancer cells can alter the splicing of a large number of genes by deregulating a handful of splicing factors. While this might seem to be an overkill, evidence does point to the fact that among the thousands of changes, some have distinct and significant effects on the transformation process. For example, SRSF2 mutants in MDS lead to mis-splicing of hundreds of pre-mRNAs, but one of them, the EZH2 pre-mRNA, encoding a transcriptional regulator that

isoforms with various alternative first exons can be generated for p53 pre-mRNA. In this case, the first exon is what is usually exon 5 in the canonical transcript, leading to the production of p53 protein lacking TADs, PXXP, and part of DBD. This truncated p53 is expected to be dominant negative. A similar protein can be encoded by transcripts in which intron 2 is retained, leading to the usage of start codon in exon 4 rather than the canonical start codon in exon 2. On the other hand, retention of intron 9 and/or inclusion of the cryptic intronic exon 9, i9, change the reading frame causing the loss of the encoded amino acids from exons 10 and 11. The resulting p53 proteins lack OD and NRD. These truncated p53 proteins could compete with wild type p53 for DNA binding but are not functional as they cannot oligomerize.

is required for maintaining the repressed state of many genes during hematopoiesis, stands out. Hematopoietic cells expressing SRSF2 mutants show higher inclusion of a highly conserved "poison" exon in the EZH2 mRNA, leading to degradation of the mRNA by nonsense-mediated decay and loss-of-function of the EZH2 gene (Kim et al., 2015).

hnRNP proteins also play their share in cancer progression. For example, mis-regulation of a number of hnRNP proteins have been linked to HCC tumor progression, whereas the overexpression of hnRNP A1 in particular has been linked to tumor invasion and metastasis (Zhou Z. J. et al., 2013). The detailed contribution of the RNA splicing-dependent effects of mis-regulation of many hnRNPs in cancer is still under intense investigation by several laboratories and should shed some light on mechanisms as well as potential novel therapeutic targets.

Of note here is that mutations in splicing factors in MDS patients typically cause distinct and sometimes non-overlapping splicing defects, suggesting an alternate underlying mechanism. Indeed, a recent study has uncovered that mutations on distinct splicing factors in MDS commonly cause elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway (Chen et al., 2018). These effects can lead to deregulated transcription pause release, raising the possibility that the MDS phenotype is related to a transcriptional defect rather than a splicing one.

### CHANGES IN UPSTREAM SIGNALING PATHWAYS THAT DEREGULATE SPLICING FACTORS

In order to ensure that several splicing factors and other cellular processes are deregulated, the signaling pathways that relay extracellular signals to splicing factors are often targeted in cancer. The SR protein family is often deregulated as the function of SR proteins tightly depends on their phosphorylation status, which itself is regulated by upstream kinases. For example, the splicing of the cassette exons in Caspase 9 pre-mRNA is regulated by the splicing factor SRSF1 leading to either caspase 9a or caspase 9b mRNAs. SRSF1 is itself phosphorylated upon activation of multiple signaling pathways, including the PI3K/AKT pathway. Since AKT signaling is often constitutively activated in cancers, such as lung cancer, this leads to constitutive phosphorylation of SRSF1 and deregulated expression of Caspase 9a/9b (Shultz et al., 2010).  This deregulated Caspase 9a/9b ratio has marked consequences on apoptosis and contributes to the ability of cancer cells to resist cell death.

Interestingly, several key components of signaling pathways that are typically deregulated in cancer can themselves be alternatively spliced to produce cancer-specific isoforms. For example, the inclusion of exon 6 in the pre-mRNA of the First Apoptosis Signal (Fas) receptor produces an isoform that encodes a membrane bound receptor that plays a key role in relaying extracellular signal that lead to programmed cell death. On the other hand, the Fas isoform with exon 6 being skipped encodes a soluble protein that does not induce apoptosis upon relevant signaling. Epidermal Growth Factor Receptor (EGFR), Insulin Receptor (INSR), Receptor d'Origine Nantais (RON), and Vascular Endothelial Growth Factor Receptor (VEGFR) are among several receptor tyrosine kinases whose splicing is altered in cancer leading to tumor progression or reduced response to therapy (reviewed in, Abou-Fayçal et al., 2017). In the case of VEGFR, one intron retention leads to the production of a shorter and decoy receptor that is dominant negative (Kendall et al., 1996; Vorlová et al., 2011). Similarly, alternative splicing in EGFR pre-mRNA produces several isoforms, some of which are dominant negative whereas others are constitutively active, leading to enhanced tumorgenicity, migration and invasion (Guillaudeau et al., 2012a,b; Piccione et al., 2012; Zhou M. et al., 2013; Zhou Z. J. et al., 2013; Padfield et al., 2015).

### ABERRATIONS IN SPLICEOSOMAL COMPONENTS THAT ARE LINKED TO CANCER

It is remarkable that loss-of-function mutations in core components of the spliceosome are not compatible with life, which speaks to the critical role the spliceosome plays in all cells. However, components of the spliceosome can be mutated without complete loss-of-function leading to widespread alterations in splicing and disease.

Patients with MDS, chronic myelomonocytoic leukemia (CMML), or chronic lymphocytic leukemia (CLL) acquire mutations in the spliceosomal components SF3B1, SF1, PRPF40B, and U2AF35 besides mutations in the splicing factor SRSF2 and ZRSR2, a component of U11/U12 di-snRNP, (Armstrong et al., 2018). Interestingly, SF3B1 and U2AF35 mutation tend to be missense mutations and mutually exclusive, again suggesting that cells with severe aberrations in spliceosome function are not viable (Armstrong et al., 2018). These mutations are drivers in cancer and they strongly correlate with prognosis and clinical phenotype.

On the other hand, several genetic diseases are linked to mutations in core components of the spliceosome. These include retinitis pigmentosa, a progressive neurodegeneration of Rod photoreceptors in the retina, which is linked to mutations in PRPF31, PRPF8, BRR2, PRPF4, or PRPF3. Spinal Muscular Atrophy (SMA) is a severe neurodegenerative disease caused by mutations in SMN1 gene, which encodes a protein that functions in the biogenesis of spliceosomal snRNPs and reduced SMN function in cells has been shown to lead to widespread aberrations in splicing. Mutations in one of the snRNA components of the minor spliceosome, U4atac, have been identified and linked to severe mental retardation and dwarfism, microcephalic osteodysplastic primordial dwarfism type 1 (TALS/MOPD). Despite their low abundance in cells, minor introns are highly conserved and serve as critical molecular switches for the expression of genes that harbor them (Younis et al., 2013). Some of these genes are bona fide oncogenes and tumor suppressor genes, suggesting a role for deregulating minor intron splicing in cancer (Unpublished data).

### SYSTEMATIC APPROACHES FOR IDENTIFYING SPLICING ABERRATIONS THAT ARE LINKED TO CANCER

Genetic alterations or mutations in cancer patients that affect the splicing of one gene are relatively easy to study, track, and even propose therapeutic tactics based on fixing the splicing of that one pre-mRNA. However, a major challenge emerges when the alteration is in a splicing factor or core component of the spliceosome as these lead to global (thousands) alterations in splicing affecting a wide range of genes. Still more challenging are cases where the expression of the splicing factors is altered without an obvious underlying genetic mutation. Two of the many challenges are: (1) identifying amongst the thousands of splicing alterations those that significantly contribute to cancer progression, and (2) therapeutically target the splicing factors without having massive side effects that sometimes are worse than the actual disease itself. In order to successfully address these points, it is important to develop a systematic and standardized approach to gain sufficient understanding of splicing deregulation in cancer and their impact on cancer.

Before the advent of next generation sequencing of RNA (referred to here as RNA-seq), transcriptome profiling to identify global splicing changes relied mostly on Gene Expression Exon Microarrays, that contain probes for almost all exons and many introns. While these arrays were a major advance over traditional microarrays with limited number of probes per gene, they tend to be very hard to interpret and generate a lot of false positives if not properly analyzed and replicated. Also, the lack of a large number of probes in introns causes these microarrays to miss a major category of splicing aberration that is intron retention. Nowadays, the gold standard for transcriptome profiling that includes both information on genome-wide expression level changes as well as splicing disruptions is RNA-seq. While this method is both quantitative and qualitative, it has its own challenges as well. As a start, generating the libraries for RNAseq from high quality RNA is expensive, tedious, and require well trained personnel. The data generated is massive in size (several gigabytes per sample) and requires powerful computing machines for storage as well as analysis. The analysis itself is a major bottleneck. Several off-the-shelf pipelines exist for mapping raw reads and analysis of differential expression of genes. However, these only scratch the surface and do not fully take advantage of the wealth of data generated by any welldesigned RNA-seq experiment. For example, there are several publicly available algorithms that attempt to identify splicing alterations from RNA-seq, but our personal experience with these is that they all fail at capturing the real picture as most of them use statistical models that are not suitable for biological systems, leading to identification of an endless list of statistically significant but small changes that have no or little impact on the phenotype. Thus, many laboratories have opted for their own inhouse pipelines that are suited for their own analysis but remain far from suited to apply globally. The major challenge in the analysis of RNA-seq data does not detract from the fact that it has been widely used to generate important databases of splicing alterations in many cancers. The list of splicing aberrations in cancer will grow and our understanding of the molecular basis of these changes as well as their contribution to cancer will improve tremendously in the coming years as our ability to standardize the analysis pipeline improves. In fact, we propose that identifying the right splicing isoforms can be so powerful, they should be used as novel biomarkers for many cancer types and subtypes.

