REVIEW

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

ABSTRACTThe cell biology field has outstanding working knowledge of thefundamentals of membrane-trafficking pathways, which are of criticalimportance in health and disease. Current challenges includeunderstanding how trafficking pathways are fine-tuned forspecialized tissue functions in vivo and during development. Inparallel, the ENCODE project and numerous genetic studies haverevealed that alternative splicing regulates gene expression in tissuesand throughout development at a post-transcriptional level. ThisReview summarizes recent discoveries demonstrating that alternativesplicing affects tissue specialization and membrane-traffickingproteins during development, and examines how this regulation isaltered in human disease. We first discuss how alternative splicing ofclathrin, SNAREs and BAR-domain proteins influences endocytosis,secretion and membrane dynamics, respectively. We then focus onthe role of RNA-binding proteins in the regulation of splicing ofmembrane-trafficking proteins in health and disease. Overall, our aimis to comprehensively summarize how trafficking is molecularlyinfluenced by alternative splicing and identify future directionscentered on its physiological relevance.

KEYWORDS: RNA-binding proteins, Alternative splicing, Membranedynamics, Trafficking

IntroductionMembrane trafficking controls multiple cellular functions.Trafficking to and from the plasma membrane modulates cellcommunication during organ development and function.Trafficking from the cell exterior comprises internalization of ionchannels, receptors and ligands to control homeostasis andsignaling. Trafficking from the inside controls the transport ofnewly synthetized proteins from the endoplasmic reticulum to theirfinal destinations. Numerous human diseases are caused bymutations in membrane-trafficking genes (Dowling et al., 2008;Sigismund et al., 2012), highlighting the physiological importanceof these proteins.Membrane-trafficking genes are developmentally and tissue-

specifically regulated by alternative splicing (Brinegar et al., 2017;Dillman et al., 2013; Giudice et al., 2014; Hannigan et al., 2017;Irimia et al., 2014), a post-transcriptional mechanism used by singlegenes to produce multiple transcripts and, thus, several proteinisoforms with different features. Brain, heart and skeletal muscle,together with the testes, are the organs where most of the tissue-specific and conserved alternative splicing takes place (Merkin

et al., 2012). Tissue-specific exons encode disordered segmentswithin proteins that function in microtubule-based transport,endocytosis and membrane deformation (Buljan et al., 2012; Elliset al., 2012) (Box 1).

In humans, 90-95% of genes undergo alternative splicing,expanding protein function beyond genetic diversity (Pan et al.,2008;Wang et al., 2008). Splicing of intronic regions is regulated bythe strength of the splice sites; strong splice sites lead to constitutivesplicing, whereas weak splice sites are used in a context-dependentmanner (alternative splicing). Usage of weak splice sites is regulatedby cis-regulatory sequences, trans-acting factors such as RNA-binding proteins (RBPs) and epigenetics (Kornblihtt et al., 2013).Depending on splice site locations, different types of alternativesplicing events are produced, which comprise insertion ofalternative cassette exons or mutually exclusive exons, selectionbetween alternative 5′- or 3′-splice sites, poly-adenylation sites andintron retention (Fig. 1). Alternative splicing can dramaticallyimpact protein function or affect the expression, localization, and/orstability of mRNAs (Irimia and Blencowe, 2012). Coordination ofalternative splicing contributes to cell differentiation, lineagedetermination, tissue identity acquisition and, ultimately, organdevelopment (Baralle and Giudice, 2017; Wang et al., 2008). Thephysiological relevance of splicing is evident from the vast numberof mutations in cis-regulatory elements, RBPs or spliceosomecomponents, which cause a broad spectrum of human diseases(Scotti and Swanson, 2016).

Here, we review the molecular connection between alternativesplicing and membrane trafficking from both physiological anddisease perspectives. First, we discuss how alternative splicingimpacts membrane-trafficking proteins involved in clathrin-mediated endocytosis (CME), secretory pathways and membranedynamics. Then, we discuss the role of RBPs in controllingalternative splicing of trafficking proteins and how this regulationcontributes to health and disease. The final section identifies themajor questions still outstanding in the field.

Alternative splicing regulation and CMECME is one of the most common mechanisms that cells employ toabsorb nutrients, hormones or proteins from the exterior andinvolves clathrin-coated vesicles. In addition, CME regulates theprotein content of the plasma membrane, monitors external cuesfrom the surrounding environment, modulates signaling pathwaysand directs protein recycling and degradation (McMahon andBoucrot, 2011). Loss of function of central components of theCME machinery, such as clathrin, AP2, epsin or dynamin, isembryonically lethal and alterations in other CME proteins areoften present in cancer, neurological disorders, genetic syndromesand muscle pathologies. CME occurs in multiple steps (Fig. 2):(1) the formation of clathrin-coated vesicles starts by membraneinvagination (nucleation); (2) proteins are recruited to the nucleationsite by the AP2 complex, which together with other cargo-specificadaptors mediates cargo selection; (3) clathrin polymerizationstabilizes the curvature of the forming vesicle; (4) dynamin,

1Department of Cell Biology & Physiology, School of Medicine, The University ofNorth Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 2McAllister HeartInstitute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599,USA. 3Curriculum in Genetics and Molecular Biology (GMB), The University ofNorth Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.*These authors contributed equally to this work

‡Author for correspondence ( jimena_giudice@med.unc.edu)

J.G., 0000-0002-3330-7784

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endophilin and BAR-domain proteins drive vesicle scission fromthe membrane; (5) vesicles are uncoated to fuse with targetendosomes; (6) cargo is transported through early endosomes intoeither recycling vesicles, or late endosomes and lysosomes fordegradation (McMahon and Boucrot, 2011). Several of the CMEproteins undergo alternative splicing (highlighted in red in Fig. 2)and some are discussed below.

Clathrin chainsNon-assembled clathrin comprises a three-legged structurecalled a triskelion (Unanue et al., 1981), which is formed bythree heavy chain polypeptides bound to light chain subunits

arranged along the triskelion leg (Brodsky, 2012). Humans havetwo genes encoding clathrin heavy chain, CLTC (also known asCHC or CHC17) and CLTCL1 (also known as CHC22) (seeTable S1, for a list of official and alternative gene and proteinnames) (Kedra et al., 1996; Vassilopoulos et al., 2009). CLTCpredominates, except in skeletal muscle where the two proteinsare equally expressed.

