Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

Marcella Vacca,Andrea Carpi,Maria Strazzullo,Ornella Rossetto,Lara Albania,Maurizio D'Esposito,Floriana Della Ragione,Nicola De Franceschi,Francesco Filippini,Valeria Rossi

doi:10.1186/1471-2199-12-26

Abstract

BackgroundThe control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.ResultsAlternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level.ConclusionsPrevious evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.

Highlights

The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells
Preliminary experiments with primers pairing to the untranslated regions of the gene amplified multiple fragments, the longest of which corresponding to VAMP7a; we found no intron retention, suggesting alternative splicing (AS) of SYBL1 is based on exon skipping
AS at the SYBL1 locus results in a more sophisticated modulation of the domain architecture: two non-sensitive factor attachment protein receptors (SNAREs) isoforms consist of the Longin domain (LD) alone (VAMP7i) or “membrane-anchored” LD (VAMP7j), and two Δ-longin isoforms (VAMP7d/h, this work and VAMP7c, [16]) share synaptobrevin-like architecture with different N-terminal extensions

Summary

Introduction

The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble Nethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. Splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle (v-SNAREs) and the target membrane (t-SNARES) are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes [8]. The LD of VAMP7 regulates membrane fusion; it is crucial to neurite outgrowth, as overexpression of a “deregulated” fragment missing the LD (Δ-longin) increases neurite outgrowth whereas reverse effect (outgrowth inhibition) is obtained when expressing the LD alone [14,15]. VAMP7 has been found to interact with the positive regulator of neurite growth Varp, a guanine nucleotide exchange factor (GEF) of the small GTPase Rab and a binding partner of Rab 32 and Rab 38 [23,24]

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