Once enough molecular understanding of the splicing aberrations is gained and their impact on cancer is proven,

### REFERENCES

innovative RNA-based therapies are required to correct the splicing alterations or induce splicing changes in cancer cells that make them more susceptible to traditional chemotherapy. Only recently, RNA-based therapies, which include a range of mechanisms such as antisense oligonucleotides, RNAi, antimiRNA, miRNA mimics, aptamers, ribozymes, and others, seemed far-fetched and unpractical. However, the recent success of antisense oligonucleotides in correcting the splicing of exon 7 of SMN gene in SMA patients and its approval by the FDA speaks to the power of such therapies. In order to start applying such strategies to cancer, it is better to focus first on a few targets with large effects on phenotype. For example, given the large contribution of mis-splicing of genes involved in apoptosis on the ability of cancer cells to resist cell death, these genes are the low hanging fruit. In addition, it is noted that splicing alterations would rarely be the sole driver in cancer progression, we thus do not suggest the use of RNA-based therapies to overcome splicing aberrations as an alternate to traditional therapies, but rather a combination therapy for more effective treatment (we anticipate the effects to be synergistic) with less unfavorable side effects.

### AUTHOR CONTRIBUTIONS

EE curated and compiled information toward completion of this review. IY wrote the review.

### ACKNOWLEDGMENTS

This publication was made possible by the generous support of the Qatar Foundation through Carnegie Mellon University in Qatar's Seed Research program. The statements made herein are solely the responsibility of the authors.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 El Marabti and Younis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Alternative Splicing in Neurogenesis and Brain Development

#### Chun-Hao Su1 , Dhananjaya D1,2 and Woan-Yuh Tarn1,2 \*

*1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 2 Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan*

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

#### Edited by:

*Naoyuki Kataoka, The University of Tokyo, Japan*

#### Reviewed by:

*Hidehito Kuroyanagi, Tokyo Medical and Dental University, Japan Ihab Younis, Carnegie Mellon University in Qatar, Qatar*

> \*Correspondence: *Woan-Yuh Tarn<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Molecular Biosciences*

Received: *17 November 2017* Accepted: *25 January 2018* Published: *12 February 2018*

#### Citation:

*Su C-H, D D and Tarn W-Y (2018) Alternative Splicing in Neurogenesis and Brain Development. Front. Mol. Biosci. 5:12. doi: 10.3389/fmolb.2018.00012* Keywords: alternative splicing, splicing factors, neurogenesis, neuronal differentiation, neuronal migration, neuronal development

### INTRODUCTION

Alternative splicing is a crucial step of post-transcriptional gene expression that substantially increases transcriptome diversity and is critical for diverse cellular processes, including cell differentiation and development as well as cell reprogramming and tissue remodeling. Our understanding of the physiological significance and disease implications of alternative splicing has been greatly improved by genetic approaches and RNA deep sequencing. In this review, we focus on alternative splicing in neuronal differentiation from stem/progenitor cells, neuronal migration and functional development of neurons (**Figure 1**).

### Alternative Splicing and Its Role in Development

Approximately 95% of human multi-exon genes undergo alternative splicing of precursor mRNAs (pre-mRNAs) (Pan et al., 2008; Wang et al., 2008). In mammals, alternative splicing involves differential use of intron splice sites or the inclusion/exclusion of exons. Alternatively spliced mRNAs may generate protein isoforms with distinct and perhaps antagonistic functions or with altered stability or subcellular localization (Dredge et al., 2001; Matlin et al., 2005). In addition, alternative splicing may introduce premature termination codons into the resulting

mature mRNAs, leading to mRNA downregulation via nonsensemediated decay (Lareau et al., 2007). Alternative splicing is governed by the interplay between trans-acting splicing regulators and cis-elements of pre-mRNAs (Matera and Wang, 2014). In general, a splicing activator may enhance splice-site recognition or utilization by the spliceosome, whereas a splicing suppressor may prevent the association of spliceosomal factors with pre-mRNAs or compete off splicing activators. Moreover, alternative splicing is also influenced by transcription rate, histone modifications, and chromatin structure (Kornblihtt et al., 2004, 2013; Luco et al., 2010). Alternative splicing may occur in a tissue- or developmental-specific manner or in response to cellular signals and no doubt plays critical roles in many cellular processes (Nilsen and Graveley, 2010).

Alternative splicing provides a means to differentiate gene expression between cell types during development. Tissuespecific regulation of alternative splicing involves the coordinated actions of splicing factors. Cell type-specific or timely expression of certain splicing regulators is important for precise control of alternative splicing. For example, the RNA-binding protein CUGBP and ETR-3-like factor 1 (CELF1) and muscleblind-like 1 (MBNL1) exhibit switched expression during heart development to regulate splicing of cardiac mRNAs (Kalsotra et al., 2008). Forced expression of embryonic CELF1 or ablation of MBNL1 in the adult mouse heart reverts splicing toward embryonic/early postnatal patterns (Kalsotra et al., 2008). Similarly, switching of splicing regulators also occurs in the developing brain (see below). Thus, temporal control of alternative splicing is critical for fetal-to-adult transitions during development. Coordinated splicing networks contribute substantially to the development of various tissues and organs as well as their physiology.

Splicing abnormalities are linked to human genetic diseases, including brain disorder (Raj and Blencowe, 2015; Vuong et al., 2016). For example, familial dysautonomia is caused by a 5′ splice site mutation of the IKBKAP gene (Slaugenhaupt et al., 2001). This mutation reduces IKBKAP expression via alternative splicing-coupled nonsense-mediated decay, and hence downregulates a set of cell migration-related genes (Anderson et al., 2001; Yoshida et al., 2015). Gene abnormalities in the splicing factor RBFOX1 gene have been linked to autism spectrum disorder and additional neuromuscular abnormalities (Barnby et al., 2005; Martin et al., 2007; Conboy, 2017). The associations between splicing defects and human disease have been reviewed extensively elsewhere, and will not be emphasized in this review.

### Experimental Insights into the Role of Alternative Splicing in Brain Development

Emerging new technologies for RNA studies have greatly enhanced our knowledge of alternative splicing in development. Capture of specific mRNA ribonucleoproteins followed by highthroughput sequencing or splicing microarrays has identified dynamic alternative splicing programs during cell differentiation or development and also revealed the tissue-specific or developmentally regulated RNA-binding landscapes of splicing factors (Rossbach et al., 2014). Use of knockout and transgenic mice has identified the targets and physiological roles of neuronal splicing regulators and revealed how their defects impact brain development and neuronal function (**Table 1**). Moreover, genetic tagging with a reporter provides a tool for isolating specific cell types for transcriptome comparison (Wang et al., 2011). For example, by using Tbr2 promoter-driven green fluorescent protein as a tracer, neural progenitor cells (NPCs) can be distinguished from neurons in the developing brain (Zhang et al., 2016). Recently, single-cell profiling techniques enabled the resolution of population heterogeneity and revealed insights into cellular differentiation and development (Darmanis et al., 2015). Computational analysis of deep-sequencing data and annotated databases helped establish the correlation between genetic mutations, splicing variants, and disease (Kircher et al., 2014; Mort et al., 2014). Recently, an unbiased "deep-learning" computational method provided a more powerful link between rare single-nucleotide variations and neurological disorders such as spinal muscular atrophy and autism spectrum disorder (Xiong et al., 2015). Advanced sequencing tools would likely facilitate the detection of cell type- and stimulus-dependent splicing changes and perhaps the identification of previously unrecognized splicing products such as circular RNAs during neuronal development (van Rossum et al., 2016).

### Neuronal Differentiation Involves Coordinated Changes in the Expression of Splicing Factors

Genome-wide transcriptome analysis has revealed an exceptionally high level of alternative splicing in the mammalian brain (Yeo et al., 2004). The nervous system adopts alternative splicing for cell differentiation, morphogenesis, the formation of complex neuronal networks, and the establishment/plasticity of delicate synapses (Norris and Calarco, 2012; Zheng and Black, 2013). Splicing regulation may involve some neuron-specific splicing factors and their interplay with ubiquitous factors (Raj and Blencowe, 2015; Vuong et al., 2016). A switch from predominant expression of PTBP1 to its neuronal paralog PTBP2 (nPTB), which occurs during differentiation of progenitor cells into postmitotic neurons, is important for the stem cell-toneuron transition (Boutz et al., 2007; Vuong et al., 2016). PTBP1 is downregulated by the neuron-specific microRNA miR-124 (Makeyev et al., 2007). Notably, PTBP1 suppresses the inclusion of exon 10 of PTBP2, producing an exon 10-skipped mRNA that is susceptible to nonsense-mediated decay (**Figure 2**). Thus, PTBP1 restricts the level of PTBP2 in non-neuronal cells or NPCs. RBM4 is a ubiquitous RNA-binding protein, but its level is elevated during neuronal differentiation of mouse embryonal carcinoma P19 cells (Tarn et al., 2016). Interestingly, RBM4 acts in the same manner as PTBP1 to suppress exon 11/10 of PTBP1/PTBP2 in myoblast cells, and it downregulates PTBP1/PTBP2 levels (Lin and Tarn, 2011; **Figure 2**). However, during neuronal differentiation of mesenchymal stem cells, RBM4 induces the skipping of mammalian-specific exon 9 of PTBP1, which produces a functional PTBP1 isoform with compromised splicing activity compared with full-length PTBP1 (Su et al., 2017). Therefore, RBM4 attenuates the activity of PTBP1 in splicing regulation (Su et al., 2017; **Figure 2**). Notably, PTBP2 does not contain an exon equivalent of exon 9 of PTBP1, so PTBP2 is likely resistant to regulation by RBM4 during stem cell differentiation. On the other hand, the neural-specific SR-related protein of 100 kDa (nSR100/SRRM4) promotes exon 10 inclusion of PTBP2 and thus maintains PTBP2 level in neurons (Calarco et al., 2009).

PTBP1 and PTBP2 regulate overlapping but distinct repertoires of splicing events. PTBP1 suppresses the splicing of a subset of neural targets to inhibit neuronal differentiation. PTBP2 expression is elevated in differentiating neuronal cells and activates certain neural targets that promote differentiation (Boutz et al., 2007). Nevertheless, PTBP2 is downregulated as cells mature and undergo synaptogenesis. This sequential downregulation of PTBP1 and PTBP2 is important for two transitions of splicing regulation throughout neuronal differentiation and maturation and for functional expression of postsynaptic density protein-95 (PSD-95) via splicing control (Zheng et al., 2012). Both RBM4 and PTBP1 have preference for CU-rich cis-elements and hence antagonize each other during splicing regulation; thus, in general, they function oppositely in cell differentiation.