By self-assembly, the triskelia form a polyhedral lattice that coatsthe transport vesicles (Brodsky et al., 2001; Ungewickell andHinrichsen, 2007). While CLTC constitutes the backbone of thevesicle lattice, the light chains regulate clathrin recruitment in thecell (Majeed et al., 2014). In humans and mice, the CLTC genecontains 33 exons, of which exon 31 is postnatally regulated byalternative splicing in cardiomyocytes and skeletal muscles, with itbeing skipped in neonates and included in adults (Brinegar et al.,2017; Giudice et al., 2014, 2016). CLTC regulates the formation andmaintenance of myofiber architecture (Vassilopoulos et al., 2014);thus, the tissue-specificity of CLTC splicing suggests that it mightbe important for muscle structure. Alternative splicing is not limitedto CLTC, but also regulates the clathrin light chain genes CLTA andCLTB (also known as LCA/CLCa and LCB/CLCb, respectively). Incomparison with the ubiquitous isoforms, brain splice variants ofCLTA contain 18 or 30 additional residues, and a further differentbut homologous 18 residues were found in CLTB (Jackson et al.,1987; Kirchhausen et al., 1987). Alternative splicing regulatesCLTA exon 5, which encodes the 18 residues and CLTA exon 6,which encodes the 12 residues, as well as CLTB exon 5, whichencodes 18 residues. In mouse hearts, Clta exon 6 is skipped inneonates and included in adults, whereas Clta exon 5 and Cltb exon6 are skipped at all developmental stages (Giudice et al., 2014).Although CLTA and CLTB are regulated by alternative splicing in atissue- and developmental-stage-specific manner, we still lack acomplete description of the tissue expression patterns of transcriptand protein isoforms and their functions.

Dynamin and DAB2In addition to the clathrin triskelion, other CME proteins are alsoalternatively spliced, including the adaptor protein disabled-2(DAB2), which controls endocytosis of the low-densitylipoprotein receptor family (Keyel et al., 2006; Maurer andCooper, 2006). DAB2 has two tissue-specific splice isoformsknown as P96 and P67 (Xu et al., 1995). P96 contains two clathrin-binding sites and one AP2-binding site, which are absent in P67,explaining why P96 binds to clathrin and AP2 and localizes atclathrin-coated pits, whereas P67 does not (Buljan et al., 2012;Morris and Cooper, 2001). DAB2 depletion results in alterations inthe surface levels of integrins that give rise to migration and

Box 1. Alternative splicing of trafficking proteins is tissuespecific and affects short disordered motifsBrain, heart and skeletal muscle, as well as the testes, are the organswhere most of the tissue-specific and conserved alternative splicingtakes place (Merkin et al., 2012). Tissue-specific alternative exonsencode regions of proteins that tend to be centrally located within protein-protein interaction networks (Buljan et al., 2012; Ellis et al., 2012) andcomprise short intrinsically disordered motifs that confer functionaldiversity onto splice variants (Weatheritt and Gibson, 2012; Weatherittet al., 2012). In general, the pairs of variants tend to behave like distinctproteins in terms of their interaction with other proteins, and theinteraction partners that are specific to each splice isoform tend to behighly tissue specific (Yang et al., 2016). Tissue-specific alternativeexons are present in genes encoding proteins that control microtubule-based transport, endocytosis, membrane deformation and endosomeformation (Buljan et al., 2012; Ellis et al., 2012). Table S2 summarizesseveral of these tissue-specific alternative exons in membrane-trafficking genes that are predicted to impact protein-proteininteractions and below we highlight two examples that have beenexperimentally demonstrated.

(1) Growth factor receptor bound protein-2 (GRB2) is involved ininternalization of receptor tyrosine kinases. GRB2 homodimerizesthrough an interaction between its SH2 and SH3 domains (Maignanet al., 1995), which is important for signaling (McDonald et al., 2008).Exon 4 of GRB2 is alternatively spliced and encodes a region thatoverlaps the SH2 domain. When exon 4 is skipped, the self-interaction ofGRB2 is lost, suggesting that alternative splicing controls GRB2homodimerization and thus signaling activity (Ellis et al., 2012).

(2) Kinesin-1 is a molecular motor protein associated withmicrotubules; it is formed by two heavy chains that control motoractivity and two light chains (KLC1 and KLC2) involved in cargo binding.KLC1 contains five alternative exons (13-17). Inclusion of exon 15reduces the interaction of KLC1 with Marlin1, which controls transport ofthe GABA-B receptor towards dendrites (Vidal et al., 2007). Therefore,regulation of exon 15 splicing might control distribution of the GABA-Breceptor, and thus neurotransmission (Ellis et al., 2012).

Proximal polyA Distal polyA

A Cassette exon C Intron retention

D Alternative 3� splice site F Alternative polyA selection

B Mutually exclusive exons

E Alternative 5� splice site

Fig. 1. Types of alternative splicing events. The typesof splicing events are defined by the location of thealternative splice sites. (A) Cassette exons can be eitherincluded or spliced out. (B) Mutually exclusive exons areconsecutive exons that are included in a mutuallyexclusive manner, i.e. when one of them is included theother one is skipped and vice versa. (C) An intron or aportion of an intronic region can be included in the mRNA.(D,E) When more than one splice site exists at the end orbeginning of an exon, alternative 5′ or 3′ splice sites canbe selected. (F) The polyA tail at the 3′ end of an RNA canbe added in some genes at different poly-adenylation sites(polyA), leading to the production of more than onetranscript. Constitutive exons are shown in gray boxes,introns as lines and alternative regions as colored boxes.

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polarization defects, which can be rescued by re-expression of P96,but not P67 (Teckchandani et al., 2009).In mammals, there are three dynamin genes (DNM1, DNM2 and

DNM3), which encode members of a family of GTPases involved inmembrane fission and the release of clathrin-coated vesicles from theplasmamembrane. The three genes are highly regulated by alternativesplicing, giving rise to more than 25 splice isoforms. The neuron-specific DNM1 is essential for synaptic vesicle endocytosis and isalternatively spliced at two regions: the middle domain and theC-terminus. Splicing results in either DNM1ax or DNM1bxisoforms, where ‘a’ and ‘b’ are the middle domain variants and ‘x’is one of the three alternative terminal exons. In mice, a spontaneousmissense mutation (fitful) (p.A408T) that only affects the DNM1axisoforms (the ‘a’ exon is spliced out in the DNM1bx variants) altersendocytosis, DNM1 self-assembly, which is required for dynaminfunction and, ultimately, synaptic transmission (Boumil et al., 2010).DNM2 is ubiquitously expressed and has several splice variants.

