How Alternative Splicing Affects Membrane-Trafficking Dynamics R

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REVIEW How alternative splicing affects membrane-trafficking dynamics R. Eric Blue1, Ennessa G. Curry1,*, Nichlas M. Engels1,*, Eunice Y. Lee1 and Jimena Giudice1,2,3,‡

ABSTRACT et al., 2012). Tissue-specific exons encode disordered segments The cell biology field has outstanding working knowledge of the within proteins that function in microtubule-based transport, fundamentals of membrane-trafficking pathways, which are of critical endocytosis and membrane deformation (Buljan et al., 2012; Ellis importance in health and disease. Current challenges include et al., 2012) (Box 1). understanding how trafficking pathways are fine-tuned for In humans, 90-95% of genes undergo alternative splicing, specialized tissue functions in vivo and during development. In expanding protein function beyond genetic diversity (Pan et al., parallel, the ENCODE project and numerous genetic studies have 2008; Wang et al., 2008). Splicing of intronic regions is regulated by revealed that alternative splicing regulates gene expression in tissues the strength of the splice sites; strong splice sites lead to constitutive and throughout development at a post-transcriptional level. This splicing, whereas weak splice sites are used in a context-dependent Review summarizes recent discoveries demonstrating that alternative manner (alternative splicing). Usage of weak splice sites is regulated splicing affects tissue specialization and membrane-trafficking by cis-regulatory sequences, trans-acting factors such as RNA- proteins during development, and examines how this regulation is binding proteins (RBPs) and epigenetics (Kornblihtt et al., 2013). altered in human disease. We first discuss how alternative splicing of Depending on splice site locations, different types of alternative clathrin, SNAREs and BAR-domain proteins influences endocytosis, splicing events are produced, which comprise insertion of secretion and membrane dynamics, respectively. We then focus on alternative cassette exons or mutually exclusive exons, selection ′ ′ the role of RNA-binding proteins in the regulation of splicing of between alternative 5 -or3-splice sites, poly-adenylation sites and membrane-trafficking proteins in health and disease. Overall, our aim intron retention (Fig. 1). Alternative splicing can dramatically is to comprehensively summarize how trafficking is molecularly impact protein function or affect the expression, localization, and/or influenced by alternative splicing and identify future directions stability of mRNAs (Irimia and Blencowe, 2012). Coordination of centered on its physiological relevance. alternative splicing contributes to cell differentiation, lineage determination, tissue identity acquisition and, ultimately, organ KEY WORDS: RNA-binding proteins, Alternative splicing, Membrane development (Baralle and Giudice, 2017; Wang et al., 2008). The dynamics, Trafficking physiological relevance of splicing is evident from the vast number of mutations in cis-regulatory elements, RBPs or spliceosome Introduction components, which cause a broad spectrum of human diseases Membrane trafficking controls multiple cellular functions. (Scotti and Swanson, 2016). Trafficking to and from the plasma membrane modulates cell Here, we review the molecular connection between alternative communication during organ development and function. splicing and membrane trafficking from both physiological and Trafficking from the cell exterior comprises internalization of ion disease perspectives. First, we discuss how alternative splicing channels, receptors and ligands to control homeostasis and impacts membrane-trafficking proteins involved in clathrin- signaling. Trafficking from the inside controls the transport of mediated endocytosis (CME), secretory pathways and membrane newly synthetized proteins from the endoplasmic reticulum to their dynamics. Then, we discuss the role of RBPs in controlling final destinations. Numerous human diseases are caused by alternative splicing of trafficking proteins and how this regulation mutations in membrane-trafficking genes (Dowling et al., 2008; contributes to health and disease. The final section identifies the Sigismund et al., 2012), highlighting the physiological importance major questions still outstanding in the field. of these proteins. Membrane-trafficking genes are developmentally and tissue- Alternative splicing regulation and CME specifically regulated by alternative splicing (Brinegar et al., 2017; CME is one of the most common mechanisms that cells employ to Dillman et al., 2013; Giudice et al., 2014; Hannigan et al., 2017; absorb nutrients, hormones or proteins from the exterior and Irimia et al., 2014), a post-transcriptional mechanism used by single involves clathrin-coated vesicles. In addition, CME regulates the genes to produce multiple transcripts and, thus, several protein protein content of the plasma membrane, monitors external cues isoforms with different features. Brain, heart and skeletal muscle, from the surrounding environment, modulates signaling pathways together with the testes, are the organs where most of the tissue- and directs protein recycling and degradation (McMahon and specific and conserved alternative splicing takes place (Merkin Boucrot, 2011). Loss of function of central components of the CME machinery, such as clathrin, AP2, epsin or dynamin, is embryonically lethal and alterations in other CME proteins are 1Department of Cell Biology & Physiology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 2McAllister Heart often present in cancer, neurological disorders, genetic syndromes Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, and muscle pathologies. CME occurs in multiple steps (Fig. 2): USA. 3Curriculum in Genetics and Molecular Biology (GMB), The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. (1) the formation of clathrin-coated vesicles starts by membrane *These authors contributed equally to this work invagination (nucleation); (2) proteins are recruited to the nucleation

‡ site by the AP2 complex, which together with other cargo-specific Author for correspondence ( [email protected]) adaptors mediates cargo selection; (3) clathrin polymerization

J.G., 0000-0002-3330-7784 stabilizes the curvature of the forming vesicle; (4) dynamin, Journal of Cell Science