Besides the above, the neuron-specific splicing regulator Nova-1 can negatively autoregulate its own expression by suppressing exon 4 inclusion (Dredge et al., 2005). A study revealed that RBM4 promotes Nova-1 exon 4 inclusion during differentiation and maturation of brown adipocytes (Lin J. C. et al., 2016), but whether this regulation occurs in neurons is unclear. Moreover, all three Rbfox family members exploit a conserved mechanism of splicing autoregulation to produce a splice isoform with a truncated RNA-recognition motif; this isoform has dominant-negative activity in splicing (Damianov and Black, 2010). The splicing switch of RBFOX3 from the truncated isoform to the full-length protein occurs in a development-dependent manner, and the TABLE 1 | Examples of the function of neuronal splicing regulators in neuronal differentiation and brain development.

*PSD-95: postsynaptic density protein 95. Dnm1: dynamin1. Flna: filamin A. Dab1: disabled homolog-1.*

*hnRNP: heterogeneous nuclear ribonucleoprotein. Snap25: synaptosomal-associated protein 25.*

*ApoER2: apolipoprotein E receptor 2. TRF2: telomeric repeat-binding factor 2.*

latter is necessary for late neuronal differentiation (Kim et al., 2013).

Together, precise timing and level control of splicing regulators is critical for dynamic alternative splicing regulation during cell differentiation and development.

### Alternative Splicing in Self-renewal and Differentiation of Stem Cells

Alternative splicing also plays a critical role in self-renewal of pluripotent cells as well as in cell-fate determination and reprogramming (Graveley et al., 2011; Ye and Blelloch, 2014). Genome-wide RNA sequencing (RNA-seq) studies have revealed that stem cells and differentiated cells exhibit different splicing profiles (Pritsker et al., 2005). Fine-tuning the expression of several stemness-related transcription factors such as Oct4, Nanog, Sox2, and Tcf3 is important for pluripotency maintenance (Chen et al., 2008; Kim et al., 2008). In particular, different isoforms of Tcf3 and Oct4 influence self-renewal of stem cells (Atlasi et al., 2008; Salomonis et al., 2010). The forkhead box transcription factor FoxP1 plays a hierarchical role in the transcription network of pluripotency; the switching of its mutually exclusive exons controls pluripotency and reprogramming of embryonic stem cells (Gabut et al., 2011). Several splicing factors modulate alternative splicing in embryonic stem cells and contribute positively (such as Rbfox2 and SRSF2) or negatively (such as MBNL1/2) to maintaining the stem cell splicing program (Ye and Blelloch, 2014). Thus, alternative splicing plays a critical role in the decision between stem cell self-renewal and differentiation.

Alternative splicing modulates the activity of certain histone modification enzymes in neuronal cells and hence influences the epigenetic status (Fiszbein and Kornblihtt, 2016). The histone methyltransferase G9a is a suppressor of pluripotency-related genes (Kellner and Kikyo, 2010). During neuronal differentiation of neuroblastoma neuro-2a cells, alternative exon inclusion of G9a promotes its nuclear localization and hence increases the dimethylation of histone 3 lysine 9 (H3K9me2). Thus, the regulation of G9a alternative splicing is necessary for efficient neuronal differentiation (Fiszbein et al., 2016). More intriguingly, alternative splicing also modulates the activity of the demethylase LSD1 (Laurent et al., 2015). Therefore, the balanced methylation of H3K9 is likely important for regulating gene expression profiles during neuronal differentiation.

### Alternative Splicing in Differentiation of Neuronal Stem/Progenitor Cells

Transcriptome profiling demonstrated the dynamic nature of alternative splicing events in different cell types, brain regions, and developmental stages (Johnson et al., 2009; Zhang et al., 2014; Yan et al., 2015). RNA-seq analysis of purified NPCs and differentiating neurons in the mouse cortex revealed an alternative splicing switch for a set of neuron-specific exons during differentiation (Zhang et al., 2016). Analysis of human cerebral organoids and fetal neocortex also revealed different splicing patterns in intermediate progenitor cells, redial

level by promoting exon 10 skipping of *PTBP2* mRNA (Left). During neuronal differentiation, PTBP1 level is downregulated by miR-124, whereas RBM4-induced exon 9 skipping of *PTBP1* mRNA generates an isoform with reduced splicing activity, which compromises the splicing effect of PTBP1 during neural differentiation (Right). (B) Exclusion of exon 11/10 (red box) of *PTBP1/PTBP2* generates splicing isoforms with a premature translation-termination codon, and such isoforms are subjected to degradation via alternative splicing-coupled nonsense-mediated decay. RBM4 promotes exon 9 (blue box) skipping, which is specific to PTBP1.

glial cells, immature neurons, and neurons during cortical development (Camp et al., 2015; Zhang et al., 2016). Therefore, splicing regulation establishes cell type- and stage-specific gene expression profiles during neurogenesis and brain development, which rely on proper expression and function of splicing regulators (Raj and Blencowe, 2015; Vuong et al., 2016; Baralle and Giudice, 2017).

Among neuronal splicing regulators, PTBP1 is exclusively expressed in embryonic stem cells and NPCs, whereas PTBP2 and Rbfox proteins are mainly expressed in neurons. A recent report showed that PTBP1 and Rbfox antagonistically modulate neuronal fate via their roles in regulating alternative exon selection (Zhang et al., 2016). Rbfox switches the centrosomal isoform of Ninein to the non-centrosomal form as a result of alternative splicing and hence influences centriolar dynamics and promotes NPC differentiation. On the other hand, PTBP1 suppresses a premature stop codon-containing exon of filamin A (Flna) in NPCs and hence maintains apical progenitors. Genetic mutations that generate aberrant Flna splice isoforms in NPCs are linked to periventricular nodular heterotopia, a neuronal migration disorder. Thus, a better understanding of the mechanisms of neuronal alternative splicing may provide plausible treatment strategies for neuronal disorders.

The Notch receptors play a critical role in fate decisions of various stem/progenitor cells, and Numb is a critical effector of Notch signaling. Alternative splicing of exons 3 and 9 of Numb generate four different isoforms, which differentially modulate Notch activity. The detail of how alternative splicing of Numb modulates cell differentiation is not completely known. Rbfox3 can regulate alternative splicing of Numb, and Rbfox3 depletion impairs neurogenesis in the hippocampal dentate gyrus (Kim et al., 2013; Lin Y. S. et al., 2016). Our recent study showed that RBM4 determines the selection of two alternative exons, and its overexpression preferentially produces a Numb isoform with the highest potential to promote Mash1 expression and subsequent differentiation of neuronal progenitor cells. Moreover, additional splicing regulators of Numb have been implicated in either cancer progression or tumor suppression (Bechara et al., 2013; Zong et al., 2014). Thus, it is conceivable that fine-tuning the expression of Numb isoforms during fate decision of neuronal progenitor cells may constitute a combinatorial effect of multiple splicing regulators.

### Different Alternative Splicing Patterns in Neurons and Glia

Brain tissues comprise a variety of cell types including neural precursor cells, neurons, and various subtypes of neuroglia. Tantalizing issues remain as to whether and how alternative splicing influences neural fate determination and which splicing regulators are involved (Raj and Blencowe, 2015). Expression of specific alternatively spliced isoforms in distinct neurons has been reported in Caenorhabditis elegans and Drosophila (Lah et al., 2014; Norris et al., 2014). For example, UNC75 and EXC7 (respective homologs of mammalian CELF and Hu/ELAV) differentially modulate alternative splicing of unc-16 in GABAergic motor neurons and cholinergic motor neurons (Norris et al., 2014). The energy requirement of different types of brain cells varies; the oxidative and glycolytic pathways predominate in neurons and astrocytes, respectively (Magistretti and Allaman, 2015). Transcriptome profiling has revealed distinct pyruvate kinase M (PKM) splice isoforms, i.e., PKM1 and PKM2 in neurons and glial cells, respectively (Zhang et al., 2014). The PKM1 and PKM2 isoforms result from mutually exclusive exon selection. Selective expression of PKM isoforms is also critical for regulating glucose metabolism in muscle and cancer (Christofk et al., 2008). Gradual switching of embryonic PKM2 to adult PKM1 occurs during mouse brain development and during neuronal differentiation of human mesenchymal stem cells (Su et al., 2017). RBM4 antagonizes PTBP1 activity and hence promotes the PKM2-to-PKM1 switch. Overexpression of RBM4 or PKM1 increases oxygen consumption and accordingly facilitates neuronal differentiation. These results support the high energy demand of neurons. Because neuroenergetics is dynamic and changes in response to neuronal activity such as glutamatergic stimulation and hypoxia (Bélanger et al., 2011), whether the expression of the splice isoforms of certain synthetic enzymes, including PKMs, is coordinately changed remains to be investigated. PKM is involved not only in cell metabolism but also in the modulation of gene expression. PKM2 acts coordinately with β-catenin during gene activation underlying the epithelial-tomesenchymal transition and thus promotes cell proliferation and tumorigenesis (Yang et al., 2011). A recent report demonstrated that the RNA binding protein Quaking maintains neural stem cell functions during early brain development by preventing the PKM2 switch to PKM1 (Hayakawa-Yano et al., 2017).

