© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 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 1 REVIEW Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 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