In particular, two alternative regions generate four splice variants,DNM2aa, DNM2ab, DNM2ba and DNM2bb (Cao et al., 1998;Cook et al., 1994; Sontag et al., 1994). All isoforms localize toclathrin-coated pits at the plasma membrane but only DNM2ba andDNM2bb localize to the Golgi. All isoforms rescue the endocytosisdefects in Dyn2-depleted cells, but DNM2ba and DNM2bb weremore effective than the DNM2aa and DNM2bb variants with regardto rescuing the export of the neutrophin receptor p75 from the trans-Golgi network (Liu et al., 2008).In summary, the regulation of CME factors by alternative splicing

has been demonstrated to be tissue- and developmental-stage-specific. However, further studies are required to better describe the

molecular details of splicing events and their physiologicalimplications.

Alternative splicing of SNARE proteinsThe SNARE machinery controls the interaction between vesicularv-SNAREs (VAMPs) and target t-SNAREs (such as SNAPs andsyntaxins) during vesicle fusion (Fig. 3A). Several components ofthe SNARE machinery undergo alternative splicing, as discussedbelow.

SNAPsSynaptosome-associated protein-25 (SNAP25) bridges synapticvesicles to the plasma membrane during exocytosis. In highervertebrates, two mutually exclusive exons, 5a and 5b, give riseto the SNAP25-a and SNAP25-b isoforms (Bark and Wilson,1994) (Fig. 3B). These variants differ by nine centrally locatedresidues, two of which alter the relative positioning of clusteredcysteines whose palmitoylation controls membrane anchoring(Vogel and Roche, 1999). Snap25 null mice exhibit defects inprimed vesicle pools and fast calcium-triggered release inchromaffin cells. Here, re-expression of SNAP25-b results inlarger primed vesicle pools compared with those seen upon re-expression of SNAP25-a, suggesting that alternative splicingregulates the ability of SNAP25 to stabilize primed vesicles(Sørensen et al., 2003). A developmental switch from the fetalSNAP25-a isoform to adult SNAP25-b occurs in brain (Barket al., 1995), and its impairment induces lethality in mice threeto five weeks after birth (Bark et al., 2004). The exclusiveexpression of the SNAP25-a isoform through replacement of

Lysosome

6. Endosomal processing

Recyclingendosome

5. Uncoating

2. Cargo selection

Nucleus

3. Coat assembly 4. Vesicle scission

1. NucleationEPS15

ITSN1/2FCHO1/2

DAB2EPS15R

CLTCCLTA, CLTB

EPN1/ 2AP2

HIP1RSNAP91

CLTCCLTA, CLTB

NUMBAAK

BAR proteinsDynamin

CLTCCLTA, CLTBEndophilin

BAR proteinsDynamin

SNX9Cortactin

CLTCCLTA, CLTBSynaptojanin

HSC70DNAJC6

GAKActin

Fig. 2. Regulatory mechanisms for alternative splicing impact numerous components of the CME. CME occurs in multiple steps: (1) clathrin-coatedvesicles are formed; (2) proteins are recruited to the nucleation site by the AP2 complex, which together with other cargo-specific adaptors mediates cargoselection; (3) the clathrin triskelion facilitates vesicle assembly and clathrin polymerization stabilizes the curvature of the forming vesicle; (4) dynamin, endophilinand BAR-domain proteins drive vesicle scission from the membrane; (5) vesicles are uncoated to fuse with target endosomes; (6) cargo is transported throughearly-endosomes into either recycling vesicles, or late endosomes and lysosomes for degradation. Alternative splicing regulates several of the proteins thatcontrol 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, epidermalgrowth 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, endocyticadapter protein; SNAP91, synaptosome-associated protein-91; SNX9, sorting nexin 9.

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exon 5b with a copy of exon 5a leads to developmental defects,spontaneous seizures, impaired short-term synaptic plasticity,morphological alterations in adult hippocampus, changes inneuropeptide expression and impairment of spatial learning(Johansson et al., 2008). Furthermore, when provided with a high-fat diet, these mice develop a metabolic syndrome accompanied byweight gain, thus linking the neuronal defect in the exocytosismachinery with dyslipidemia and disrupted glucose homeostasis

(Valladolid-Acebes et al., 2015). A lack of the SNAP25-b isoformin endocrine β-cells increases insulin secretion and alters calciumdynamics (Daraio et al., 2017). Recently, hippocampal lysates haverevealed that SNAP25-a is less efficient than SNAP25-b in formingcomplexes with Munc18-1 and the Gβ1 and Gβ2 subunits ofheterotrimeric G-proteins. As these interactions play important rolesin presynaptic inhibition, the results suggest a less inhibitory role ofSNAP25-a (Daraio et al., 2018).

C

D

BMajor functional isoform

Larger primed vesicle poolsand increased secretion

VAMP7 gene

SNAP25 gene1 2 3 4 5a 5b 6 7 8 9

AUG stop

SNAP25-a

SNAP25-b

VAMP7-a

VAMP7-b

VAMP7-c

AA Cargorelease

Trafficking

Tetheringand docking

Primingand fusion

v-SNAREs t-SNAREsSNAPs

SyntaxinsVAMPs

Golgi

Nucleus

SNAP25 splicing affectsstrength of exocytic bursts

Syntaxins

Secretory vesicle

Plasma membrane SNAPs

CELF syntaxin splicingnetwork affects axon repair

STX3 splicing affects protein domains

VAMP7 splicing affects protein structure and localization

VAMPsCargo

N CLD

N CTMSM

AUG stop

STX3-A 3ab 5 6 7 8 9a10a 11a

Major isoform in non-neuronal tissue,involved in epithelial cell membrane trafficking

AUG

STX3-B

stop

Main isoform in the retina where it is required for vesicle exocytosis

11b

AUG stop

STX3-D 1 2 43ab 3c Truncated protein

AUG

STX3-C

stop

No SNARE or transmembrane domains

No interaction with AP3, no targeting to late endosomes

VAMP7-i

VAMP7-j

VAMP7-d

VAMP7-h

Truncated protein

N CTMSM Truncated protein

N CTMSM

N CLD TM

N CLD Premature stop codon, only nuclear isoform

N CLD TMSM Main isoform in most tissues

+ exon 5b

+ exon 5a 5a

5b

1 2 4

3ab 5 6 7 8 9b10b 11b1 2 4

3c 5 6 7 8 9b10b 11b1 2 4

3 5 6 7 81 2 4

8

stop

3 5 6 7 81 2 4

stop

3 5 7 81 2 4

8

stop

5 6 7 81 2 4stop

5 6 7 81 2 4stop

3 5 6 7 81 2 4

8

stop

3 6 7 81 2 4

stop

3 7 81 2 4

Fig. 3. Alternative splicing of SNARE-related proteins. (A) Steps of vesicle-mediated exocytosis that require the assembly of the SNARE complex. TheSNARE machinery controls the interaction between vesicular v-SNAREs and target t-SNAREs during vesicle fusion. (B-D) Multiple components of the SNAREmachinery undergo alternative splicing, including synaptosome associated protein-25 (SNAP25) (B), syntaxin-3 (STX3) (C) and vesicle associated membraneprotein-7 (VAMP7) (D). CELF, CUGBP Elav-like family member-1; LD, Longin domain; SM, SNARE motif; TM, transmembrane domain.