### Alternative Splicing in Neuronal Migration and Brain Development

The mammalian cerebral cortex has a highly organized sixlayered structure consisting of a variety of neuron subtypes (Molyneaux et al., 2007). Positioning of newborn neurons that originate from the ventricular zone and subventricular zone in the embryonic cortical plate occurs in a birth date-dependent "inside-out" manner (Cooper, 2008; Gao and Godbout, 2013). Several signaling cascades regulate neuronal migration in the cortical plate, including the Reelin-Disabled homolog 1 (Dab1) pathway (Franco et al., 2011; Gao and Godbout, 2013). Upon binding to the very low density lipoprotein receptor (VLDLR) or apolipoprotein E receptor 2 (ApoER2), Reelin induces differential phosphorylation of the cytosolic adaptor protein Dab1 and elicits subsequent downstream events that link Dab1 to the control of neuronal migration. Reeler mutant mice and mice with spontaneous or targeted mutations of Dab1 or either of the receptors exhibit similar phenotypes characterized by ataxia, tremors, and a reeling gait (D'Arcangelo et al., 1995; Howell et al., 1997; Sheldon et al., 1997; Trommsdorff et al., 1999). Differential exon selection of Dab1 occurs during brain development, resulting in multiple splice isoforms (Gao et al., 2012). Nova2 suppresses the inclusion of mouse Dab1 exon 9b/c (Yano et al., 2010). Nova2 knockout causes neuronal migration defects in both the cerebral cortex and cerebellum due to increasing aberrant exon 9 b/c-containing Dab1. Differential selection of exons 7 and 8 of Dab1 is also intriguing because these two exons encode a domain containing critical tyrosines that are targets of Reelin-mediated phosphorylation. Moreover, ApoER2 also undergoes alternative splicing. The exon 19-containing domain of ApoER2 is important for synapse formation and function via its interaction with PSD-95 (Beffert et al., 2005; Hinrich et al., 2016). Exon 19 inclusion is reduced in the brain of Alzheimer's patients. It has been shown that SRSF1 inhibits exon 19 inclusion of ApoER2 and that blocking SRSF1-binding sites using an antisense oligonucleotide has therapeutic potential (Hinrich et al., 2016). Reelin signaling also plays a role in dendritic spine formation and modulates synaptic plasticity in the developing and adult brain (D'Arcangelo, 2014). Therefore, imbalance of splicing factors likely affects neuronal migration and cortical lamination.

### Alternative Splicing in Neurologic Functions

Alternative splicing also regulates neurologic functions such as axon guidance and synaptogenesis. A number of neuronal mRNAs undergo alternative exon selection to generate isoforms in response to neuronal stimulation. Synaptic activity promotes exon 19 inclusion of ApoER2, which then binds Reelin and enhances long-term potentiation (Beffert et al., 2005). Moreover, alternative splicing of the synaptic cell-adhesion molecules neurexins and neuroligins generates multiple isoforms, and interactions between the various isoforms modify their activity toward glutamatergic and GABA-mediated synaptogenesis. Therefore, alternative splicing can shape the strength and functions of synapses. PTBP2 and Sam68 are involved in splicing regulation of neurexins (Resnick et al., 2008; Iijima et al., 2011). Notably, Sam68 activity is regulated by depolarizationinduced calcium/calmodulin-dependent kinase IV, indicating that neuronal activity controls the diversity of neurexins via splicing regulation and hence influences synaptic functions (Iijima et al., 2011). Moreover, alternative splicing also regulates the dynamics of neuronal transcriptomes. In pilocarpinestimulated neurons, exclusion of a cryptic "poison" exon of the sodium channel Scn9a mRNA increases the SCN9A level (Eom et al., 2013). A more recent report revealed that neurons can rapidly regulate the expression of several dendritic mRNAs by removing introns that are retained in existing transcripts stored in the nucleus (Mauger et al., 2016). Thus, rapid and signal-responsive splicing regulation is critical for neurological functions.

### Perspectives

The combination of various genetic tools and RNA-seq has advanced our knowledge of the impact of alternative splicing on neural development and function. Recently, the use of cell-surface or genetically engineered fluorescent protein markers and fluorescence-activated cell sorting has enabled the isolation of stem/progenitor cells and specific neuronal types (Zhang et al., 2016). Using Cre recombinase-expressing mouse lines, one can manipulate the temporal expression of a splicing regulator or wild-type or disease-related mutant in specific types of neurons and investigate changes in the transcriptome or splicing patterns or isolate target mRNA ribonucleoproteins (Möröy and Heyd, 2007). Single-cell RNAseq has begun to clarify cell-to-cell transcriptome variability. Since mammalian brains comprise complex and diverse neuronal cell types, to decipher alternative splicing patterns at the single-neuron level still remains challenged. More recently, a single-cell topological data analysis revealed timeseries gene expression changes of individual cells throughout murine embryonic stem cell differentiation into motor neurons (Rizvi et al., 2017). With the aid of new technologies, future investigations will paint a more comprehensive picture and define the dynamic scope of how splicing programming determines stem/progenitor cell fate determination and differentiation into the various brain cell types as well as neural circuit development. Emerging in situ sequencing and single-cell fluorescence in situ hybridization strategies (Liu and Trapnell, 2016) may allow revealing topological changes of alternative splicing in a brain network and perhaps unveiling pathological mechanisms at the single-cell level.

### AUTHOR CONTRIBUTIONS

C-HS, DD, and W-YT: Jointly wrote this review; W-YT: Defined the scope of the review and edited the draft. All authors read and approved the final manuscript.

### REFERENCES

### ACKNOWLEDGMENTS

The work from our laboratory that is reported in this review was supported by the Ministry of Science and Technology grant 106-2311-B-001-015.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Su, D and Tarn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Alternative Splicing Regulator RBM20 and Cardiomyopathy

#### Takeshi Watanabe1,2, Akinori Kimura3,4 and Hidehito Kuroyanagi 1,4,5 \*

*1 Laboratory of Gene Expression, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 2 Department of Psychosomatic Dentistry, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 3 Division of Pathology, Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 4 Laboratory for Integrated Research Projects on Intractable Diseases Advanced Technology Laboratories, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 5 Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, United States*

RBM20 is a vertebrate-specific RNA-binding protein with two zinc finger (ZnF) domains, one RNA-recognition motif (RRM)-type RNA-binding domain and an arginine/serine (RS)-rich region. *RBM20* has initially been identified as one of dilated cardiomyopathy (DCM)-linked genes. RBM20 is a regulator of heart-specific alternative splicing and *Rbm20*1*RRM* mice lacking the RRM domain are defective in the splicing regulation. The *Rbm20*1*RRM* mice, however, do not exhibit a characteristic DCM-like phenotype such as dilatation of left ventricles or systolic dysfunction. Considering that most of the *RBM20* mutations identified in familial DCM cases were heterozygous missense mutations in an arginine-serine-arginine-serine-proline (RSRSP) stretch whose phosphorylation is crucial for nuclear localization of RBM20, characterization of a knock-in animal model is awaited. One of the major targets for RBM20 is the *TTN* gene, which is comprised of the largest number of exons in mammals. Alternative splicing of the *TTN* gene is exceptionally complicated and RBM20 represses >160 of its consecutive exons, yet detailed mechanisms for such extraordinary regulation are to be elucidated. The *TTN* gene encodes the largest known protein titin, a multi-functional sarcomeric structural protein specific to striated muscles. As titin is the most important factor for passive tension of cardiomyocytes, extensive heart-specific and developmentally regulated alternative splicing of the *TTN* pre-mRNA by RBM20 plays a critical role in passive stiffness and diastolic function of the heart. In disease models with diastolic dysfunctions, the phenotypes were rescued by increasing titin compliance through manipulation of the *Ttn* pre-mRNA splicing, raising RBM20 as a potential therapeutic target.

Keywords: RBM20, dilated cardiomyopathy (DCM), alternative splicing, isoform switching, mutation, arginine/serine (RS)-rich region, titin, nuclear localization

### INTRODUCTION

Cardiomyopathy is a myocardial disease with cardiac dysfunction. Cardiomyopathy is roughly classified as genetic cardiomyopathy including hypertrophic cardiomyopathy (HCM), and mixed (genetic and acquired) cardiomyopathy such as dilated cardiomyopathy (DCM; Dadson et al., 2017). HCM is a disease in which hypertrophy of the ventricle occurs despite the absence of high blood pressure or valvular disease that cause ventricular hypertrophy (Elliott, 2014). More than half

#### Edited by:

*Naoyuki Kataoka, The University of Tokyo, Japan*

#### Reviewed by:

*Ihab Younis, Carnegie Mellon University in Qatar, Qatar Claudia Ghigna, Istituto di genetica molecolare (IGM), Italy*

> \*Correspondence: *Hidehito Kuroyanagi<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Molecular Biosciences*

Received: *02 October 2018* Accepted: *09 November 2018* Published: *28 November 2018*

#### Citation:

*Watanabe T, Kimura A and Kuroyanagi H (2018) Alternative Splicing Regulator RBM20 and Cardiomyopathy. Front. Mol. Biosci. 5:105. doi: 10.3389/fmolb.2018.00105* of the HCM patients carry mutations in one of eight sarcomere genes (Sabater-Molina et al., 2018). DCM is another common form of cardiomyopathy, affecting ∼1 in 250–500 in general population (McKenna et al., 2017) and characterized by left ventricular dilatation and systolic dysfunction in the absence of abnormal loading conditions or coronary artery disease (Rampersaud et al., 2011; McCartan et al., 2012). Mortality rate of DCM is high as a result of heart failure (Kirk et al., 2009). Among the idiopathic DCM cases, 20–35% are familial, with autosomal dominant inheritance in most cases (Kimura, 2016). A next generation sequencing method has recently identified more than 400 potentially causative mutations in 60 genes both in familial and sporadic DCM cases (Pérez-Serra et al., 2016). These DCM-associated genes can be classified into various functional groups such as muscle contraction, Ca2+ handling, and nuclear function. Such molecular genetic complexity makes it difficult to elucidate the mechanisms bringing about the common phenotypes of DCM (Hershberger et al., 2013). Among the DCM-linked mutations, 25% were mapped to the TTN gene (Herman et al., 2012; Hershberger et al., 2013; Fatkin and Huttner, 2017). The human TTN gene has 364 exons, the largest number of exons in a single gene in mammals, 363 of which are coding. The TTN gene encodes the largest known protein titin, a multi-functional sarcomeric structural protein specific to striated muscles (Gigli et al., 2016). Titin plays a major role in passive tension of cardiomyocytes (Hidalgo and Granzier, 2013). The TTN pre-mRNA undergoes extensive alternative splicing, leading to tissue-specific and developmentally regulated titin isoforms.