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arranged along the triskelion leg (Brodsky, 2012). Humans have Box 1. Alternative splicing of trafficking proteins is tissue two genes encoding clathrin heavy chain, CLTC (also known as specific and affects short disordered motifs CHC or CHC17) and CLTCL1 (also known as CHC22) (see Brain, heart and skeletal muscle, as well as the testes, are the organs Table S1, for a list of official and alternative gene and protein where most of the tissue-specific and conserved alternative splicing names) (Kedra et al., 1996; Vassilopoulos et al., 2009). CLTC takes place (Merkin et al., 2012). Tissue-specific alternative exons predominates, except in skeletal muscle where the two proteins encode regions of proteins that tend to be centrally located within protein- are equally expressed. protein interaction networks (Buljan et al., 2012; Ellis et al., 2012) and comprise short intrinsically disordered motifs that confer functional By self-assembly, the triskelia form a polyhedral lattice that coats diversity onto splice variants (Weatheritt and Gibson, 2012; Weatheritt the transport vesicles (Brodsky et al., 2001; Ungewickell and et al., 2012). In general, the pairs of variants tend to behave like distinct Hinrichsen, 2007). While CLTC constitutes the backbone of the proteins in terms of their interaction with other proteins, and the vesicle lattice, the light chains regulate clathrin recruitment in the interaction partners that are specific to each splice isoform tend to be cell (Majeed et al., 2014). In humans and mice, the CLTC gene highly tissue specific (Yang et al., 2016). Tissue-specific alternative contains 33 exons, of which exon 31 is postnatally regulated by exons are present in genes encoding proteins that control microtubule- based transport, endocytosis, membrane deformation and endosome alternative splicing in cardiomyocytes and skeletal muscles, with it formation (Buljan et al., 2012; Ellis et al., 2012). Table S2 summarizes being skipped in neonates and included in adults (Brinegar et al., several of these tissue-specific alternative exons in membrane- 2017; Giudice et al., 2014, 2016). CLTC regulates the formation and trafficking genes that are predicted to impact protein-protein maintenance of myofiber architecture (Vassilopoulos et al., 2014); interactions and below we highlight two examples that have been thus, the tissue-specificity of CLTC splicing suggests that it might experimentally demonstrated. be important for muscle structure. Alternative splicing is not limited (1) Growth factor receptor bound protein-2 (GRB2) is involved in internalization of receptor tyrosine kinases. GRB2 homodimerizes to CLTC, but also regulates the clathrin light chain genes CLTA and through an interaction between its SH2 and SH3 domains (Maignan CLTB (also known as LCA/CLCa and LCB/CLCb, respectively). In et al., 1995), which is important for signaling (McDonald et al., 2008). comparison with the ubiquitous isoforms, brain splice variants of Exon 4 of GRB2 is alternatively spliced and encodes a region that CLTA contain 18 or 30 additional residues, and a further different overlaps the SH2 domain. When exon 4 is skipped, the self-interaction of but homologous 18 residues were found in CLTB (Jackson et al., GRB2 is lost, suggesting that alternative splicing controls GRB2 1987; Kirchhausen et al., 1987). Alternative splicing regulates homodimerization and thus signaling activity (Ellis et al., 2012). CLTA exon 5, which encodes the 18 residues and CLTA exon 6, (2) Kinesin-1 is a molecular motor protein associated with microtubules; it is formed by two heavy chains that control motor which encodes the 12 residues, as well as CLTB exon 5, which activity and two light chains (KLC1 and KLC2) involved in cargo binding. encodes 18 residues. In mouse hearts, Clta exon 6 is skipped in KLC1 contains five alternative exons (13-17). Inclusion of exon 15 neonates and included in adults, whereas Clta exon 5 and Cltb exon reduces the interaction of KLC1 with Marlin1, which controls transport of 6 are skipped at all developmental stages (Giudice et al., 2014). the GABA-B receptor towards dendrites (Vidal et al., 2007). Therefore, Although CLTA and CLTB are regulated by alternative splicing in a regulation of exon 15 splicing might control distribution of the GABA-B tissue- and developmental-stage-specific manner, we still lack a receptor, and thus neurotransmission (Ellis et al., 2012). complete description of the tissue expression patterns of transcript and protein isoforms and their functions. endophilin and BAR-domain proteins drive vesicle scission from Dynamin and DAB2 the membrane; (5) vesicles are uncoated to fuse with target In addition to the clathrin triskelion, other CME proteins are also endosomes; (6) cargo is transported through early endosomes into alternatively spliced, including the adaptor protein disabled-2 either recycling vesicles, or late endosomes and lysosomes for (DAB2), which controls endocytosis of the low-density degradation (McMahon and Boucrot, 2011). Several of the CME lipoprotein receptor family (Keyel et al., 2006; Maurer and proteins undergo alternative splicing (highlighted in red in Fig. 2) Cooper, 2006). DAB2 has two tissue-specific splice isoforms and some are discussed below. known as P96 and P67 (Xu et al., 1995). P96 contains two clathrin- binding sites and one AP2-binding site, which are absent in P67, Clathrin chains explaining why P96 binds to clathrin and AP2 and localizes at Non-assembled clathrin comprises a three-legged structure clathrin-coated pits, whereas P67 does not (Buljan et al., 2012; called a triskelion (Unanue et al., 1981), which is formed by Morris and Cooper, 2001). DAB2 depletion results in alterations in three heavy chain polypeptides bound to light chain subunits the surface levels of integrins that give rise to migration and

A Cassette exon B Mutually exclusive exons C Intron retention Fig. 1. Types of alternative splicing events. The types of splicing events are defined by the location of the alternative splice sites. (A) Cassette exons can be either included or spliced out. (B) Mutually exclusive exons are consecutive exons that are included in a mutually exclusive manner, i.e. when one of them is included the other one is skipped and vice versa. (C) An intron or a D Alternative 3Ј splice site E Alternative 5Ј splice site F Alternative polyA selection portion of an intronic region can be included in the mRNA. (D,E) When more than one splice site exists at the end or beginning of an exon, alternative 5′ or 3′ splice sites can be selected. (F) The polyA tail at the 3′ end of an RNA can be added in some genes at different poly-adenylation sites (polyA), leading to the production of more than one transcript. Constitutive exons are shown in gray boxes,

Proximal polyA Distal polyA introns as lines and alternative regions as colored boxes. Journal of Cell Science

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2. Cargo selection 3. Coat assembly 4. Vesicle scission CLTC CLTC CLTC CLTA, CLTB CLTA, CLTB CLTA, CLTB EPN1/ 2 NUMB Endophilin 1. Nucleation AP2 AAK BAR proteins EPS15 HIP1R BAR proteins Dynamin ITSN1/2 SNAP91 Dynamin SNX9 FCHO1/2 Cortactin DAB2 EPS15R

5. Uncoating CLTC CLTA, CLTB Synaptojanin HSC70 Nucleus DNAJC6 GAK Recycling Actin endosome

Lysosome

6. Endosomal processing

Fig. 2. Regulatory mechanisms for alternative splicing impact numerous components of the CME. CME occurs in multiple steps: (1) clathrin-coated vesicles are formed; (2) proteins are recruited to the nucleation site by the AP2 complex, which together with other cargo-specific adaptors mediates cargo selection; (3) the clathrin triskelion facilitates vesicle assembly and clathrin polymerization stabilizes the curvature of the forming vesicle; (4) dynamin, endophilin and BAR-domain proteins drive vesicle scission from the membrane; (5) vesicles are uncoated to fuse with target endosomes; (6) cargo is transported through early-endosomes into either recycling vesicles, or late endosomes and lysosomes for degradation. Alternative splicing regulates several of the proteins that control different CME steps, and the known splicing-regulated proteins are shown in red. AAK, 5′-AMP-activated protein kinase catalytic subunit α-1; AP2, adaptor-related protein complex-2; CLTA, clathrin light chain-a; CLTB, clathrin light chain-b; CLTC, clathrin heavy chain; DNAJC6, DNAJ heat shock protein family (Hsp40) member C6; DAB2, disabled-2; EPN1/2, epsin-1 and epsin-2; EPS15, epidermal growth factor receptor pathway substrate-15; EPS15R, epidermal growth factor receptor pathway substrate 15 recombinant; FCHO1/2, FCH-domain only 1 and FCH-domain only 2; GAK, Cyclin G-associated kinase; HIP1R, Huntington interacting protein 1 related; HSC70, heatshock protein family A (Hsp70) member 8; ITSN1/2, intersectin- 1 and intersectin-2; NUMB, endocytic adapter protein; SNAP91, synaptosome-associated protein-91; SNX9, sorting nexin 9. polarization defects, which can be rescued by re-expression of P96, molecular details of splicing events and their physiological but not P67 (Teckchandani et al., 2009). implications. In mammals, there are three dynamin genes (DNM1, DNM2 and DNM3), which encode members of a family of GTPases involved in Alternative splicing of SNARE proteins membrane fission and the release of clathrin-coated vesicles from the The SNARE machinery controls the interaction between vesicular plasma membrane. The three genes are highly regulated by alternative v-SNAREs (VAMPs) and target t-SNAREs (such as SNAPs and splicing, giving rise to more than 25 splice isoforms. The neuron- syntaxins) during vesicle fusion (Fig. 3A). Several components of specific DNM1 is essential for synaptic vesicle endocytosis and is the SNARE machinery undergo alternative splicing, as discussed alternatively spliced at two regions: the middle domain and the below. C-terminus. Splicing results in either DNM1ax or DNM1bx isoforms, where ‘a’ and ‘b’ are the middle domain variants and ‘x’ SNAPs is one of the three alternative terminal exons. In mice, a spontaneous Synaptosome-associated protein-25 (SNAP25) bridges synaptic missense mutation (fitful) (p.A408T) that only affects the DNM1ax vesicles to the plasma membrane during exocytosis. In higher isoforms (the ‘a’ exon is spliced out in the DNM1bx variants) alters vertebrates, two mutually exclusive exons, 5a and 5b, give rise endocytosis, DNM1 self-assembly, which is required for dynamin to the SNAP25-a and SNAP25-b isoforms (Bark and Wilson, function and, ultimately, synaptic transmission (Boumil et al., 2010). 1994) (Fig. 3B). These variants differ by nine centrally located DNM2 is ubiquitously expressed and has several splice variants. residues, two of which alter the relative positioning of clustered In particular, two alternative regions generate four splice variants, cysteines whose palmitoylation controls membrane anchoring DNM2aa, DNM2ab, DNM2ba and DNM2bb (Cao et al., 1998; (Vogel and Roche, 1999). Snap25 null mice exhibit defects in Cook et al., 1994; Sontag et al., 1994). All isoforms localize to primed vesicle pools and fast calcium-triggered release in clathrin-coated pits at the plasma membrane but only DNM2ba and chromaffin cells. Here, re-expression of SNAP25-b results in DNM2bb localize to the Golgi. All isoforms rescue the endocytosis larger primed vesicle pools compared with those seen upon re- defects in Dyn2-depleted cells, but DNM2ba and DNM2bb were expression of SNAP25-a, suggesting that alternative splicing more effective than the DNM2aa and DNM2bb variants with regard regulates the ability of SNAP25 to stabilize primed vesicles to rescuing the export of the neutrophin receptor p75 from the trans- (Sørensen et al., 2003). A developmental switch from the fetal Golgi network (Liu et al., 2008). SNAP25-a isoform to adult SNAP25-b occurs in brain (Bark In summary, the regulation of CME factors by alternative splicing et al., 1995), and its impairment induces lethality in mice three has been demonstrated to be tissue- and developmental-stage- to five weeks after birth (Bark et al., 2004). The exclusive specific. However, further studies are required to better describe the expression of the SNAP25-a isoform through replacement of Journal of Cell Science

