Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms
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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 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.
Alternative 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.
Previous 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.
KeywordsAlternative Splice Subcellular Localization Splice Variant Domain Architecture Snare Motif
The human gene SYBL1 (synaptobrevin-like 1) expression is finely regulated at different layers. Even if located in the Xq/Yq pseudoautosomal region, SYBL1 is subject to inactivation  and its allelic expression is controlled by multiple epigenetic mechanisms . Moreover, SYBL1 expression is altered in human pathologies characterized by DNA methylation derangement , such as ICF syndrome [4, 5] and hyperhomocysteinemia . SYBL1 encodes the v-SNARE protein VAMP7, an important modulator of intracellular trafficking also known as T etanus neurotoxin I nsensitive VAMP (TI-VAMP) . 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 . Since the hydrophobic heptad register of their α-helical coiled-coil region (SNARE motif) is interrupted at the so-called "zero layer" by a conserved R or Q residue, they are often referred to as R- or Q-SNAREs, respectively . SNAREs can significantly vary in sequence length (e.g. from <100 amino acids of some synaptobrevins to >1100 residues among tomosyns) because of their modular domain architecture. In addition to SNARE motifs they can show further regions, such a carboxy (C)-terminal transmembrane region, motifs allowing post-translational addition of lipid anchors  and a variable or conserved amino (N)-terminal domain. The N-terminal Longin domain (LD) of longins  was found to play multiple regulatory roles (reviewed in ). Longins are the only R-SNAREs conserved in all Eukaryotes, whereas brevins are limited to bilateria and hence are absent from whole taxa (e.g., plants) . Longins group into three subfamilies, prototyped by Ykt6, Sec22b and VAMP7 . In yeast, the LD of Ykt6p can regulate membrane fusion by inhibiting Ykt6p participation to the fusion bundle, by competitive intramolecular binding to the SNARE motif . The LD of VAMP7 also regulates membrane fusion; furthermore, 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]. In addition to regulating membrane fusion, LDs serve as a dominant signal for subcellular targeting. For example, in animals LD targets VAMP7 to late endosomes by binding to the δ-subunit of the AP-3 complex . In plants, several VAMP7 proteins are targeted to their different subcellular localizations by their LDs . The LD targets Ykt6 to its subcellular localization, likely by masking other localization signals . In Sec22b, export from the endoplasmic reticulum is mediated by binding to Sec23/24, a process depending on a conformational epitope created by intramolecular LD-SNARE motif binding; such binding also results in preventing promiscuous, unspecific binding by sequestering the N-terminal half of the SNARE motif . Also VAMP7 adopts a closed conformation based on intramolecular LD-SNARE motif binding . Hrb, a clathrin adaptor and ArfGAP, binds the LD of VAMP7 by its unstructured SNARE-like motif, outcompeting the SNARE motif of VAMP7 for the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when this longin is incorporated into a cis-SNARE complex [21, 22]. VAMP7 has been found to also interact with the positive regulator of neurite growth Varp, a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21 and a binding partner of Rab 32 and Rab 38 [23, 24].
Like other highly regulated processes typical of eukaryotic cells, subcellular trafficking needs fine tuning of protein functions and structures. Modulation of domain architecture and creation or deletion of sequence motifs can in turn be achieved by alternative splicing (AS): this way, a relatively large proteome can be created starting from a limited number of genes . Indeed, AS has been shown to occur in several trafficking-related genes, including SNARE genes, resulting in the production of isoforms with different subcellular localization and trafficking properties. Developmentally regulated AS of SNAP-23 and SNAP-25 modulates interactions with accessory factors and subcellular localization [26, 27]; AS also regulates interaction properties in the large t-SNARE family of syntaxins, by varying their N-terminal and/or C-terminal regions . In R-SNAREs, AS can finely tune vesicle subcellular targeting by varying the extreme C-terminal region in splice variants of VAMP1 [29, 30], and tissue-specific AS of mammalian tomosyn is involved in the regulation of neuronal secretion [31, 32]. In most Eukaryotes, the involvement of VAMP7 in multiple trafficking events at different subcellular compartments is mediated by gene amplification and variation . In mammals, VAMP7 is encoded by a single gene and yet it regulates multiple subcellular pathways [33, 34]: hence, AS is a likely strategy to achieve such complexity in regulation. Thus, we have started investigation on longins (the most conserved R-SNAREs) by a thorough analysis for the occurrence of AS at the human gene SYBL1.