RBM20, encoding RNA Binding Motif Protein-20 (RBM20) has initially been identified as one of the DCM-linked genes (Brauch et al., 2009). Genetic abnormalities in RBM20 have been identified in about 2–3% of familial and sporadic DCM cases (Li et al., 2010; Refaat et al., 2012; Kayvanpour et al., 2017). Recently, RBM20 has been identified as a crucial RNA-binding protein that controls the splicing of TTN (Guo et al., 2012). However, roles of RBM20 in the pathophysiology of DCM is still unclear. Only a few studies have addressed molecular mechanisms of splicing regulation by RBM20, and are controversial. In this review, we summarize the literature on RBM20 and discuss effects of mutations found in the DCM patients. We also summarize recent attempts to manipulate the RBM20 functions in various disease models. Finally, we will discuss open questions about the functions of RBM20 and its relevance to DCM.

### STRUCTURE OF RBM20

RBM20 is a vertebrate-specific RNA-binding protein (Zerbino et al., 2018). The human RBM20 gene resides on chromosome 10 and is composed of 14 exons. Human RBM20 protein consists of 1,227 amino acid residues and is relatively large for a splicing regulator, yet it has only three conserved recognizable functional domains: two zinc finger (ZnF) domains and one RNA-Recognition Motif (RRM)-type RNA-binding domain (**Figure 1**). Sequence alignment of RBM20 proteins from various vertebrate species revealed three other conserved regions (Guo et al., 2012; Murayama et al., 2018; Zahr and Jaalouk, 2018): a leucine (L) rich region at the N-terminus, an arginine/serine (RS)-rich region just downstream from the RRM domain and a glutamate (E) rich region between the RS-rich region and the ZnF2 domain (**Figure 1**).

Vertebrates have two proteins homologous to RBM20; matrin3, and ZNF638 have two RRM domains sandwiched by two ZnF domains and these domains are most related to those of RBM20 (Coelho et al., 2016). RBM20 is highly expressed in the heart and the skeletal muscle (Filippello et al., 2013), whereas matrin3 and ZNF638 are widely expressed across different cell types (Coelho et al., 2016).

### RBM20 MUTATIONS IN DCM PATIENTS

The RBM20 mutations identified so far in familial as well as sporadic DCM cases are listed in **Table 1**. The list clearly revealed that almost all of the RBM20 mutations are heterozygous missense mutations and are enriched in a hot spot composed of an arginine-serine-arginine-serine-proline (RSRSP) stretch at aa 634–638 in the RS-rich region (**Table 1**; **Figure 1B**). This situation is unusual considering that most of the missense mutations were mapped to the RRM domains in our previous genetic screening for loss- or reduction-of-function mutants for splicing factors (Kuroyanagi et al., 2006, 2007, 2013). We will discuss later how these mutations would affect the function of RBM20.

### ALTERNATIVE SPLICING OF THE TTN GENE

Single titin protein spans half of sarcomere, with its N- and Ctermini in the Z-disk and the M-band, respectively (**Figure 2**). It is composed of four structural and functional regions located in Z-disk, I-band, A-band, and M-band (**Figure 2A**; Wang et al., 1979). Titin is attached to the Z-disk and the thick filament via its Z-disk and A-band segments, respectively (Wang et al., 1979). The I-band region is not attached to any of the solid structures and therefore functions as a molecular spring that generates passive tension when sarcomeres are stretched during diastole (Horowits et al., 1986). In the elastic I-band region of titin, there are six domains from the N-terminus as follows: proximal immunoglobulin (Ig) repeat domain, N2Bunique element, middle Ig repeat domain, N2A-unique element, proline-glutamate-valine-lysine (PEVK) domain and distal Ig repeat domain (**Figure 2A**; Labeit et al., 1990; Bang et al., 2001; Lange et al., 2005).

The TTN gene can potentially produce an mRNA of more than 100 kb. Deduced from its sequence, the predicted fulllength mRNA would produce a protein composed of ∼39,000 amino acid residues whose molecular weight (MW) is 4.2 MDa (Bang et al., 2001; Guo and Sun, 2017). However, when SDS-PAGE was performed on rat myocardium samples, six titin isoforms of roughly 3.0 to 3.9 MDa were mainly identified (Zhu and Guo, 2017). While almost all exons encoding the Zdisk, A-band and M-band regions are constitutively included,

E-rich, glutamate-rich region; L-rich, leucine-rich region; RRM, RNA-recognition motif domain; RS-rich, arginine/serine-rich region; ZnF, zinc finger domains. (B) Amino acid sequence alignment of the RS-rich region of RBM20 proteins from human, mouse, rat, chicken and frog. Amino acid residues that match the human RBM20 residues are shaded. The RSRSP stretch is in red. Asterisks indicate evolutionarily conserved arginine (R), serine (S), and proline (P) residues.

many exons encoding the elastic I-band region are alternatively spliced in tissue-specific and developmentally regulated manners (**Figure 2B**). Exons encoding the proximal and distal Ig repeat domains are constitutively included in all isoforms. Exon 49, encoding the N2B-unique element, is a long exon (2,646 bp in human) and is specifically included in the heart but excluded in the skeletal muscle. Exon 50 is a constitutive exon and encodes an Ig domain. Exons 51–101 are alternatively spliced and encode the middle Ig repeat domain. Exons 102–108 and exons 109–224 encode the N2A-unique element and PEVK domain, respectively (Lewinter et al., 2007). Among these exons, alternative splicing of exons encoding the middle Ig repeat domain (exons 51– 100) and the PEVK domain (exons 116–218) are exceptionally complicated (Guo et al., 2010). In the myocardium, two major titin isoforms are expressed; the shorter N2B isoform contains only the N2B-unique element among the variable domains; the longer N2BA isoforms including fetal types called fetal cardiac titin (FCT) contain the N2B- and N2A-unique elements and a variable length of the middle Ig repeat domain (**Figure 1**). In the skeletal muscle, the N2A isoform containing all of the variable domains except for the N2B-unique element is expressed (Lewinter et al., 2007; Guo and Sun, 2017; **Figure 2B**).

RBM20 is the major regulator of the heart-specific TTN premRNA splicing. In the spontaneous Rbm20 mutant rat strain lacking a 95-kb region spanning from exons 2–14, titin N2B is no longer expressed while N2BA is predominantly expressed in heterozygotes, and an extraordinarily large isoform N2BA-G is exclusively expressed in homozygotes (Guo et al., 2012); **Figure 2B**. The N2BA isoform is also predominantly expressed in the heart of a DCM patient carrying a heterozygous missense mutation S635A in the RBM20 gene (Guo et al., 2012). In a human heart-failure cohort, low expression of endogenous RBM20 was correlated with the splicing pattern of the TTN gene (Maatz et al., 2014). These reports indicated that splicing control of many of the TTN exons is extremely sensitive to the amount of functional RBM20 protein.

### RBM20 REGULATES HEART-SPECIFIC ALTERNATIVE SPLICING

High-throughput RNA sequencing (RNA-seq) of cardiac transcriptomes from the Rbm20-null rats and human DCM patients with and without mutations in RBM20 revealed 31 genes whose alternative splicing is RBM20-dependent in both rats and humans (Guo et al., 2012). Crosslinking and immunoprecipitation coupled with RNA-seq (CLIP-seq) experiments of endogenous RBM20 in rat cardiomyocytes identified 80 direct target exons in 18 genes (Maatz et al., 2014). RBM20 predominantly represses cassette exons including those in the Ttn and Ryr2 genes by binding to upstream and/or downstream intron(s) of the target exons (Li et al., 2013; Maatz et al., 2014). Mutually exclusive exons are also enriched among the RBM20 target exons (Guo et al., 2012; Maatz et al., 2014). For instance, RBM20 represses exons 15 and 16 and promotes inclusion of exon 14 in the Camk2d gene encoding Ca2+/calmodulin-dependent protein kinase II-δ (CaMKII-δ); RBM20 represses exons 5–7 and promotes inclusion of exon 4 in the Ldb3 gene encoding Lim domain binding protein 3 (LDB3).

A UCUU core element has been identified as a precise RNA recognition element (RRE) for RBM20 by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) experiments with epitope-tagged human RBM20 in human embryonic kidney 293 (HEK293) cells and by the CLIPseq experiments with rat cardiomyocytes (Maatz et al., 2014). In the Ttn pre-mRNA, RBM20-binding sites were identified in many of the introns between exon 50 and exon 219 but were almost excluded from the constitutively spliced regions (Maatz et al., 2014). The introns in the alternatively spliced regions were retained in the wild-type rat heart (Li et al., 2013), suggesting that RBM20 represses exons 51–218 by inhibiting excision of most if not all of the introns in this region.

TABLE 1 | *RBM20* mutations identified in DCM patients and their symptoms other than ventricular dilatation.

*Note that residues within the RSRSP stretch in the RS-rich region are in red.*

*aEffect of RBM20 mutations on alternative splicing regulation of the TTN gene was assessed by using TTN splicing reporters (Guo et al., 2012; Murayama et al., 2018).*

*bThe patient also has an A698T mutation in LDB3.*

*cEffect of RBM20 mutations on RBM20 nuclear localization and phosphorylation of the RSRSP stretch was assessed by using mouse cDNAs with equivalent mutations (Murayama et al., 2018).*

*d Incomplete penetrance.*

*eThe patient is homozygous for the RBM20 mutation due to uniparental disomy, whereas his mother is heterozygous and asymptomatic.*

*AF, atrial fibrillation; HF, heart failure; SD, sudden death; VA, ventricular arrhythmias.*

### THE RSRSP STRETCH IN THE RS-RICH REGION IS CRUCIAL FOR NUCLEAR LOCALIZATION OF RBM20

Recently, it has been reported that both of the two serine residues in the RSRSP stretch are constitutively phosphorylated in cells and that single amino acid substitutions in the stretch disrupted nuclear localization of full-length RBM20 protein (Murayama et al., 2018; **Figure 3**). Moreover, Rbm20S637A knock-in mouse mimicking the S635A mutation showed a remarkable effect on the titin isoform expression like in the Rbm20-null rat strain (Murayama et al., 2018). These findings indicated that the RSRSP stretch is a crucial part of the RBM20 nuclear localization signal (NLS) and that the DCM-linked mutation in the RSRSP stretch fully disrupted the alternative splicing control by RBM20.