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Cargo Plasma membrane SNAPs A release SNAP25 splicing affects strength of exocytic bursts

Priming Nucleus and fusion Tethering and docking Syntaxins Golgi CELF syntaxin splicing Trafficking network affects axon repair STX3 splicing affects protein domains v-SNAREs t-SNAREs Cargo VAMPs SNAPs VAMPs VAMP7 splicing affects protein Syntaxins Secretory vesicle structure and localization

B SNAP25-a + exon 5a SNAP25 gene 5a Major functional isoform 1 2 3 4 5a 5b 6 7 8 9 SNAP25-b + exon 5b 5b Larger primed vesicle pools and increased secretion stop C AUG AUG stop 11b STX3-A 1 243ab 5 6 7 8 9a10a 11a STX3-C 1 243c 5 6 7 8 9b10b 11b Major isoform in non-neuronal tissue, No SNARE or transmembrane domains involved in epithelial cell membrane trafficking

AUG stop AUG stop

STX3-B 1 243ab 5 6 7 8 9b10b 11b STX3-D 123ab 3c 4 Truncated protein Main isoform in the retina where it is required for vesicle exocytosis

AUG stop D VAMP7 gene 1 24356 78

stop

VAMP7-a 1 24356 788 N LD SM TM C Main isoform in most tissues

stop

VAMP7-b 1 243578 N LD C

stop

VAMP7-c 1 24 5 6 788 N SM TM C No interaction with AP3, no targeting to late endosomes stop

VAMP7-d 1 245 6 78 N SM TM C Truncated protein

stop

VAMP7-h 1 24356 78 N SM TM C Truncated protein

stop

VAMP7-i 1 243 6 788 N LD C Premature stop codon, only nuclear isoform

stop VAMP7-j 1 24378 N LD TM C

Fig. 3. Alternative splicing of SNARE-related proteins. (A) Steps of vesicle-mediated exocytosis that require the assembly of the SNARE complex. The SNARE machinery controls the interaction between vesicular v-SNAREs and target t-SNAREs during vesicle fusion. (B-D) Multiple components of the SNARE machinery undergo alternative splicing, including synaptosome associated protein-25 (SNAP25) (B), syntaxin-3 (STX3) (C) and vesicle associated membrane protein-7 (VAMP7) (D). CELF, CUGBP Elav-like family member-1; LD, Longin domain; SM, SNARE motif; TM, transmembrane domain. exon 5b with a copy of exon 5a leads to developmental defects, (Valladolid-Acebes et al., 2015). A lack of the SNAP25-b isoform spontaneous seizures, impaired short-term synaptic plasticity, in endocrine β-cells increases insulin secretion and alters calcium morphological alterations in adult hippocampus, changes in dynamics (Daraio et al., 2017). Recently, hippocampal lysates have neuropeptide expression and impairment of spatial learning revealed that SNAP25-a is less efficient than SNAP25-b in forming (Johansson et al., 2008). Furthermore, when provided with a high- complexes with Munc18-1 and the Gβ1andGβ2subunitsof fat diet, these mice develop a metabolic syndrome accompanied by heterotrimeric G-proteins. As these interactions play important roles weight gain, thus linking the neuronal defect in the exocytosis in presynaptic inhibition, the results suggest a less inhibitory role of machinery with dyslipidemia and disrupted glucose homeostasis SNAP25-a (Daraio et al., 2018). Journal of Cell Science