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SNAP23 is a SNAP25 homologue that is almost ubiquitouslyexpressed and has been implicated in insulin sensitivity (Boströmet al., 2007), lipid-raft actions (Puri and Roche, 2006; Yoon et al.,2016) and endothelial exocytosis of the von Willebrand factor (Zhuet al., 2015). The human SNAP23 gene contains eight exons anddifferent splice isoforms have been reported; for instance, SNAP23-a differs from SNAP23-b in 53 residues encoded by exon 6(Mollinedo and Lazo, 1997). This region contains sites for post-translational fatty acid acylation, suggesting differences inmembrane interaction between the isoforms. In vitro, bothisoforms bind to syntaxin-6 (STX6), but SNAP23-b has anapparently higher affinity (Lazo et al., 2001). In addition, theSNAP23-c variant lacks exons 5 to 7, giving rise to a truncated 50-residue protein. Another isoform, SNAP23-d, lacks exons 6 and 7,and, here, a frame-shift changes the C-terminus of the protein. TheSNAP23-e isoform also lacks exons 6 and 7, but it uses analternative 5′-splice site within exon 8 and shares the same last eightresidues as SNAP23-a and SNAP23-b (Shukla et al., 2001). Allisoforms, except SNAP23-c, contain the cysteine-rich domain thatis palmitoylated. Transfected SNAP23-a and SNAP23-b mainlylocalize to the plasma membrane, while the other isoforms alsoexhibit intracellular localizations (Shukla et al., 2001).

SyntaxinsIn the brain, alternative splicing of syntaxins is regulated by theCUGBP Elav-like family (CELF) of RBPs. In Caenorhabditiselegans, the CELF homolog UNC-75 promotes the expression ofthe neuronal syntaxin isoform UNC-64A, which includes exon 8a,and represses the non-neuronal variant UNC-64B, which includesexon 8b. These isoforms differ in their C-terminal hydrophobicmembrane anchors (Ogawa et al., 1998; Saifee et al., 1998). Themutants lacking unc-75 only express UNC-64B and show axonregeneration and locomotion defects similar to those observed inunc-64 loss-of-function mutants. While overexpression of eitherUNC-64A or UNC-64B isoforms rescues axon regenerationdefects, only UNC-64A rescues locomotion defects (Chen et al.,2016; Norris et al., 2014). CELF proteins also control axonregeneration in rodents. The Celf2-knockout mouse exhibits axonregeneration defects and mis-splicing of several syntaxins (Stx1a,Stx2, Stx16 and Stx18) and syntaxin-binding proteins (Stxbp1,Stxbp2 and Stxbp5), suggesting that CELF-regulated splicing ofsyntaxins is an evolutionarily conserved mechanism involved inaxon regeneration (Chen et al., 2016; Norris et al., 2014).STX3 is highly expressed in spleen, lung, kidney, retina and

brain, and functions in vesicle trafficking. The mouse Stx3 genecontains several mutually exclusive exons: 3ab and 3c, 9a and 9b,10a and 10b, and 11a and 11b, generating four splice variants(shown in Fig. 3C) that differ in their domain organization andbiochemical properties (Ibaraki et al., 1995). STX3-A and STX3-Bshare the N-terminus but differ in the second half of the SNAREdomain and the C-terminal transmembrane domain. In STX3-D, theinclusion of both exons 3ab and 3c introduces a premature stop-codon and thus produces a truncated protein (Curtis et al., 2008).STX3-A is the only isoform detected in the kidney with proposedfunctions in epithelial cell membrane trafficking, whereas STX3-Bis the exclusive variant found in retinal ribbon synapses (Curtiset al., 2008) where it is required for synaptic vesicle exocytosis(Curtis et al., 2010). Recently, a translatome study in mouse retinalcells revealed that axons and soma use different last exons in theStx3 gene, leading to differences in the C-terminus of the encodedSTX3 proteins and the 3′UTR. Notably, the axon-specific exon issufficient to promote axonal mRNA translation, providing evidence

for a potential mechanism underlying the selective and dynamicmRNA translation in axons (Shigeoka et al., 2016).

VAMPsHuman VAMP7 ( previously known as SYBL1) encodes thev-SNARE protein VAMP7, which controls intracellulartrafficking (Galli et al., 1998). In the full-length VAMP7-aisoform, exons 2 to 4 encode the N-terminal Longin domain,which negatively regulates membrane fusion and neurite outgrowth(Martinez-Arca et al., 2000, 2001), whereas exons 5 to 8 encode thetransmembrane region and the SNAREmotif (Fig. 3D). Skipping ofexon 3 and the use of an alternative 5′-splice site in exon 2 lead tothe truncated proteins VAMP7-d and VAMP7-h, respectively(Vacca et al., 2011). VAMP7-c uses the same 5′-splice site inexon 2 as VAMP7-h, but lacks exon 3. Thus, in comparison withVAMP7-a, VAMP7-c lacks 40 residues within the Longin domain,losing the interaction with the adaptor protein AP3 and its targetingto late endosomes (Martinez-Arca et al., 2003). Splicing of exons 5and 6 generates three additional isoforms with intact Longindomains, but altered SNARE domains. VAMP7-b lacks exon 6, andthus has a different C-terminus than VAMP7-a. In VAMP7-i,skipping of exon 5 introduces a premature stop-codon, and thus onlythe Longin domain is expressed. In VAMP7-j, skipping of bothexons maintains the original reading frame; therefore, in addition tothe Longin domain, VAMP7-j contains a short hinge region, and theoriginal transmembrane and intra-vesicular tail regions (Vacca et al.,2011) (Fig. 3D). The different VAMP7 isoforms exhibit differentialsubcellular and tissue localizations. VAMP7-b is widely distributed,whereas VAMP7-i is the only nuclear isoform. Furthermore,VAMP7-a is the main isoform in most tissues, whereas othervariants show some tissue specificity (Vacca et al., 2011).