Many of splicing factors such as SR-protein family members are known to have RS-rich regions or RS domains consisting of multiple serine-arginine (SR) and arginine-serine (RS) dipeptides (Zahler et al., 1992). The RS domains of the SR proteins are extensively phosphorylated on the serine

FIGURE 2 | Structure of the titin protein isoforms. (A) Schematic domain structure of the titin protein. Names and positions of the domains are indicated. Corresponding exons are indicated below each domain. Distal Ig, distal Ig repeat domain; Middle Ig, middle Ig repeat domain; N2A, N2A-unique element; N2B, N2B-unique element; Proximal Ig, proximal Ig repeat domain. (B) Schematic structures of the titin isoforms. Names of the isoforms and the tissues that mainly express the isoforms are indicated on the left. Dotted lines indicate highly variable alternatively spliced regions. FCT, fetal cardiac titin.

FIGURE 3 | Missense mutations in the RSRSP stretch disrupt the normal functions of RBM20. (Left) In the wild-type, two serine residues in the RSRSP stretch are phosphorylated and the RBM20 protein is localized in the nucleus, where RBM20 regulates alternative pre-mRNA splicing of its target genes so that cardiac isoforms of mRNAs are produced. The mRNAs are translated into cardiac protein isoforms with specialized functions. (Right) In the *RBM20* missense mutant with a substitution in the RSRSP stretch, the mutant RBM20 proteins are no longer imported into the nucleus. Pre-mRNAs of the RBM20-target genes are processed into non-cardiac isoforms of mRNAs, which are then translated into non-cardiac protein isoforms, which may lack the specialized functions and/or exert aberrant functions. The mutant RBM20 proteins retained in the cytoplasm may also exert aberrant functions. P, phosphorylation.

residues and this phosphorylation plays an important role in regulating subcellular localization, protein-protein interaction and splicing regulation activities of the SR proteins (Xiao and Manley, 1997; Yeakley et al., 1999). It is therefore reasonable to suggest that the RBM20-mediated heart-specific alternative splicing is dynamically regulated during development and under pathological conditions via dynamic phosphorylation/dephosphorylation of the RSRSP stretch.

Many RBM20-interacting proteins have been identified in HEK293 cells by quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics experiments and some of these interactions were affected by the S635A mutation (Maatz et al., 2014). Totally distinct subcellular localization of the wild-type and mutant RBM20 proteins (Murayama et al., 2018) might be the major cause of this distinct interactomes. Even with the information about the RBM20 interactome, it is still unclear how the interaction with these proteins leads to repression or switching of its target exons.

### THE RRM DOMAIN OF RBM20 IS CRUCIAL FOR SPLICING REGULATION IN VIVO

Molecular mechanisms of RBM20-mediated alternative splicing have been analyzed mostly by utilizing TTN reporter minigenes expressed in non-cardiac cells. However, the results from such studies utilizing distinct reporter minigenes are controversial as to which of the conserved domains are crucial for the regulation. Mutations in the RSRSP stretch but not in the RRM domain affected repression of the 5′ PEVK exons (Guo et al., 2012). The RRM domain and the E-rich region were crucial for repressing exon 242 (Liss et al., 2018). The RSRSP stretch and the E-rich region but not putative RNA-binding domains RRM, ZnF1, or ZnF2 were crucial for repression of a chimeric exon 51/218 (Murayama et al., 2018). Full-length RBM20 (Maatz et al., 2014) or the RRM domain alone (Dauksaite and Gotthardt, 2018) does not necessarily bind to any RNA molecules containing UCUU element(s) in electrophoretic mobility shift assays (EMSAs). Therefore, other RNA element(s) and/or other RNA-binding protein(s) might be involved in the recognition of the authentic target pre-mRNAs by RBM20.

Functions of the RRM domain in vivo has been assessed by deleting exons 6 and 7 of the Rbm20 gene in the mouse. N2BA and N2BA-G isoforms of titin proteins predominated in the left ventricles (LVs) of the Rbm201RRM heterozygotes and homozygotes, respectively, and alternative splicing of the Camk2d and Ldb3 genes was evidently affected as in the Rbm20-deficient rats (Methawasin et al., 2014), indicating that the RRM domain is crucial for the splicing regulation of these genes in vivo. However, the heterozygous or homozygous Rbm201RRM mice did not show any significant differences in cardiac chamber geometry and dimensions compared to wildtype controls (Methawasin et al., 2014), suggesting that switching of the titin isoforms to N2BA-G together with the splicing change in Camk2d and Ldb3 per se does not cause DCMlike phenotypes such as LV chamber dilatation and systolic dysfunction. This is consistent with that there is not a reported familial case where a missense mutation is mapped to the RRM domain (**Table 1**). Interestingly, the phenotypes of the Rbm201RRM mice are distinct from those of the Rbm20-deficient rats (Guo et al., 2012) and Rbm20 knockout (KO) mice in which exons 4 and 5 are deleted for a frame-shift (van den Hoogenhof et al., 2018): the heterozygous and homozygous Rbm20 null rats and mice showed LV dilatation in addition to drastic splicing changes in the Ttn, Camk2d and Ldb3 genes. These observations suggest that RBM20(1RRM) protein retains some regulatory functions. Elucidation of transcripts differentially affected between the Rbm201RRM and Rbm20 KO mice would lead to identification of genes crucial for the progression of the DCM-like phenotypes in the rodent models.

The only RBM20 missense mutation outside of the RSRSP stretch in familial DCM cases with complete penetrance was mapped to a highly conserved glutamate (E) residue in the Erich region (Beqqali et al., 2016; van den Hoogenhof et al., 2018; **Table 1**). The E913K mutation has been shown to decrease the amount of total RBM20 protein and to affect the TTN splicing in the heart of a patient heterozygous for the mutation (Beqqali et al., 2016), suggesting that the E-rich region is crucial for the stability of RBM20. Other missense mutations outside of the RSRSP stretch were identified only in sporadic cases (**Table 1**) and no experimental evidence of altered splicing has been demonstrated. Therefore, it is unclear whether these mutations affected RBM20 functions and hence caused the DCM phenotypes.

The only RBM20 non-sense mutation in the DCM patients reported so far is G1031X in sporadic cases (**Table 1**). This mutation is in exon 11 and likely causes non-sense-mediated mRNA decay (NMD) of mature mRNAs (Schweingruber et al., 2013), leading to haploinsufficiency of RBM20. Notably, one of the patients is homozygous for the G1031X mutation due to uniparental disomy, whereas his mother is asymptomatic even with the heterozygous G1031X mutation (Murayama et al., 2018). It is therefore under debate whether heterozygous non-sense mutations in RBM20 leading to haploinsufficiency would cause the DCM phenotypes.

### ANALYSIS OF THE RBM20 FUNCTIONS WITH PLURIPOTENT STEM CELLS

Expression profiling throughout in vitro cardiogenesis in embryoid bodies (EBs) derived from mouse embryonic stem cells (mESCs) revealed that Rbm20 became expressed as early as Nkx2-5, a marker for cardiac progenitors, consistent with Rbm20 induction during in vivo cardiogenesis between E7.5 and E8.5 (Beraldi et al., 2014). Even though Rbm20 is maximally expressed at day 9 of in vitro differentiation, the transition of the titin isoforms was apparent at day 24, which was suppressed by Rbm20 knockdown (Beraldi et al., 2014), suggesting that RBM20-mediated splicing regulation is reproduced in the in vitro cardiogenesis.

In vitro differentiation of human induced-pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) from familial DCM patients carrying different RBM20 mutations have been utilized for gene expression profiling during in vitro cardiogenesis. Cytological analysis of the hiPSC-CMs revealed that the RBM20 mutations disorganized sarcomere structures (Wyles et al., 2016b; Streckfuss-Bömeke et al., 2017). The RBM20 hiPSC-CMs were defective in Ca2+ handling machinery with prolonged Ca2+ levels in the cytoplasm and higher Ca2+ spike amplitude (Wyles et al., 2016b; Streckfuss-Bömeke et al., 2017), consistent with an increased risk of malignant ventricular arrhythmias in DCM patients with RBM20 mutations than those with TTN mutations (van den Hoogenhof et al., 2018). The RBM20 hiPSC-CMs have also been utilized for demonstrating their increased susceptibility to β-adrenergic stress and therapeutic rescue by a β-blocker carvedilol and a Ca2+ channel blocker verapamil (Wyles et al., 2016a).

### REGULATION OF TITIN COMPLIANCE BY RBM20

Titin-based passive tension in the cardiomyocytes occupies a large proportion of the passive stiffness of the whole myocardium (Rivas-Pardo et al., 2016). It has a negative correlation with molecular weight or amino acid sequence length of titin's spring region. For instance, N2B has a shorter elastic region compared to N2BA, thus giving higher passive stiffness to the cardiomyocytes. It is therefore believed that the ratio of the titin isoforms as well as the total amount of titin protein influence the myocardial passive stiffness (Lahmers et al., 2004). The ratio of the N2B isoform to the N2BA isoform varies from species to species (Neagoe et al., 2003) and from ventricles to atria (Fukuda et al., 2003). It is also dynamically regulated during development (Lahmers et al., 2004; Opitz et al., 2004; Warren et al., 2004; Opitz and Linke, 2005; **Figure 2B**) and under pathological conditions. Amount of the N2BA isoform was increased in heart failure with DCM, heart failure with reduced ejection fraction (HFrEF) and chronic ischemic cardiomyopathy (Makarenko et al., 2004; Nagueh et al., 2004; Borbély et al., 2008), and a reduction of the N2BA isoform was observed in diastolic dysfunction resulting from hypertensive heart disease (Warren et al., 2003).