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SNAP23 is a SNAP25 homologue that is almost ubiquitously for a potential mechanism underlying the selective and dynamic expressed and has been implicated in insulin sensitivity (Boström mRNA translation in axons (Shigeoka et al., 2016). et al., 2007), lipid-raft actions (Puri and Roche, 2006; Yoon et al., 2016) and endothelial exocytosis of the von Willebrand factor (Zhu VAMPs et al., 2015). The human SNAP23 gene contains eight exons and Human VAMP7 (previously known as SYBL1) encodes the different splice isoforms have been reported; for instance, SNAP23- v-SNARE protein VAMP7, which controls intracellular a differs from SNAP23-b in 53 residues encoded by exon 6 trafficking (Galli et al., 1998). In the full-length VAMP7-a (Mollinedo and Lazo, 1997). This region contains sites for post- isoform, exons 2 to 4 encode the N-terminal Longin domain, translational fatty acid acylation, suggesting differences in which negatively regulates membrane fusion and neurite outgrowth membrane interaction between the isoforms. In vitro, both (Martinez-Arca et al., 2000, 2001), whereas exons 5 to 8 encode the isoforms bind to syntaxin-6 (STX6), but SNAP23-b has an transmembrane region and the SNARE motif (Fig. 3D). Skipping of apparently higher affinity (Lazo et al., 2001). In addition, the exon 3 and the use of an alternative 5′-splice site in exon 2 lead to SNAP23-c variant lacks exons 5 to 7, giving rise to a truncated 50- the truncated proteins VAMP7-d and VAMP7-h, respectively residue protein. Another isoform, SNAP23-d, lacks exons 6 and 7, (Vacca et al., 2011). VAMP7-c uses the same 5′-splice site in and, here, a frame-shift changes the C-terminus of the protein. The exon 2 as VAMP7-h, but lacks exon 3. Thus, in comparison with SNAP23-e isoform also lacks exons 6 and 7, but it uses an VAMP7-a, VAMP7-c lacks 40 residues within the Longin domain, alternative 5′-splice site within exon 8 and shares the same last eight losing the interaction with the adaptor protein AP3 and its targeting residues as SNAP23-a and SNAP23-b (Shukla et al., 2001). All to late endosomes (Martinez-Arca et al., 2003). Splicing of exons 5 isoforms, except SNAP23-c, contain the cysteine-rich domain that and 6 generates three additional isoforms with intact Longin is palmitoylated. Transfected SNAP23-a and SNAP23-b mainly domains, but altered SNARE domains. VAMP7-b lacks exon 6, and localize to the plasma membrane, while the other isoforms also thus has a different C-terminus than VAMP7-a. In VAMP7-i, exhibit intracellular localizations (Shukla et al., 2001). skipping of exon 5 introduces a premature stop-codon, and thus only the Longin domain is expressed. In VAMP7-j, skipping of both Syntaxins exons maintains the original reading frame; therefore, in addition to In the brain, alternative splicing of syntaxins is regulated by the the Longin domain, VAMP7-j contains a short hinge region, and the CUGBP Elav-like family (CELF) of RBPs. In Caenorhabditis original transmembrane and intra-vesicular tail regions (Vacca et al., elegans, the CELF homolog UNC-75 promotes the expression of 2011) (Fig. 3D). The different VAMP7 isoforms exhibit differential the neuronal syntaxin isoform UNC-64A, which includes exon 8a, subcellular and tissue localizations. VAMP7-b is widely distributed, and represses the non-neuronal variant UNC-64B, which includes whereas VAMP7-i is the only nuclear isoform. Furthermore, exon 8b. These isoforms differ in their C-terminal hydrophobic VAMP7-a is the main isoform in most tissues, whereas other membrane anchors (Ogawa et al., 1998; Saifee et al., 1998). The variants show some tissue specificity (Vacca et al., 2011). mutants lacking unc-75 only express UNC-64B and show axon Based on all the studies discussed above, it is clear that alternative regeneration and locomotion defects similar to those observed in splicing regulates different members of the SNARE machinery and unc-64 loss-of-function mutants. While overexpression of either so contributes to the control of exocytosis. UNC-64A or UNC-64B isoforms rescues axon regeneration defects, only UNC-64A rescues locomotion defects (Chen et al., Alternative splicing impacts membrane dynamics 2016; Norris et al., 2014). CELF proteins also control axon Cellular membranes are flexible and extensively remodeled during regeneration in rodents. The Celf2-knockout mouse exhibits axon numerous processes, including cell movement, cell division, regeneration defects and mis-splicing of several syntaxins (Stx1a, endocytosis, muscle contraction and repair, transverse tubule Stx2, Stx16 and Stx18) and syntaxin-binding proteins (Stxbp1, (T-tubule) and sarcomere formation, mitochondria fusion and Stxbp2 and Stxbp5), suggesting that CELF-regulated splicing of fission, as well as axon and dendrite growth. During membrane syntaxins is an evolutionarily conserved mechanism involved in remodeling, reversible curvature changes and areas of high tension axon regeneration (Chen et al., 2016; Norris et al., 2014). exist for limited periods of time and are controlled by trafficking STX3 is highly expressed in spleen, lung, kidney, retina and proteins, such as dynamin, amphiphysin, endophilin and epsin brain, and functions in vesicle trafficking. The mouse Stx3 gene (McMahon and Gallop, 2005). BAR-domain proteins are contains several mutually exclusive exons: 3ab and 3c, 9a and 9b, cytoplasmic molecules with membrane-bending properties (Mim 10a and 10b, and 11a and 11b, generating four splice variants and Unger, 2012). Below, we describe two BAR-domain proteins (shown in Fig. 3C) that differ in their domain organization and that are regulated by alternative splicing: CDC42-interacting biochemical properties (Ibaraki et al., 1995). STX3-A and STX3-B protein-4 (TRIP10, also known as CIP4) and bridging integrator share the N-terminus but differ in the second half of the SNARE protein-1 (BIN1, also known as amphiphysin-2). domain and the C-terminal transmembrane domain. In STX3-D, the inclusion of both exons 3ab and 3c introduces a premature stop- TRIP10 codon and thus produces a truncated protein (Curtis et al., 2008). TRIP10 controls centrosome and Golgi polarization in migratory STX3-A is the only isoform detected in the kidney with proposed cells (Tonucci et al., 2015), E-cadherin trafficking during epithelial functions in epithelial cell membrane trafficking, whereas STX3-B morphogenesis (Zobel et al., 2015), cell growth and invasion in is the exclusive variant found in retinal ribbon synapses (Curtis cancer metastasis (Chander et al., 2012; Rolland et al., 2014; et al., 2008) where it is required for synaptic vesicle exocytosis Truesdell et al., 2014), hypertrophy in neonatal cardiomyocytes (Curtis et al., 2010). Recently, a translatome study in mouse retinal (Rusconi et al., 2013), and GLUT4 trafficking in adipocytes (Chang cells revealed that axons and soma use different last exons in the et al., 2002). The N-terminus of TRIP10 contains the FCH domain Stx3 gene, leading to differences in the C-terminus of the encoded and two coiled-coil domains, and mediates the interaction with STX3 proteins and the 3′UTR. Notably, the axon-specific exon is AKAP350, tubulin and phospholipids. Its internal GTP-binding sufficient to promote axonal mRNA translation, providing evidence homology region mediates binding to the GTPases TC10 (Chang Journal of Cell Science