Based on all the studies discussed above, it is clear that alternativesplicing regulates different members of the SNARE machinery andso contributes to the control of exocytosis.

Alternative splicing impacts membrane dynamicsCellular membranes are flexible and extensively remodeled duringnumerous processes, including cell movement, cell division,endocytosis, muscle contraction and repair, transverse tubule(T-tubule) and sarcomere formation, mitochondria fusion andfission, as well as axon and dendrite growth. During membraneremodeling, reversible curvature changes and areas of high tensionexist for limited periods of time and are controlled by traffickingproteins, such as dynamin, amphiphysin, endophilin and epsin(McMahon and Gallop, 2005). BAR-domain proteins arecytoplasmic molecules with membrane-bending properties (Mimand Unger, 2012). Below, we describe two BAR-domain proteinsthat are regulated by alternative splicing: CDC42-interactingprotein-4 (TRIP10, also known as CIP4) and bridging integratorprotein-1 (BIN1, also known as amphiphysin-2).

TRIP10TRIP10 controls centrosome and Golgi polarization in migratorycells (Tonucci et al., 2015), E-cadherin trafficking during epithelialmorphogenesis (Zobel et al., 2015), cell growth and invasion incancer metastasis (Chander et al., 2012; Rolland et al., 2014;Truesdell et al., 2014), hypertrophy in neonatal cardiomyocytes(Rusconi et al., 2013), and GLUT4 trafficking in adipocytes (Changet al., 2002). The N-terminus of TRIP10 contains the FCH domainand two coiled-coil domains, and mediates the interaction withAKAP350, tubulin and phospholipids. Its internal GTP-bindinghomology region mediates binding to the GTPases TC10 (Chang

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et al., 2002) and CDC42 (Aspenström, 1997) (Fig. 4A, top). TheSrc-homology-3 (SH3) domain controls the interaction of TRIP10with the Wiskott-Aldrich syndrome protein (WASP) and DNM2,thus connecting actin polymerization with membrane deformationto promote GLUT4 trafficking (Hartig et al., 2009) and initiationand scission of endocytic vesicles (Feng et al., 2010). Exon 10 of theTRIP10 gene is alternatively spliced generating a 545-residueisoform (CIP4a) when it is skipped and a 601-residue variant(known as CIP4h or CIP4/2) when it is included (Fig. 4A). Theisoform lacking exon 10 is the most ubiquitously expressed. TRIP10variant containing exon 10 was identified in a two-hybrid screeningas a TC10-interacting protein (Chang et al., 2002;Wang et al., 2002)and found to be expressed in adult skeletal muscles and hearts (Fenget al., 2010; Giudice et al., 2014, 2016). CIP4b (also known as Felic)is a TRIP10 isoform that is exclusively expressed in human prostatecancer and lymphoblast cells and is generated by a 29-nucleotideinsertion of intron 13 that destroys the SH3 domain (Wang et al.,2002). Furthermore, aberrant splicing in renal carcinoma generatesthe CIP4-V variant by a 19-nucleotide retention of intron 9, whichintroduces a premature stop codon (Tsuji et al., 2006) (Fig. 4A).CIP4-V lacks the CDC42-binding region and the SH3 domain, andits overexpression induces the formation of ubiquitylatedaggresomes and defects in β-catenin trafficking that ultimatelyreduce cell adhesion and contribute to cancer progression andmetastasis (Tsuji et al., 2006).

BIN1 in brain and cancerBIN1 is a ubiquitous BAR-domain protein that is highly expressedin striated muscles and brain (Butler et al., 1997; Sakamuro et al.,1996). BIN1 regulates a number of cellular processes, includingendocytosis, cytoskeleton organization, DNA repair, the cell cycle,tumor suppression, membrane invagination and T-tubuleorganization (Prokic et al., 2014). BIN1 has multiple domains(Fig. 4B): (1) the BAR domain (exons 1-10), which binds tomembrane lipids and senses membrane curvature; (2) a polybasicsegment encoded by the muscle-specific exon 11 that mediates itsaffinity to phosphoinositides and T-tubules (Kojima et al., 2004;Lee et al., 2002); (3) a brain-specific clathrin- and AP2-bindingdomain (CLAP) (exons 13-16) (Ramjaun and McPherson, 1998);(4) a MYC-binding domain (MBD) (exons 17-18) that conferstumor-suppressor functions; and (5) the SH3-domain (exons 19-20), which mediates binding to proline-rich motifs. BIN1 containsseven alternatively spliced exons and mis-splicing occurs in diseasecontexts. In the brain, exon 7 and exons 13-17 are included (Prokicet al., 2014); here, inclusion of exon 7 promotes transferrinendocytosis and interaction between BIN1 with DNM2 (Ellis et al.,2012) (Fig. 4C). In melanoma and breast cancer, the aberrantinclusion of exon 13 disrupts the tumor-suppressor functions ofBIN1 (Ge et al., 1999), and exogenous expression of a BIN1 variantlacking exon 13 restores its binding with MYC and thus its tumor-suppressor functions (Anczuków et al., 2012).

BIN1 in striated musclesVesicle-mediated transport, membrane remodeling and cytoskeletalgenes are globally regulated by alternative splicing in mouse striatedmuscles during the first four postnatal weeks (Brinegar et al., 2017;Giudice et al., 2014). This is the period of maturation of thesarcoplasmic reticulum and T-tubules that are necessary forexcitation-contraction coupling and adult contractility. In striatedmuscle cells, T-tubules facilitate the access of environmental signalsand the propagation of membrane depolarization deep into cellsduring excitation-contraction coupling. Growing evidence

demonstrates that trafficking proteins have structural roles instriated muscles. For example, CLTC regulates the formation andmaintenance of the costameres (Vassilopoulos et al., 2014), whichare the lateral attachment sites between the sarcolemma and thesarcomere. Another example from a recent study in Drosophilamelanogaster revealed that autophagy is required for T-tubuleremodeling through a mechanism that implicates the GTPase RAB2(Fujita et al., 2017). Overall, these and other studies have revealedunconventional but crucial roles of trafficking proteins in musclecell architecture.