In the Rbm201RRM heterozygote mice, the compliance of titin proteins is increased and diastolic stiffness of a LV chamber is reduced without significant effect on the chamber geometry or dimensions; beneficial effects on diastolic function dominated under conditions of exercise over an unfavorable effect on end-systolic elastance (Methawasin et al., 2014). Splicing changes in the Ldb3 and Camk2d genes in the Rbm201RRM heterozygotes caused minimal effects on the LDB3 isoforms and no apparent effects on phosphorylation of known CaMKIIδ targets (Methawasin et al., 2014). The Rbm201RRM mice have therefore been utilized to prove a concept that increasing the titin compliance is beneficial to disease models where the mice suffer from lowered diastolic stiffness of the hearts (see below).

### RBM20 AS A POTENTIAL THERAPEUTIC TARGET

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome that includes diastolic dysfunction, exercise intolerance and concentric hypertrophic remodeling. Deletion of Ttn exons 251–269, corresponding to the I-band– A-band junction (IAjxn) of titin, increases strain on the spring region and causes an HFpEF-like syndrome in mice (Granzier et al., 2014). Upon constitutive or inducible, heartspecific heterozygous deletion of the RBM20 RRM domain in the Ttn1IAjxn mice, compliant titin isoforms were expressed, diastolic function was normalized, exercise performance was improved and pathological hypertrophy was attenuated (Bull et al., 2016). HFpEF model mice can also be prepared by performing transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) pellet implantation; inducible, heart-specific heterozygous deletion of the RRM domain in this model mice has also been shown to ameliorate diastolic dysfunction and to recover exercise intolerance (Methawasin et al., 2016).

Deletion of Ttn exon 49, corresponding to the N2B-unique element, results in small hearts with reduced sarcomere length and increased passive tension leading to diastolic dysfunction in mice (Radke et al., 2007). Heterozygous deletion of the RBM20 RRM domain from the Ttn N2B KO mice restored the cardiac dimension and improved the diastolic function (Hinze et al., 2016).

Deletion of Ttn constitutive exons 30–38, corresponding to nine proximal Ig domains in the spring region, increased diastolic stiffness leading to diastolic dysfunction (Chung et al., 2013) and caused mild kyphosis, a phenotype associated with skeletal muscle myopathy (Buck et al., 2014) in mice. RBM20 was upregulated at the protein level in the Ttn IG KO soleus muscle leading to further shortening of titin and heterozygous deletion of the RBM20 RRM domain from the Ttn IG KO mice restored the length of titin in the soleus (Buck et al., 2014).

These examples demonstrated that diastolic dysfunctions could be rescued by increasing titin compliance through manipulation of Ttn pre-mRNA splicing in the animal models, and raised the Ttn splicing regulator RBM20 as a potential therapeutic target. Recently, high-throughput screening of small compounds were performed with RBM20-sensitive TTN splicing reporters and cardenolides have been shown to inhibit RBM20 mediated repression of TTN exons at least in part by reducing RBM20 protein level in cultured cells (Liss et al., 2018).

Extracellular and intracellular signals affecting the titin isoform ratios via RBM20 have also been investigated. Thyroid hormone T3 can promote developmental titin isoform transitions in primary rat cardiomyocytes via the phosphatidylinositol-3-kinase (PI3K)/AKT pathway (Krüger et al., 2008) and the effect of T3 on titin is dependent on RBM20 (Zhu et al., 2015). Western blot analysis of the cardiomyocytes with anti-RBM20 antibody detected two bands with apparent molecular weight of 135 kDa and 145 kDa and the amounts of the two isoforms were differentially affected by T3 and/or a PI3K inhibitor, implying that the activity of RBM20 is regulated by post-translational modification(s) (Zhu et al., 2015). Insulin can also promote the developmental transition of the titin isoforms in primary rat cardiomyocytes by increasing the amount of RBM20 proteins through the PI3K-Akt-mTOR kinase axis (Zhu et al., 2017).

### REGULATION OF CIRCULAR RNA PRODUCTION FROM THE TTN GENE BY RBM20

Circular RNAs (circRNAs) can be generated by "back-splicing" of pre-mRNAs (Li et al., 2018) and some of the circRNAs have physiological functions as miRNA sponges in vivo (Hansen et al., 2013; Memczak et al., 2013). The TTN gene has been shown to produce a variety of circRNAs mostly from the alternatively spliced exons (Li et al., 2013; Khan et al., 2016). Recent RNAseq experiments identified thousands of circRNAs in the human heart (Khan et al., 2016) and >1,000 circRNAs in the mouse heart (Aufiero et al., 2018). However, only a small subset of circRNAs expressed in the heart are evolutionarily conserved (Aufiero et al., 2018), implying that most of the cardiac circRNAs are nonfunctional. Forty-three of the human cardiac circRNAs including those from the TTN and CAMK2D genes were differentially expressed in heart samples from DCM patients compared with those from controls (Khan et al., 2016). Thirty-eight of the mouse cardiac circRNAs were differentially expressed in the Rbm20 KO mice, 11 of which were generated from the Ttn gene in an RBM20-dependent manner (Aufiero et al., 2018). One may therefore say that RBM20 switches from N2BA-G production to circRNA production from the TTN gene, although physiological and pathological functions of the circRNAs from the TTN and other genes remain to be elucidated.

### OPEN QUESTIONS AND FUTURE RESEARCH DIRECTIONS

Although it has been genetically shown that the RBM20 mutations cause DCM, subsequent cardiac transcriptome analyses and animal models have not yet specified RBM20 regulated genes whose aberrant splicing are critically linked to each of the DCM symptoms such as systolic dysfunction, left ventricle dilatation and a risk of ventricular arrhythmia. RBM20 mutant protein with a missense mutation in the RSRSP stretch may exert aberrant functions in the cytoplasm (**Figure 3**). Identification of such critical splice variants or aberrant effects may lead to development of new therapeutics for DCM symptoms not restricted to those caused by the RBM20 mutations. To validate the candidate events, it is necessary to genetically restore cardiac isoforms and/or reduce aberrant isoforms in an appropriate animal model that show DCM-like phenotypes as in the RBM20-linked DCM patients. Ventricular

### REFERENCES

arrhythmia has not been reported for the 1RRM or KO mouse models, and therefore another animal model that phenocopies the RBM20-linked DCM is awaited. A recent large-scale genomewide association study (GWAS) of >1 million people including 60,620 atrial fibrillation cases have identified RBM20 as one of genes near risk variants (Nielsen et al., 2018), suggesting its implication in atrial cardiomyopathy.

TTN exons 51–124 and alternative splicing events in CAMK2D and LDB3 are hyper-sensitive to reduction in the amount of functional RBM20 protein, whereas TTN exons 125–218 are much less affected by the heterozygous mutations (Guo et al., 2012; Beqqali et al., 2016; Murayama et al., 2018). Biochemical and biophysical analysis of the splicing control of TTN by RBM20 should address the following points: (1) how tens of consecutive cassette exons can be synchronously repressed depending on the amount of a single factor RBM20, and (2) how other tens of consecutive cassette exons can be almost completely repressed by less amount of RBM20. Application of recent technical progress in direct sequencing of full-length cDNA/mRNA to the extremely long TTN transcripts would elucidate precise splicing patterns of the RBM20-dependent isoforms, which will help understanding the regulation mechanisms.

RBM20 is expressed at early cardiogenesis, yet a compliant titin FCT isoform is expressed in the embryonic heart. RBM20 is also expressed in the skeletal muscle, yet the splicing patterns of the TTN mRNAs are totally different between these tissues. So far, it is unknown how the activity of RBM20 is regulated during development and in different tissues. Elucidating such mechanisms will lead to further understanding of heart-specific alternative splicing regulation as well as to future possible therapeutics that manipulate the activity of RBM20.

### AUTHOR CONTRIBUTIONS

TW, AK, and HK drafted the manuscript, and revised critically the manuscript for important intellectual content.

## FUNDING

The study leading to this article was supported by Grants-in-Aid for Scientific Research (KAKENHI, Grant Numbers JP15H01350, JP17H05596, JP17H03633, and JP15KK0252) from Japan Society for the Promotion of Science (JSPS) (to HK) and a grant from Takeda Science Foundation (to HK).

and its interaction with obscurin identify a novel Z-line to Iband linking system. Circ. Res. 89, 1065–1072. doi: 10.1161/hh2301. 100981

Beqqali, A., Bollen, I. A. E., Rasmussen, T. B., van den Hoogenhof, M. M., van Deutekom, H. W. M., Schafer, S., et al. (2016). A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank– Starling mechanism. Cardiovasc. Res. 112, 452–463. doi: 10.1093/cvr/ cvw192

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Watanabe, Kimura and Kuroyanagi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Importance of Functional Loss of FUS in FTLD/ALS

Shinsuke Ishigaki 1,2 \* and Gen Sobue3,4 \*

*1 Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2 Department of Therapeutics for Intractable Neurological Disorders, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3 Brain and Mind Research Center, Nagoya University, Nagoya, Japan, 4 Research Division of Dementia and Neurodegenerative Disease, Nagoya University Graduate School of Medicine, Nagoya, Japan*

Fused in sarcoma (FUS) is an RNA binding protein that regulates RNA metabolism including alternative splicing, transcription, and RNA transportation. FUS is genetically and pathologically involved in frontotemporal lobar degeneration (FTLD)/amyotrophic lateral sclerosis (ALS). Multiple lines of evidence across diverse models suggest that functional loss of FUS can lead to neuronal dysfunction and/or neuronal cell death. Loss of FUS in the nucleus can impair alternative splicing and/or transcription, whereas dysfunction of FUS in the cytoplasm, especially in the dendritic spines of neurons, can cause mRNA destabilization. Alternative splicing of the *MAPT* gene at exon 10, which generates 4-repeat Tau (4R-Tau) and 3-repeat Tau (3R-Tau), is one of the most impactful targets regulated by FUS. Additionally, loss of FUS function can affect dendritic spine maturations by destabilizing mRNAs such as Glutamate receptor 1 (GluA1), a major AMPA receptor, and Synaptic Ras GTPase-activating protein 1 (SynGAP1). Moreover, FUS is involved in axonal transport and morphological maintenance of neurons. These findings indicate that a biological link between loss of FUS function, Tau isoform alteration, aberrant post-synaptic function, and phenotypic expression might lead to the sequential cascade culminating in FTLD. Thus, to facilitate development of early disease markers and/or therapeutic targets of FTLD/ALS it is critical that the functions of FUS and its downstream pathways are unraveled.