5 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 et al., 2002) and CDC42 (Aspenström, 1997) (Fig. 4A, top). The demonstrates that trafficking proteins have structural roles in Src-homology-3 (SH3) domain controls the interaction of TRIP10 striated muscles. For example, CLTC regulates the formation and with the Wiskott-Aldrich syndrome protein (WASP) and DNM2, maintenance of the costameres (Vassilopoulos et al., 2014), which thus connecting actin polymerization with membrane deformation are the lateral attachment sites between the sarcolemma and the to promote GLUT4 trafficking (Hartig et al., 2009) and initiation sarcomere. Another example from a recent study in Drosophila and scission of endocytic vesicles (Feng et al., 2010). Exon 10 of the melanogaster revealed that autophagy is required for T-tubule TRIP10 gene is alternatively spliced generating a 545-residue remodeling through a mechanism that implicates the GTPase RAB2 isoform (CIP4a) when it is skipped and a 601-residue variant (Fujita et al., 2017). Overall, these and other studies have revealed (known as CIP4h or CIP4/2) when it is included (Fig. 4A). The unconventional but crucial roles of trafficking proteins in muscle isoform lacking exon 10 is the most ubiquitously expressed. TRIP10 cell architecture. variant containing exon 10 was identified in a two-hybrid screening BIN1 induces membrane tubulation in muscle cells, and as a TC10-interacting protein (Chang et al., 2002; Wang et al., 2002) misregulation of its alternative splicing contributes to T-tubule and found to be expressed in adult skeletal muscles and hearts (Feng alterations seen in muscular dystrophies and cardiac diseases (Böhm et al., 2010; Giudice et al., 2014, 2016). CIP4b (also known as Felic) et al., 2013; Fugier et al., 2011; Hong et al., 2014). In patients with is a TRIP10 isoform that is exclusively expressed in human prostate myotonic dystrophy (DM), exon 11 is skipped, contributing to cancer and lymphoblast cells and is generated by a 29-nucleotide muscle weakness and T-tubule defects (Fugier et al., 2011). insertion of intron 13 that destroys the SH3 domain (Wang et al., Reintroduction of the BIN1 isoform that contains exon 11 restores 2002). Furthermore, aberrant splicing in renal carcinoma generates T-tubule organization in myofibers isolated from DM patients, the CIP4-V variant by a 19-nucleotide retention of intron 9, which whereas, by contrast, the variant lacking exon 11 is unable to introduces a premature stop codon (Tsuji et al., 2006) (Fig. 4A). recover the phenotype (Fugier et al., 2011). Further evidence for the CIP4-V lacks the CDC42-binding region and the SH3 domain, and importance of exon 11 comes from the human BIN1 mutation its overexpression induces the formation of ubiquitylated (IVS10-1G>A) that affects the acceptor splice site in a aggresomes and defects in β-catenin trafficking that ultimately consanguineous family with rapidly progressive and fatal reduce cell adhesion and contribute to cancer progression and centronuclear myopathy (Böhm et al., 2013). Interestingly, the metastasis (Tsuji et al., 2006). same splice site is mutated (IVS10-2A>G) in a spontaneous dog model of the disease. Humans and dogs carrying these mutations BIN1 in brain and cancer lack exon 11 and exhibit a similar histopathology with BIN1 is a ubiquitous BAR-domain protein that is highly expressed ultrastructural and membrane-triad defects (Böhm et al., 2013). In in striated muscles and brain (Butler et al., 1997; Sakamuro et al., the heart, exons 7 and 11 are skipped, and exons 13 and 17 are 1996). BIN1 regulates a number of cellular processes, including developmentally regulated, contributing to T-tubule organization endocytosis, cytoskeleton organization, DNA repair, the cell cycle, (Hong et al., 2014). In cardiomyocytes, T-tubules contain dense tumor suppression, membrane invagination and T-tubule protective inner membrane folds, which are formed by the BIN1 organization (Prokic et al., 2014). BIN1 has multiple domains isoform containing exons 13 and 17 (Hong et al., 2014). (Fig. 4B): (1) the BAR domain (exons 1-10), which binds to Accordingly, in Bin1-depleted mice, T-tubule folding is membrane lipids and senses membrane curvature; (2) a polybasic decreased, leading to free diffusion of local calcium and segment encoded by the muscle-specific exon 11 that mediates its potassium ions, which prolongs the action potential and increases affinity to phosphoinositides and T-tubules (Kojima et al., 2004; susceptibility to ventricular arrhythmias. These T-tubule defects can Lee et al., 2002); (3) a brain-specific clathrin- and AP2-binding be rescued by expression of the BIN1 isoform that contains exons 13 domain (CLAP) (exons 13-16) (Ramjaun and McPherson, 1998); and 17, which facilitates the folding of the T-tubule membrane (4) a MYC-binding domain (MBD) (exons 17-18) that confers (Hong et al., 2014) and so creates a local region of ion accumulation tumor-suppressor functions; and (5) the SH3-domain (exons 19- that restricts ion flux. 20), which mediates binding to proline-rich motifs. BIN1 contains Therefore, the fact that T-tubule maturation and alternative seven alternatively spliced exons and mis-splicing occurs in disease splicing of membrane-trafficking genes follow similar contexts. In the brain, exon 7 and exons 13-17 are included (Prokic developmental time-courses in striated muscles, suggests that et al., 2014); here, inclusion of exon 7 promotes transferrin splicing contributes to T-tubule biogenesis. In support of this endocytosis and interaction between BIN1 with DNM2 (Ellis et al., notion, developmental splicing networks coordinately revert to 2012) (Fig. 4C). In melanoma and breast cancer, the aberrant neonatal patterns in disease contexts, such as heart failure, inclusion of exon 13 disrupts the tumor-suppressor functions of cardiomyopathies, congenital muscular dystrophy and DM, as BIN1 (Ge et al., 1999), and exogenous expression of a BIN1 variant discussed below. lacking exon 13 restores its binding with MYC and thus its tumor- suppressor functions (Anczuków et al., 2012). Role of RBPs in alternative splicing of membrane-trafficking proteins in health and disease BIN1 in striated muscles In this section, we discuss how alternative splicing of trafficking Vesicle-mediated transport, membrane remodeling and cytoskeletal proteins is regulated by RBPs and misregulated in neurological and genes are globally regulated by alternative splicing in mouse striated muscle disease (Fig. 5). muscles during the first four postnatal weeks (Brinegar et al., 2017; Giudice et al., 2014). This is the period of maturation of the CELF and MBNL splicing networks in DM1 sarcoplasmic reticulum and T-tubules that are necessary for The muscleblind-like (MBNL) and CELF family of RBPs excitation-contraction coupling and adult contractility. In striated antagonistically regulate alternative splicing in striated muscles muscle cells, T-tubules facilitate the access of environmental signals (Wang et al., 2015) and are associated with DM, which is an and the propagation of membrane depolarization deep into cells inherited neuromuscular disease and the most common adult during excitation-contraction coupling. Growing evidence muscular dystrophy. DM patients suffer from skeletal myopathy, Journal of Cell Science

6 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465

A TRIP10 gene + Exon 10 N FCH Coiled-coil SH3 C TRIP10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (601 aa) – Exon 10 N FCH Coiled-coil SH3 C TRIP10 (545 aa)

AKAP350 TC10 WASP 9 10 13 14 Tubulin CDC42 DNM2 Phospholipids GAPEX5 Binding partners 9 10 13 14

19 nt 29 nt Premature stop codon Premature stop codon

N C N C CIP4-V CIP4b (341 aa) (456 aa)

Ubiquitylated aggresomes Human prostate cancer cells Cell adhesion Human lymphoblast cells Defects in -catenin trafficking Alternative exons B Brain 7 11 13 14-16 17 + – + + +

+ – + + –

BIN1 gene Skeletal muscle – + – – + 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20C – + – – –

N BAR PI CLAP MBDM SH3 C Heart Adult – – + – – CLIP1 T-tubules CLTC MYC DNM1/2 BIN1, MTM1 PIPs AP1 MYCN RIN2/3, MTM1 Fetal – – + – + AMPH1, DNM2 AP2 SH3GLB1 Curvature sensor SH3GL2 SNX4, BIN1 Cancer cells Interactions – – + – +

MYC

Ubiquitous – – – – + – – – – – C + Exon 7 – Exon 7

DNM2 Binding BIN1-DNM2 No binding BIN1-DNM2

DNM2

Transferrin receptor

Transferrin Transferrin Transferrin endocytosis endocytosis

Fig. 4. Alternative splicing of the BAR-domain proteins TRIP10 and BIN1. (A) The TRIP10 (CDC42-interacting protein-4) gene is regulated by alternative splicing. Alternative splicing of exon 10 gives rise to two protein isoforms, one containing 601 amino acids (aa) and the other 545 aa. Retention of portions of intron 9 or 13 generates CIP4-V and CIP4b (Felic) variants, respectively, which are present in the context of cancer. (B) The bridging integrator protein-1 BIN1 is also highly regulated by alternative splicing in a tissue- and developmental-stage-specific manner. BIN1 domain structure is shown on the left with the diverse splice isoforms in different tissues illustrated on the right. (C) Mis-splicing of exon 7 of BIN1 affects its interaction with dynamin-2 (DNM2) and thus endocytosis, as illustrated here for the model cargo transferrin. AKAP350, A-kinase anchoring protein-9; AMPH1, amphiphysin; AP1/2, adaptor complex-1/2; CDC42, cell division cycle 42; CLAP, clathrin- and AP2-binding domain; CLIP1, CAP-Gly domain containing linker protein-1; CLTC, clathrin heavy chain; DM, myotonic dystrophy; DNM1, dynamin-1; GAPEX5, GTPase activating protein and VPS9 domains 1; MBD, MYC-binding domain; MTM1, myotubularin-1; MYC, MYC proto-oncogene; bHLH transcription factor; nt, nucleotides; PI, phosphoinositide binding domain; PIPs, phosphoinositides; RIN2, Ras and Rab interactor 2; RIN3, Ras and Rab interactor 2; SH3, Src-homology-3 domain; SH3GL2, SH3-domain containing GRB2-like or endophilin A1; SH3GLB1, SH3-domain containing GRB2 like or endophilin B1; SNX4, sorting nexin-4; TC10-Ras homolog family member-Q; T-tubules, transverse tubules; WASP, Wiskott-Aldrich syndrome protein family. Journal of Cell Science