BIN1 induces membrane tubulation in muscle cells, andmisregulation of its alternative splicing contributes to T-tubulealterations seen in muscular dystrophies and cardiac diseases (Böhmet al., 2013; Fugier et al., 2011; Hong et al., 2014). In patients withmyotonic dystrophy (DM), exon 11 is skipped, contributing tomuscle weakness and T-tubule defects (Fugier et al., 2011).Reintroduction of the BIN1 isoform that contains exon 11 restoresT-tubule organization in myofibers isolated from DM patients,whereas, by contrast, the variant lacking exon 11 is unable torecover the phenotype (Fugier et al., 2011). Further evidence for theimportance of exon 11 comes from the human BIN1 mutation(IVS10-1G>A) that affects the acceptor splice site in aconsanguineous family with rapidly progressive and fatalcentronuclear myopathy (Böhm et al., 2013). Interestingly, thesame splice site is mutated (IVS10-2A>G) in a spontaneous dogmodel of the disease. Humans and dogs carrying these mutationslack exon 11 and exhibit a similar histopathology withultrastructural and membrane-triad defects (Böhm et al., 2013). Inthe heart, exons 7 and 11 are skipped, and exons 13 and 17 aredevelopmentally regulated, contributing to T-tubule organization(Hong et al., 2014). In cardiomyocytes, T-tubules contain denseprotective inner membrane folds, which are formed by the BIN1isoform containing exons 13 and 17 (Hong et al., 2014).Accordingly, in Bin1-depleted mice, T-tubule folding isdecreased, leading to free diffusion of local calcium andpotassium ions, which prolongs the action potential and increasessusceptibility to ventricular arrhythmias. These T-tubule defects canbe rescued by expression of the BIN1 isoform that contains exons 13and 17, which facilitates the folding of the T-tubule membrane(Hong et al., 2014) and so creates a local region of ion accumulationthat restricts ion flux.

Therefore, the fact that T-tubule maturation and alternativesplicing of membrane-trafficking genes follow similardevelopmental time-courses in striated muscles, suggests thatsplicing contributes to T-tubule biogenesis. In support of thisnotion, developmental splicing networks coordinately revert toneonatal patterns in disease contexts, such as heart failure,cardiomyopathies, congenital muscular dystrophy and DM, asdiscussed below.

Role of RBPs in alternative splicing of membrane-traffickingproteins in health and diseaseIn this section, we discuss how alternative splicing of traffickingproteins is regulated by RBPs and misregulated in neurological andmuscle disease (Fig. 5).

CELF and MBNL splicing networks in DM1The muscleblind-like (MBNL) and CELF family of RBPsantagonistically regulate alternative splicing in striated muscles(Wang et al., 2015) and are associated with DM, which is aninherited neuromuscular disease and the most common adultmuscular dystrophy. DM patients suffer from skeletal myopathy,

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A

B

BAR CLAP MBD SH3N

C1 2 3 5 6 7 84 9 1011 12 13 14 15 16 17 18 19 20

TRIP10 gene1 2 3 5 6 7 84 9 10 11 12 13 14 15

AKAP350Tubulin

Phospholipids

TC10CDC42

WASPDNM2

GAPEX5

9 10

9 10

Premature stop codon

CIP4-V(341 aa)

Ubiquitylated aggresomesCell adhesion

Defects in -catenin trafficking

13 14

13 14

29 nt19 nt

CIP4b(456 aa)

Premature stop codon

TRIP10(601 aa)

Human prostate cancer cellsHuman lymphoblast cells

TRIP10(545 aa)

– Exon 10

+ Exon 10

CNCN

N

N

C

C

SH3FCH Coiled-coil

SH3FCH Coiled-coil

Binding partners

PI CLAP MPI

CLIP1BIN1, MTM1

AMPH1, DNM2 Curvature sensor

T-tubulesPIPs

CLTCAP1AP2

Interactions

MYCMYCN

DNM1/2RIN2/3, MTM1

SH3GLB1SH3GL2

SNX4, BIN1

Brain

Skeletal muscle

Heart

Cancer cells

Ubiquitous

+ – + + +

+ – + + –

– + – – +

– + – – –

– – – –

– – + – +

– – + – +

– – – – + – – – – –

7 11 13 14-16 17

Alternative exons

Adult

Fetal

MYC

C

+ Exon 7 – Exon 7

DNM2

DNM2

Transferrin

Transferrinreceptor

C

Transferrinendocytosis

Transferrinendocytosis

Binding BIN1-DNM2 No binding BIN1-DNM2

+

BIN1 gene

Fig. 4. Alternative splicing of the BAR-domain proteins TRIP10 and BIN1. (A) The TRIP10 (CDC42-interacting protein-4) gene is regulated by alternativesplicing. 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 intron9 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 alsohighly regulated by alternative splicing in a tissue- and developmental-stage-specific manner. BIN1 domain structure is shown on the left with the diverse spliceisoforms 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, asillustrated here for themodel cargo transferrin. AKAP350, A-kinase anchoring protein-9; AMPH1, amphiphysin; AP1/2, adaptor complex-1/2; CDC42, cell divisioncycle 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 Rabinteractor 2; SH3, Src-homology-3 domain; SH3GL2, SH3-domain containing GRB2-like or endophilin A1; SH3GLB1, SH3-domain containing GRB2 like orendophilin B1; SNX4, sorting nexin-4; TC10-Ras homolog family member-Q; T-tubules, transverse tubules; WASP, Wiskott-Aldrich syndrome protein family.

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cardiac arrhythmia, cataracts, hypogonadism, hyper-somnolenceand insulin resistance, among other symptoms (Harper, 2001). DM1is caused by expansion of CTG repeats in the 3′UTR region of theDMPK gene that leads to activation of CELF1 (gain of function) andsequestration of MBNL, causing its loss of function. Alterations inthe functions of CELF and MBNL result in mis-splicing of thetargets of these two RBPs, which include several trafficking genes(Fig. 5A) (Dixon et al., 2015; Freyermuth et al., 2016; Fugier et al.,2011). CELF and MBNL misregulation results in the global re-expression of the fetal splicing isoforms of several proteins that wediscuss below. This reprogramming in adult tissues takes placemainly in the heart and skeletal muscle. The fetal isoforms arefunctional in embryonic and neonatal muscles; however, adultmuscle cells are large and require a precise internal architecture forproper excitation-contraction coupling. In adults, skipping of exon7a of the muscle chloride channel CLCN1 generates the functionalfull-length protein but loss of function of MBNL in DM1 leads tothe inclusion of exon 7a, which generates a frameshift and thus non-functional CLCN1 (Charlet-Berguerand et al., 2002;Mankodi et al.,2002). Chloride channels are dispensable in neonatal smallmyofibers with undeveloped T-tubules, but their absence in largeadult myofibers disrupts the potassium counterbalance within T-tubules, resulting in depolarization and myotonia, as seen in DM1patients (Charlet-Berguerand et al., 2002; Mankodi et al., 2002).Furthermore, the alterations in MBNL and CELF function result inthe mis-splicing of the ryanodine receptor-1 (RYR1) (exon 70), theATPase sarcoplasmic/endoplasmic reticulum calcium transporting-1 (ATP2A1) (exon 22), the calcium voltage-gated channel subunit-alpha1S (CACNA1S) (exon 29) and BIN1 (exon 11), which isthought to partially contribute to muscle weakness associated withDM1 (Fugier et al., 2011; Kimura et al., 2005). In particular,aberrant skipping of exon 29 of the channel CACN1S correlateswith the severity of muscle weakness in DM patients (Tang et al.,2012). Finally, insulin insensitivity is associated with mis-splicingof exon 11 of the insulin receptor (INSR), which is due to thedysfunction of MBNL and CELF proteins (Dansithong et al., 2005;Echeverria and Cooper, 2014; Savkur et al., 2001, 2004).Heart samples from DM1 adult patients exhibit global splicing