Keywords: FUS, FTLD/ALS, tau, GluA1, SynGAP

### INTRODUCTION

Amyotrophic lateral sclerosis (ALS), characterized by selective motor neuronal loss in the central nervous system, and frontotemporal lobar degeneration (FTLD), which is distinguished by changes in character, abnormal behaviors, language impairments, and progressive dementia, have recently been recognized as two ends of the spectrum of one disease (Robberecht and Philips, 2013). This notion is supported by the genetic determinants underlying familial FTLD/ALS (Renton et al., 2014) and lines of evidence showing a pathological continuity between ALS and FTLD (Riku et al., 2014). RNA binding proteins (RBPs) such as transactive response (TAR) DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS) genetically and pathologically link the two neurodegenerative diseases to a single disease state (Van Langenhove et al., 2012). These genes are causative for familial ALS and FTLD, and are pathological hallmarks of both familial and

#### Edited by:

*Naoyuki Kataoka, The University of Tokyo, Japan*

#### Reviewed by:

*Hitomi Tsuiji, Nagoya City University, Japan Woan-Yuh Tarn, Academia Sinica, Taiwan*

#### \*Correspondence:

*Shinsuke Ishigaki<EMAIL_ADDRESS>Gen Sobue<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Molecular Biosciences*

> Received: *28 February 2018* Accepted: *17 April 2018* Published: *03 May 2018*

#### Citation:

*Ishigaki S and Sobue G (2018) Importance of Functional Loss of FUS in FTLD/ALS. Front. Mol. Biosci. 5:44. doi: 10.3389/fmolb.2018.00044* sporadic FTLD/ALS in which TDP-43 or FUS-positive inclusions are observed (Kwiatkowski et al., 2009; Lagier-Tourenne and Cleveland, 2009; Vance et al., 2009; Mackenzie et al., 2011; Strong and Volkening, 2011). Additionally, FTLD has also been classified as a tauopathy characterized by an accumulation of phosphorylated microtubule-associated protein tau (Tau) in affected neurons (Seelaar et al., 2011).

FUS was originally identified as a fusion protein that resulted from a chromosomal translocation in human myxiod liposarcomas, in which the N-terminal portion of FUS was translocated and fused to the transcription factor CHOP (Crozat et al., 1993). FUS functions as a regulator of multiple aspects of RNA metabolism, including transcription, alternative splicing, and mRNA transport, as well as DNA damage regulation (Bertolotti et al., 1996; Wang et al., 2008; Schwartz et al., 2012; Tan et al., 2012). Whole-body knockout of FUS in highly homogenous inbred C57B6 strain mice resulted in early neonatal death due to immune system defects (Hicks et al., 2000), whereas FUS KO in outbred mice had no developmental impairments (Kuroda et al., 2000). These suggest that the inbred background lacks genes required to compensate for the FUS KO effects. Similar to TDP-43 pathology, FUSrelated FTLD/ALS pathology is characterized by mislocalization of FUS to the cytoplasm and a concomitant reduction in nuclear expression in affected neurons (Neumann et al., 2009; Deng et al., 2010; Mackenzie et al., 2011). Redistribution of FUS from the nucleus to the cytoplasm implies that the loss of nuclear FUS is causal for FUS-associated ALS/FTLD. Indeed, loss of FUS leads to neuronal cell death in Drosophila and zebrafish (Kabashi et al., 2011; Wang et al., 2011). On the other hand, accumulation of FUS in the cytoplasm is strongly associated with stress granules, which are nonmembranous, cytoplasmic ribonucleoprotein (RNP) granules composed of mRNAs, translation initiation factors, ribosomes, and other RBPs. These granules are induced by various cellular stresses, such as oxidative stress, glucose starvation, mitochondrial dysfunction, and viral infection that inhibit translation initiation. The stress granule associated gain-of– toxicity hypothesis of FUS has been well reviewed elsewhere (Gao et al., 2017).

This review provides an overview of recent findings that reveal the effects of functional loss of FUS on the pathogenesis of FTLD/ALS. First, loss of FUS in the nucleus leads to imbalanced Tau isoforms due to insufficient skipping of exon 10 in the MAPT gene. Second, loss of FUS in the cytoplasm causes decreased stability in GluA1 and SynGAPα2 mRNA resulting in aberrant maturation of dendritic spines. In addition, we summarize the roles of FUS in neurite maintenance and axonal transport, and provide a briefly overview of the FUS liquid-phase-transition, which may alter its various physiological functions and contribute to the development of toxic cellular effects under pathological conditions. Thus, the functional properties of FUS may influence multiple cellular processes of neurons and/or glial cells whose dysfunction could be the most plausible explanation for neuronal toxicity mediated by loss of FUS.

## QUANTITATIVE AND QUALITATIVE LOSS OF FUNCTION OF FUS

Although recent reports have suggested that loss-of-FUSfunction in motor neurons might not contribute to motor neuron degeneration in ALS (Scekic-Zahirovic et al., 2016; Sharma et al., 2016), lines of evidence suggest that loss-of-FUS-function in cerebral neurons can contribute to neuronal dysfunction and neurodegeneration in FTLD. FUS-deficient mice generated either via silencing or FUS knock-out exhibit behavioral impairments (Kino et al., 2015; Udagawa et al., 2015). However, recovery of wild-type FUS in the FUS-silenced mice rescued the behavioral phenotypes, whereas a disease-associated mutant did not (Ishigaki et al., 2017).

Although FUS pathology is detected in both ALS and FTLD cases, the majority of disease-causing mutations within FUS are associated with ALS cases.  Moreover, a spectrum of cognitive impairments have been observed in a considerable subpopulation of ALS patients (Swinnen and Robberecht, 2014). Taken together, the clinical data and FUS-silenced mice model findings support the hypothesis that FUS dysfunction results in early cognitive impairments.

In familial and sporadic FTLD/ALS cases, which are, respectively, characterized by mutations in the FUS coding sequence or the presence of a basophilic inclusion body (BIBD), the affected motor neurons exhibit dislocation of FUS with the protein accumulating in the cytoplasm rather than the nucleus. Cytoplasmic mislocalization of FUS is presumably the first step in the disease cascade; therefore, quantitative loss-of-FUS is thought to be causal for FTLD/ALS. However, diseaseassociated mutations do not trigger complete mislocalization of FUS to the cytoplasm as a moderate amount of the protein remains localized in the nucleus (Kino et al., 2011). This implies that the FUS mutants are non-functional, and that this, in conjunction with the quantitative reduction in protein, culminates in neuronal dysfunction and FTLD/ALS pathophysiology. It has been reported that FUS binds Urich small nuclear ribonucleoproteins (snRNPs) and the SMN cmplex, which is the machinery for snRNP biogenesis, and hence compromises precursor mRNA splicing, leading to FUSassociated FTLD/ALS (Tsuiji et al., 2013; Sun et al., 2015). In our recent study, the presence of disease-associated mutations in FUS disrupted formation of a high molecular weight FUS complex by impeding interactions with a second protein, Splicing factor, proline- and glutamine-rich (SFPQ). The impaired FUS functionality suggests that the pathophysiological features of FTLD/ALS also arise from qualitative losses in FUS and SFPQ (Ishigaki et al., 2017; **Figure 1)**. Another group recently reported on the presence of possible SFPQ mutations in familial ALS cases (Thomas-Jinu et al., 2017). These findings suggest that aberrant interactions between FUS and its spliceosome binding partners in the nucleus of neurons might lead to neuronal dysfunction and

disease-associated mutations in FUS affecting this alternative splicing machinery.

subsequent neurodegeneration. However, future pathological studies examining the FUS/SFPQ nuclear interaction in both FTLD/ALS and tauopathies are necessary.

The cell selectivity of neurodegenerative diseases such as FTLD/ALS has remained a mystery. For instance, the pathology of FTLD/ALS in both motor neurons and cortical neurons involves major selective neuronal vulnerability. Glial cells, such as astrocytes and microglial cells, have also been linked with FTLD/ALS as modifiers of the non-cell-autonomous mechanism of disease pathogenesis, while cerebellar neurons are typically spared in FTLD/ALS (Boillée et al., 2006; Yamanaka et al., 2008a,b). The cell and region specific-selectivity of FTLD/ALS, however, cannot be explained by the expression pattern of FUS since it is expressed ubiquitously throughout the CNS (Kwiatkowski et al., 2009). We previously found that the profiles of FUS-mediated gene expression and alternative splicing in motor neurons are similar to those of cortical neurons, but differ from those in cerebellar neurons despite the similarity of their innate transcriptome signature. The gene expression profiles in glial cells were similar to those in motor and cortical neurons. Given that motor and cortical neurons are the major affected cell-types in FTLD/ALS, whereas glial cells are modifiers and cerebellar neurons are spared, it is possible that the FUS-regulated transcriptome profiles in each cell-type may determine the cellular fate in association with FTLD/ALS and that neuron-glia interactions may be involved in the pathogenesis (Fujioka et al., 2013). Indeed, FUS silencing caused glial cell proliferation in the brain of non-human primates (Endo et al., 2017).

Taken together, these findings indicate that both quantitative and qualitative losses of FUS function are likely involved in the pathogenesis of FTLD/ALS, and should provide clues for therapeutics that clarify the functional properties of FUS.