7 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 cardiac arrhythmia, cataracts, hypogonadism, hyper-somnolence regulate an evolutionarily conserved splicing-trafficking network and insulin resistance, among other symptoms (Harper, 2001). DM1 that includes the sortin nexin-9 (SNX9), STX2 and VPS29 is caused by expansion of CTG repeats in the 3′UTR region of the (Burguera et al., 2017) (Fig. 5B). RBFOX2 is induced in EMT DMPK gene that leads to activation of CELF1 (gain of function) and and regulates splicing of the endocytic proteins cortactin and sequestration of MBNL, causing its loss of function. Alterations in DNM2. Depletion of RBFOX2 after EMT significantly reduces cell the functions of CELF and MBNL result in mis-splicing of the invasive potential, suggesting that RBFOX2-regulated splicing targets of these two RBPs, which include several trafficking genes controls tissue invasiveness (Braeutigam et al., 2014). (Fig. 5A) (Dixon et al., 2015; Freyermuth et al., 2016; Fugier et al., 2011). CELF and MBNL misregulation results in the global re- NOVA proteins expression of the fetal splicing isoforms of several proteins that we In the pancreas, NOVA1 has been implicated in regulating splicing of discuss below. This reprogramming in adult tissues takes place a number of proteins, including the INSR, exocytosis proteins such as mainly in the heart and skeletal muscle. The fetal isoforms are SNAP25, the calcium-dependent secretion activator (CADPS), as functional in embryonic and neonatal muscles; however, adult well as Rho-GTPases, such as cell division cycle-42 (CDC42) and the muscle cells are large and require a precise internal architecture for RhoGEF protein ARHGEF12 (Villate et al., 2014) (Fig. 5B). proper excitation-contraction coupling. In adults, skipping of exon However, the functional implications of this splicing network that is 7a of the muscle chloride channel CLCN1 generates the functional regulated by NOVA1 in the β-cells were not investigated. full-length protein but loss of function of MBNL in DM1 leads to In the brain, NOVA1 regulates neuron-specific alternative splicing the inclusion of exon 7a, which generates a frameshift and thus non- and is essential for motor neuron survival after birth (Jensen et al., functional CLCN1 (Charlet-Berguerand et al., 2002; Mankodi et al., 2000). Furthermore, the Nova2-knockout mouse exhibits major 2002). Chloride channels are dispensable in neonatal small splicing defects in genes that encode proteins with well-defined myofibers with undeveloped T-tubules, but their absence in large functions at the synapse, including neurotransmitter receptors, cation adult myofibers disrupts the potassium counterbalance within T- channels, adhesion and scaffold proteins, or in axon guidance (Ule tubules, resulting in depolarization and myotonia, as seen in DM1 et al., 2005). NOVA proteins regulate splicing of all members of the patients (Charlet-Berguerand et al., 2002; Mankodi et al., 2002). spectrin–ankyrin–Protein-4.1–CASK scaffold complex, which acts Furthermore, the alterations in MBNL and CELF function result in as a linker between membrane proteins and the actin-tropomyosin the mis-splicing of the ryanodine receptor-1 (RYR1) (exon 70), the cytoskeleton, and contributes to organization of the GABAergic ATPase sarcoplasmic/endoplasmic reticulum calcium transporting- synapse (Jensen et al., 2000; Ule et al., 2003; Ule et al., 2005). The 1 (ATP2A1) (exon 22), the calcium voltage-gated channel subunit- functional implications of NOVA-regulated splicing have been alpha1S (CACNA1S) (exon 29) and BIN1 (exon 11), which is convincingly demonstrated for the reelin adaptor protein DAB1, thought to partially contribute to muscle weakness associated with where NOVA2 controls neuronal migration by regulating the splicing DM1 (Fugier et al., 2011; Kimura et al., 2005). In particular, of exons 7b and 7c of DAB1 (Yano et al., 2010). aberrant skipping of exon 29 of the channel CACN1S correlates with the severity of muscle weakness in DM patients (Tang et al., PTBP coordinates splicing trafficking networks in spermatogenesis 2012). Finally, insulin insensitivity is associated with mis-splicing and ovarian cancer of exon 11 of the insulin receptor (INSR), which is due to the Polypyrimidine-tract binding proteins (PTBPs) regulate alternative dysfunction of MBNL and CELF proteins (Dansithong et al., 2005; splicing in the brain (Vuong et al., 2016), during spermatogenesis Echeverria and Cooper, 2014; Savkur et al., 2001, 2004). (Hannigan et al., 2017; Zagore et al., 2015), in myogenesis (Hall et al., Heart samples from DM1 adult patients exhibit global splicing 2013) and in ovarian cancer (He et al., 2015). PTBP2 is required for changes in genes encoding proteins that control ion handling, cell mouse spermatogenesis and proper splicing regulation of a network of architecture and vesicle-mediated transport (Freyermuth et al., 2016). genes that encode proteins involved in nearly all steps of vesicle- One of these genes is the sodium channel protein type-5 subunit-alpha mediated trafficking. This includes subunits of the AP1 and AP2 (SCN5A), which is critical for cardiomyocyte excitability and impulse complexes and DENND1A, which is a component of clathrin-coated propagation through the conduction system. The SCN5A isoform vesicles that binds to the AP2 complex, proteins involved in tethering containing exon 6a is predominant in human fetal hearts, exons 6a vesicles to microtubules and target membranes, and factors involved and 6b are used to similar extents in infants, and the isoform in actin dynamics (Hannigan et al., 2017; Zagore et al., 2015) containing exon 6b is preferred in adulthood. The two isoforms differ (Fig. 5C). PTBP2 depletion in germ cells results in a disorganization in seven residues in one of the voltage-sensing domains, and thus in of the F-actin cytoskeleton in Sertoli cells, indicating that regulation of their electrophysiological properties. DM1 patients express the fetal alternative splicing is necessary for cellular crosstalk during germ cell isoform of SCN5A and exhibit severe cardiac conduction defects and development (Hannigan et al., 2017). arrhythmias that are reproduced when this isoform is re-expressed in In ovarian cancer cells, PTBP1 depletion has been shown to adult murine hearts (Freyermuth et al., 2016; Onkal et al., 2008). inhibit the formation of filopodia and to alter CDC42 splicing (Fig. 5C). The Cdc42 gene encodes two protein isoforms, CDC42-v1 ESRPs and RBFOX proteins and CDC42-v2, which differ in their terminal exons. While CDC42- The epithelial splicing regulatory proteins ESRP1 and ESRP2, v1 includes exon 6a, CDC42-v2 includes exon 6b. Because PTBP1 along with the RNA-binding fox-1 homolog-2 protein (RBFOX2) represses exon 6b, it reduces expression of the CDC42-v2 variant, and the neuro-oncological ventral antigen-1 (NOVA1) regulate which has been shown to have inhibitory effects on ovarian cancer splicing during epithelial-mesenchymal transition (EMT) in cell growth and invasiveness, thus functioning as a tumor suppressor development and in metastatic cancer (Lu et al., 2015). ESRP2 is (He et al., 2015). More recently, it has been shown that perturbing the a driver of liver development, where it regulates splicing of the ratio between the CDC42-v1 and CDC42-v2 variants in neurons retromer complex component VPS29, synaptojanin-2 (SYNJ2) and results in alterations in axons and dendrites because CDC42-v1 is STX2, among other trafficking genes (Bhate et al., 2015). In the required for the development of dendritic spines, whereas CDC42-v2 embryonic epidermis in humans, mice and fish, ESRP1 and ESRP2 functions in axogenesis (Yap et al., 2016) (Fig. 5D). Journal of Cell Science