changes in genes encoding proteins that control ion handling, cellarchitecture and vesicle-mediated transport (Freyermuth et al., 2016).One of these genes is the sodium channel protein type-5 subunit-alpha(SCN5A), which is critical for cardiomyocyte excitability and impulsepropagation through the conduction system. The SCN5A isoformcontaining exon 6a is predominant in human fetal hearts, exons 6aand 6b are used to similar extents in infants, and the isoformcontaining exon 6b is preferred in adulthood. The two isoforms differin seven residues in one of the voltage-sensing domains, and thus intheir electrophysiological properties. DM1 patients express the fetalisoform of SCN5A and exhibit severe cardiac conduction defects andarrhythmias that are reproduced when this isoform is re-expressed inadult murine hearts (Freyermuth et al., 2016; Onkal et al., 2008).

ESRPs and RBFOX proteinsThe epithelial splicing regulatory proteins ESRP1 and ESRP2,along with the RNA-binding fox-1 homolog-2 protein (RBFOX2)and the neuro-oncological ventral antigen-1 (NOVA1) regulatesplicing during epithelial-mesenchymal transition (EMT) indevelopment and in metastatic cancer (Lu et al., 2015). ESRP2 isa driver of liver development, where it regulates splicing of theretromer complex component VPS29, synaptojanin-2 (SYNJ2) andSTX2, among other trafficking genes (Bhate et al., 2015). In theembryonic epidermis in humans, mice and fish, ESRP1 and ESRP2

regulate an evolutionarily conserved splicing-trafficking networkthat includes the sortin nexin-9 (SNX9), STX2 and VPS29(Burguera et al., 2017) (Fig. 5B). RBFOX2 is induced in EMTand regulates splicing of the endocytic proteins cortactin andDNM2. Depletion of RBFOX2 after EMT significantly reduces cellinvasive potential, suggesting that RBFOX2-regulated splicingcontrols tissue invasiveness (Braeutigam et al., 2014).

NOVA proteinsIn the pancreas, NOVA1 has been implicated in regulating splicing ofa number of proteins, including the INSR, exocytosis proteins such asSNAP25, the calcium-dependent secretion activator (CADPS), aswell as Rho-GTPases, such as cell division cycle-42 (CDC42) and theRhoGEF protein ARHGEF12 (Villate et al., 2014) (Fig. 5B).However, the functional implications of this splicing network that isregulated by NOVA1 in the β-cells were not investigated.

In the brain, NOVA1 regulates neuron-specific alternative splicingand is essential for motor neuron survival after birth (Jensen et al.,2000). Furthermore, the Nova2-knockout mouse exhibits majorsplicing defects in genes that encode proteins with well-definedfunctions at the synapse, including neurotransmitter receptors, cationchannels, adhesion and scaffold proteins, or in axon guidance (Uleet al., 2005). NOVA proteins regulate splicing of all members of thespectrin–ankyrin–Protein-4.1–CASK scaffold complex, which actsas a linker between membrane proteins and the actin-tropomyosincytoskeleton, and contributes to organization of the GABAergicsynapse (Jensen et al., 2000; Ule et al., 2003; Ule et al., 2005). Thefunctional implications of NOVA-regulated splicing have beenconvincingly demonstrated for the reelin adaptor protein DAB1,where NOVA2 controls neuronal migration by regulating the splicingof exons 7b and 7c of DAB1 (Yano et al., 2010).

PTBP coordinates splicing trafficking networks in spermatogenesisand ovarian cancerPolypyrimidine-tract binding proteins (PTBPs) regulate alternativesplicing in the brain (Vuong et al., 2016), during spermatogenesis(Hannigan et al., 2017; Zagore et al., 2015), inmyogenesis (Hall et al.,2013) and in ovarian cancer (He et al., 2015). PTBP2 is required formouse spermatogenesis and proper splicing regulation of a network ofgenes that encode proteins involved in nearly all steps of vesicle-mediated trafficking. This includes subunits of the AP1 and AP2complexes and DENND1A, which is a component of clathrin-coatedvesicles that binds to the AP2 complex, proteins involved in tetheringvesicles to microtubules and target membranes, and factors involvedin actin dynamics (Hannigan et al., 2017; Zagore et al., 2015)(Fig. 5C). PTBP2 depletion in germ cells results in a disorganizationof the F-actin cytoskeleton in Sertoli cells, indicating that regulation ofalternative splicing is necessary for cellular crosstalk during germ celldevelopment (Hannigan et al., 2017).

In ovarian cancer cells, PTBP1 depletion has been shown toinhibit the formation of filopodia and to alter CDC42 splicing(Fig. 5C). TheCdc42 gene encodes two protein isoforms, CDC42-v1and CDC42-v2, which differ in their terminal exons. While CDC42-v1 includes exon 6a, CDC42-v2 includes exon 6b. Because PTBP1represses exon 6b, it reduces expression of the CDC42-v2 variant,which has been shown to have inhibitory effects on ovarian cancercell growth and invasiveness, thus functioning as a tumor suppressor(He et al., 2015). More recently, it has been shown that perturbing theratio between the CDC42-v1 and CDC42-v2 variants in neuronsresults in alterations in axons and dendrites because CDC42-v1 isrequired for the development of dendritic spines, whereas CDC42-v2functions in axogenesis (Yap et al., 2016) (Fig. 5D).