8 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465

A Heart and skeletal muscle B Liver, pancreas, epidermis, and EMT

CTG repeats DMPK gene 3ЈUTR ESRP STX2, SYNJ2, VPS29, CELF MBNL SNX9 gain of function loss of function

Insulin RBFOX2 resistance EMT CTTN, DNM2 Mis-splicing BIN1, CLCN1, RYR1, Channel dysfunction, CACNA1S, ATP2A1, myotonia Epidermis INSR, SCN5A T-tubule defects, muscle weakness Cancer NOVA1 Metabolism Invasiveness CDC42, CADPS1, Arrhythmia DM1 CADPS2, INSR, Diabetes SNAP25, ARHGEF12 Development Functions in β-cells? PTBP1 Ovarian cancer Development Development of CDC42 Spermatogenesis Synaptogenesis axons and dendrites ASD CELF Syntaxins, syntaxin- PTBP1 binding proteins CDC42 Filopodia formation Neuronal NOVA migration DAB1, CADPS2, CASK, ankyrins, spectrins, EPB41 Organization of actin cytoskeleton Clathrin-coated Neuronal activity Mis-splicing vesicles DNM2, CASK,BIN1, PTBP2 AP1B1, AP1S2, STX2, Splicing network involving: CTTN, SYNJ1, PTBP2 AP complex Mis-splicing STX16, STXBP1, SNAPs and SYNJs SRRM4 AP2A1, AP3S1, Actin dynamics downregulated CLTA, CLTB RBFOX1 downregulated ASD

ASD ASD C Reproductive organs D Brain and neurons

Fig. 5. RBPs regulate alternative splicing of membrane-trafficking proteins in health and disease. (A) In myotonic dystrophy type-1 (DM1), CTG repeat expansion in the 3′UTR region of the DMPK gene leads to the activation of CUGBP Elav-like family member-1 (CELF1) (gain of function) and the sequestration of muscleblind-like protein (MBNL), which causes its loss of function. Alterations in the functions of CELF and MBNL lead to mis-splicing of their targets (listed below). (B) Epithelial ESRP1 and ESRP2 along with the RNA-binding fox-1 homolog-2 protein (RBFOX2) and the neuro-oncological ventral antigen-1 (NOVA1) regulate splicing during epithelial-mesenchymal transition (EMT) in liver development and in metastatic cancers. (C) Polypyrimidine-tract binding proteins (PTBPs) regulate alternative splicing in spermatogenesis and ovarian cancer. PTBP2 is required for mouse spermatogenesis and proper splicing regulation of a network of genes encoding subunits of the AP1 and AP2 complexes, members of the SNARE complex and synaptojanins, and proteins involved in actin dynamics. In ovarian cancer cells, PTBP1 depletion inhibits the formation of filopodia and affects CDC42 (cell division cycle 42) splicing. (D) In the brain, NOVA proteins regulate neuron-specific alternative splicing events that are essential for motor neuron survival, synapse, and axon guidance. Brains of people with autism spectrum disorder (ASD) show downregulation of RBFOX1 and the serine/arginine repetitive matrix-4 (SRRM4), and thus dysregulation of the dependent alternative exons in the factors listed. AP2A1, adaptor-related protein complex-2 α1 subunit; AP3S1, adaptor-related protein complex-3 sigma-1 subunit; ARHGEF12, Rho-GEF12; ATP2A1, ATPase sarcoplasmic/endoplasmic reticulum calcium transporting-1; BIN1, bridging integrator protein-1; CACNA1S, calcium voltage-gated channel subunit alpha-1S; CADPS, calcium-dependent secretion activators; CASK, calcium-/calmodulin-dependent serine protein kinase; CLTA, clathrin light chain-a; CLTB, clathrin light chain-b;CTTN, cortactin; DAB1, reelin adaptor protein; DNM2, dynamin-2; EPB4.1, erythrocyte membrane protein band 4.1; INSR, insulin receptor; RYR1, ryanodine receptor-1; SCN5A, sodium voltage-gated channel alpha subunit-5; SNX9, sorting nexin-9; STX16, syntaxin-16; STXBP1, syntaxin binding protein-1; SYNJ1, synaptojanin-1; SYNJ2, synaptojanin-2; VPS29, retromer complex component.

RBFOX1 and SRRM4 in autism spectrum diseases RBFOX1-dependent alternative exons in genes that encode Autism spectrum disorder (ASD) is a highly heritable proteins involved in cytoskeleton organization and vesicle- neurodevelopmental condition which is characterized by genetic mediated transport such as CLTA, CLTB, STX16, STXBP1, heterogeneity. High-throughput studies have revealed common AP2A1, AP3S1, among several others (Voineagu et al., 2011) patterns of misregulated gene expression and alternative splicing (Fig. 5D). in ASD (Gupta et al., 2014; Irimia et al., 2014; Quesnel-Vallieres̀ Another RBP that is downregulated in ASD is the serine/arginine et al., 2016; Voineagu et al., 2011). Brains from ASD patients repetitive matrix-4 (SRRM4, also known as nSR100). SRRM4 exhibit a downregulation of the RBP RBFOX1 (also known as controls the inclusion of a large set of brain-specific alternative

A2BP1 and FOX1) and, accordingly, a dysregulation of the exons that are significantly enriched in genes associated with Journal of Cell Science