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RBFOX1 and SRRM4 in autism spectrum diseasesAutism spectrum disorder (ASD) is a highly heritableneurodevelopmental condition which is characterized by geneticheterogeneity. High-throughput studies have revealed commonpatterns of misregulated gene expression and alternative splicingin ASD (Gupta et al., 2014; Irimia et al., 2014; Quesnel-Valliereset al., 2016; Voineagu et al., 2011). Brains from ASD patientsexhibit a downregulation of the RBP RBFOX1 (also known asA2BP1 and FOX1) and, accordingly, a dysregulation of the

RBFOX1-dependent alternative exons in genes that encodeproteins involved in cytoskeleton organization and vesicle-mediated transport such as CLTA, CLTB, STX16, STXBP1,AP2A1, AP3S1, among several others (Voineagu et al., 2011)(Fig. 5D).

Another RBP that is downregulated in ASD is the serine/argininerepetitive matrix-4 (SRRM4, also known as nSR100). SRRM4controls the inclusion of a large set of brain-specific alternativeexons that are significantly enriched in genes associated with

CELF gain of function

NOVA1CDC42, CADPS1, CADPS2, INSR,

SNAP25, ARHGEF12

ESRPSTX2, SYNJ2, VPS29,

SNX9

Channel dysfunction,myotonia

Arrhythmia

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

D Brain and neuronsC Reproductive organs

T-tubule defects,muscle weakness

NOVADAB1, CADPS2, CASK,

ankyrins, spectrins, EPB41

Mis-splicingSTX16, STXBP1, AP2A1, AP3S1,

CLTA, CLTB

Mis-splicingDNM2, CASK,BIN1,

AP1B1, AP1S2, STX2, CTTN, SYNJ1, PTBP2

RBFOX2CTTN, DNM2

PTBP1CDC42

PTBP1CDC42Development of

axons and dendrites

Neuronal activity

Invasiveness

Filopodia formation

CELFSyntaxins, syntaxin-binding proteins

Neuronalmigration

Ovarian cancer

DM1 Diabetes

SpermatogenesisASDSynaptogenesis

Cancer

Development

Metabolism

Development

Organization of actincytoskeleton

CTG repeatsDMPK gene

3�UTR

Mis-splicing BIN1, CLCN1, RYR1, CACNA1S, ATP2A1,

INSR, SCN5A

MBNL loss of function

Insulinresistance EMT

Functions in β-cells?

ASD

ASDASD

RBFOX1downregulated

SRRM4downregulated

PTBP2Splicing network involving:

AP complexSNAPs and SYNJs

Actin dynamics

Clathrin-coatedvesicles

Epidermis

Fig. 5. RBPs regulate alternative splicing of membrane-trafficking proteins in health and disease. (A) In myotonic dystrophy type-1 (DM1), CTG repeatexpansion in the 3′UTR region of theDMPK gene leads to the activation of CUGBP Elav-like family member-1 (CELF1) (gain of function) and the sequestration ofmuscleblind-like protein (MBNL), which causes its loss of function. Alterations in the functions of CELF and MBNL lead to mis-splicing of their targets (listedbelow). (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 anetwork of genes encoding subunits of the AP1 and AP2 complexes, members of the SNARE complex and synaptojanins, and proteins involved in actindynamics. In ovarian cancer cells, PTBP1 depletion inhibits the formation of filopodia and affects CDC42 (cell division cycle 42) splicing. (D) In the brain, NOVAproteins regulate neuron-specific alternative splicing events that are essential for motor neuron survival, synapse, and axon guidance. Brains of people withautism spectrum disorder (ASD) show downregulation of RBFOX1 and the serine/arginine repetitive matrix-4 (SRRM4), and thus dysregulation of the dependentalternative 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, calciumvoltage-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 band4.1; 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.

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membrane dynamics, endocytosis and cytoskeleton remodeling(Calarco et al., 2009). SRRM4 controls the neural specificity ofalternative splicing by activating the expression of the neuronalisoform of PTBP2 (nPTBP), which includes its exon 10. Therefore,nPTB and SRRM4 act in concert to control neuronal alternativesplicing (Calarco et al., 2009). Numerous neural microexons (3-15nucleotides) are frequently misregulated in ASD brains, and this hasbeen shown to be associated with a reduced expression of SRRM4(Irimia et al., 2014). Gene ontology analysis revealed that themicroexons that are misregulated in ASD are significantly enrichedfor those present in vesicle-mediated transport genes, among a fewother categories (Irimia et al., 2014). Furthermore, mutant micelacking one functional copy of the Srrm4 gene exhibit severalautistic-like features and recapitulate the misregulated splicingpatterns that are observed in brains of peoplewith ASD (Irimia et al.,2014; Quesnel-Vallieres et al., 2016) (Fig. 5D).Taken together, it has become clear that the regulation of

trafficking proteins by alternative splicing is not limited to onespecific RBP and in most cases, RNA processing results from theaction of multiple RBPs that function in concert.

PerspectivesThe physiological relevance of alternative splicing was firstinvestigated for individual genes; however, genome-wide studieshave exponentially increased the number of splicing isoforms, andhighlighted networks with unknown and unexplored functions(Baralle and Giudice, 2017). High-resolution imaging and state-of-the-art genetic tools are now available to study trafficking withinentire organisms and recent progress in the splicing and traffickingfields pinpoints the questions that remain unanswered at theintersection between these two scientific fields. First, thephysiological roles of numerous splicing events in traffickingproteins are still unknown within in vivo contexts. The secondchallenge arises from the fact that alternative splicing is regulated ormisregulated in a coordinated manner in development and disease.Therefore, we need to investigate how splicing trafficking networksmodulate intracellular functions, thereby contributing to tissueidentity, and ultimately organ maturation and function. Finally, webelieve that expanding our knowledge of the contribution of mis-splicing of membrane-trafficking genes in disease will open newpossibilities for therapeutic approaches.

AcknowledgementsWe thank Drs Thomas Cooper (Baylor College of Medicine) and Frances Brodsky(University College London) for their feedback on the initial draft.

Competing interestsThe authors declare no competing or financial interests.

FundingThe authors are funded by National Institutes of Health/NIGMS grant (R25-GM089569) to E.G.C., Junior Faculty Development Award, Pilot & FeasibilityResearch Grant (Nutrition and Obesity Research Center, P30DK056350), and start-up funds from The University of North Carolina at Chapel Hill (to J.G.) and in part bythe March of Dimes Foundation (5-FY18-36) to J.G. Deposited in PMC for releaseafter 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.216465.supplemental

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