9 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 membrane dynamics, endocytosis and cytoskeleton remodeling Aspenström, P. (1997). A Cdc42 target protein with homology to the non-kinase (Calarco et al., 2009). SRRM4 controls the neural specificity of domain of FER has a potential role in regulating the actin cytoskeleton. Curr. Biol. 7, 479-487. alternative splicing by activating the expression of the neuronal Baralle, F. E. and Giudice, J. (2017). Alternative splicing as a regulator of isoform of PTBP2 (nPTBP), which includes its exon 10. Therefore, development and tissue identity. Nat. Rev. Mol. Cell Biol. 18, 437-451. nPTB and SRRM4 act in concert to control neuronal alternative Bark, I. C. and Wilson, M. C. (1994). Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25. Gene 139, 291-292. splicing (Calarco et al., 2009). Numerous neural microexons (3-15 Bark, I. C., Hahn, K. M., Ryabinin, A. E. and Wilson, M. C. (1995). Differential nucleotides) are frequently misregulated in ASD brains, and this has expression of SNAP-25 protein isoforms during divergent vesicle fusion events of been shown to be associated with a reduced expression of SRRM4 neural development. Proc. Natl. Acad. Sci. USA 92, 1510-1514. (Irimia et al., 2014). Gene ontology analysis revealed that the Bark, C., Bellinger, F. P., Kaushal, A., Mathews, J. R., Partridge, L. D. and Wilson, M. C. (2004). Developmentally regulated switch in alternatively spliced microexons that are misregulated in ASD are significantly enriched SNAP-25 isoforms alters facilitation of synaptic transmission. J. Neurosci. 24, for those present in vesicle-mediated transport genes, among a few 8796-8805. other categories (Irimia et al., 2014). Furthermore, mutant mice Bhate, A., Parker, D. J., Bebee, T. W., Ahn, J., Arif, W., Rashan, E. H., lacking one functional copy of the Srrm4 gene exhibit several Chorghade, S., Chau, A., Lee, J.-H., Anakk, S. et al. (2015). ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation. Nat. autistic-like features and recapitulate the misregulated splicing Commun. 6, 8768. patterns that are observed in brains of people with ASD (Irimia et al., Böhm, J., Vasli, N., Maurer, M., Cowling, B., Shelton, G. D., Kress, W., 2014; Quesnel-Vallieres̀ et al., 2016) (Fig. 5D). Toussaint, A., Prokic, I., Schara, U., Anderson, T. J. et al. (2013). Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly Taken together, it has become clear that the regulation of progressive centronuclear myopathy. PLoS Genet. 9, e1003430. trafficking proteins by alternative splicing is not limited to one Boström, P., Andersson, L., Rutberg, M., Perman, J., Lidberg, U., Johansson, specific RBP and in most cases, RNA processing results from the B. R., Fernandez-Rodriguez, J., Ericson, J., Nilsson, T., Borén, J. et al. (2007). action of multiple RBPs that function in concert. SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity. Nat. Cell Biol. 9, 1286-1293. Boumil, R. M., Letts, V. A., Roberts, M. C., Lenz, C., Mahaffey, C. L., Zhang, Z., Perspectives Moser, T. and Frankel, W. N. (2010). A missense mutation in a highly conserved The physiological relevance of alternative splicing was first alternate exon of dynamin-1 causes epilepsy in fitful mice. PLoS Genet. 6, e1001046. investigated for individual genes; however, genome-wide studies Braeutigam, C., Rago, L., Rolke, A., Waldmeier, L., Christofori, G. and Winter, J. have exponentially increased the number of splicing isoforms, and (2014). The RNA-binding protein Rbfox2: An essential regulator of EMT-driven highlighted networks with unknown and unexplored functions alternative splicing and a mediator of cellular invasion. Oncogene 33, 1082-1092. (Baralle and Giudice, 2017). High-resolution imaging and state-of- Brinegar, A. E., Xia, Z., Loehr, J. A., Li, W., Rodney, G. G. and Cooper, T. A. (2017). Extensive alternative splicing transitions during postnatal skeletal muscle the-art genetic tools are now available to study trafficking within development are required for Calcium handling functions. eLife 6, e27192. entire organisms and recent progress in the splicing and trafficking Brodsky, F. M. (2012). Diversity of clathrin function: new tricks for an old protein. fields pinpoints the questions that remain unanswered at the Annu. Rev. Cell Dev. Biol. 28, 309-336. intersection between these two scientific fields. First, the Brodsky, F. M., Chen, C.-Y., Knuehl, C., Towler, M. C. and Wakeham, D. E. (2001). Biological basket weaving: formation and function of clathrin-coated physiological roles of numerous splicing events in trafficking vesicles. Annu. Rev. Cell Dev. Biol. 17, 517-568. proteins are still unknown within in vivo contexts. The second Buljan, M., Chalancon, G., Eustermann, S., Wagner, G. P., Fuxreiter, M., challenge arises from the fact that alternative splicing is regulated or Bateman, A. and Babu, M. M. (2012). Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks. Mol. misregulated in a coordinated manner in development and disease. Cell 46, 871-883. Therefore, we need to investigate how splicing trafficking networks Burguera, D., Marquez, Y., Racioppi, C., Permanyer, J., Torres-Méndez, A., modulate intracellular functions, thereby contributing to tissue Esposito, R., Albuixech-Crespo, B., Fanlo, L., D’Agostino, Y., Gohr, A. et al. identity, and ultimately organ maturation and function. Finally, we (2017). Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes. Nat. Commun. 8, 1799. believe that expanding our knowledge of the contribution of mis- Butler, M. H., David, C., Ochoa, G.-C., Freyberg, Z., Daniell, L., Grabs, D., splicing of membrane-trafficking genes in disease will open new Cremona, O. and De Camilli, P. (1997). Amphiphysin II (SH3p9; BIN1), a possibilities for therapeutic approaches. member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle. J. Cell Biol. 137, 1355-1367. Acknowledgements Calarco, J. A., Superina, S., O’Hanlon, D., Gabut, M., Raj, B., Pan, Q., Skalska, We thank Drs Thomas Cooper (Baylor College of Medicine) and Frances Brodsky U., Clarke, L., Gelinas, D., van der Kooy, D. et al. (2009). Regulation of (University College London) for their feedback on the initial draft. vertebrate nervous system alternative splicing and development by an SR-related protein. Cell 138, 898-910. Competing interests Cao, H., Garcia, F. and Mcniven, M. A. (1998). Differential distribution of dynamin The authors declare no competing or financial interests. isoforms in mammalian cells. Mol. Biol. Cell 9, 2595-2609. Chander, H., Truesdell, P., Meens, J. and Craig, A. W. B. (2012). Transducer of Funding Cdc42-dependent actin assembly promotes breast cancer invasion and The authors are funded by National Institutes of Health/NIGMS grant (R25- metastasis. Oncogene 32, 3080-3090. GM089569) to E.G.C., Junior Faculty Development Award, Pilot & Feasibility Chang, L., Adams, R. D. and Saltiel, A. R. (2002). The TC10-interacting protein Research Grant (Nutrition and Obesity Research Center, P30DK056350), and start- CIP4/2 is required for insulin-stimulated Glut4 translocation in 3T3L1 adipocytes. up funds from The University of North Carolina at Chapel Hill (to J.G.) and in part by Proc. Natl. Acad. Sci. USA 99, 12835-12840. the March of Dimes Foundation (5-FY18-36) to J.G. Deposited in PMC for release Charlet-Berguerand, N., Savkur, R. S., Singh, G., Philips, A. V., Grice, E. A. and after 12 months. Cooper, T. A. (2002). Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol. Cell 10, 45-53. Chen, L., Liu, Z., Zhou, B., Wei, C., Zhou, Y., Rosenfeld, M. G., Fu, X.-D., Supplementary information Chisholm, A. D. and Jin, Y. (2016). CELF RNA binding proteins promote axon Supplementary information available online at regeneration in C. elegans and mammals through alternative splicing of syntaxins. http://jcs.biologists.org/lookup/doi/10.1242/jcs.216465.supplemental eLife 5, e16072. Cook, T. A., Urrutia, R. and McNiven, M. A. (1994). Identification of dynamin 2, an References isoform ubiquitously expressed in rat tissues. Proc. Natl. Acad. Sci. USA 91, Anczuków, O., Rosenberg, A. Z., Akerman, M., Das, S., Zhan, L., Karni, R., 644-648. Muthuswamy, S. K. and Krainer, A. R. (2012). The splicing factor SRSF1 Curtis, L. B., Doneske, B., Liu, X., Thaller, C., McNew, J. A. and Janz, R. (2008). regulates apoptosis and proliferation to promote mammary epithelial cell Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina. J. Comp.

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