Results and discussion
Exon skipping at the SYBL1 locus results in modulating isoform domain architecture
Exon skipping at the 5' half of the gene results in synaptobrevin-like, "non-longin" isoforms
Skipping of SYBL1 exons 5 and/or 6 produces non-SNARE, LD protein isoforms
Quantitative expression and tissue specific distribution of VAMP7/TI-VAMP isoforms
Immunodetection of isoforms by domain-specific oligoclonal antibodies
VAMP7/TI-VAMP isoforms show different subcellular localizations
Fine tuning of gene functions is an important strategy by which higher organisms can achieve their complexity without needing a proportional increase in gene number. Indeed, AS at a single locus can result in the production of a (small to large) number of splice isoforms with varying domain architecture and function(s). It is noteworthy that increased complexity in trafficking routes mediated by VAMP7 is achieved in plants by increased gene number, whereas a corresponding increase in animals is not apparent. Rather, as observed for other SNAREs and trafficking proteins, the SYBL1 locus undergoes AS and this in turn results in a number of VAMP7 isoforms with intriguing modulation in domain architecture. Further work will be needed to characterize biochemical, structural and cellular properties of each isoform. However, these "natural mutants" of VAMP7 can be now used to shed more light on the function(s) of the LD and other regions/domains. To this aim, artificial constructs have been used so far: the subcellular localization of several chimeric VAMP7 proteins was compared in plants , whereas separated VAMP7 domains were used to investigate both subcellular localization and effects on mammalian cell physiology. In particular, a synaptobrevin-like Δ-longin fragment and an LD-only constructs were found to elicit somewhat opposite effects [14, 15]. Isoforms VAMP7d/h and VAMP7i show the same domain architectures of respectively Δ-longin and LD-only fragments and are likely to mediate equal/similar effects. Noteworthy, VAMP7d/h and Δ-longin fragment co-localize with VAMP7a, while VAMP7i and LD-only do not, thus showing congruent subcellular sorting in two different cell types (neuronal and epithelial). AS at the SYBL1 locus results in a more sophisticated modulation of the domain architecture: two non-SNARE isoforms consist of the LD alone (VAMP7i) or "membrane-anchored" LD (VAMP7j), and two Δ-longin isoforms (VAMP7d/h, this work and VAMP7c, ) share synaptobrevin-like architecture with different N-terminal extensions. Finally, VAMP7b is only partially "non-SNARE" because it still keeps the N-terminal part of the SNARE, which is crucial for intramolecular binding to the LD, closed conformation and targeting . VAMP7b and VAMP7a show different subcellular localization, in agreement with evidence that other protein regions/domains can modulate subcellular targeting capacity of the LD [21, 22]. This is further suggested when comparing VAMP7i and VAMP7j, which share the LD and lack the SNARE motif. VAMP7i is found in the nucleus, whereas the transmembrane anchor in VAMP7j is likely to prevent nuclear localization. Indeed, VAMP7i consists only of the LD fold, similar to longin-like proteins  like σ AP  and sedlin . Intriguingly, sedlin has been recently reported to show nuclear localization and to interact with transcription factors (TF) and TF-binding proteins [46, 47]. Production by AS of non-SNARE isoforms further strengthens the concept that the LD can play an important role in trafficking independently on the SNARE motif, as suggested by the existence of non SNARE longins . Tissue-specific expression data suggest that AS at the SYBL1 locus is developmentally regulated along cell lineage/tissue differentiation. Alternative mRNAs encoding variants with different domain combinations are expressed at levels ranging 5-12% of VAMP7a (Figure 4) in cell lines. In differentiated tissues the aggregate level of non-SNARE, inhibitory variants is comparable or even higher than VAMP7a. Each isoform might contribute to finely tune VAMP7-mediated regulation of cell physiology: indeed, several regulatory mechanisms do not require for 1:1 interactions. The main isoform is distributed among multiple locations along its trafficking route, because of interactions mediated by all its domains. Instead, splice variants missing either the LD or the SNARE motif are likely more involved in specific interactions, thus increasing their relative weight in influencing specific trafficking events. Evidence from this work further suggests that AS may contribute to finely tune the vesicle-mediated membrane trafficking specificity, providing new insights about the biological importance of the Longin domain over the SNARE functions.
The following human cell lines were used in this work: SH-SY5Y (neuroblastoma); C63 (skin fibroblasts); HepG2 (hepatocellular carcinoma); HeLa (cervical carcinoma); Jurkat (T-cell leukaemia). All cells were grown at 37°C in a humidified atmosphere, 5% CO2. SH-SY5Y and Jurkat cells were grown in the following media (all reagents from Invitrogen), supplemented with Glutamax and 10% heat-inactivated fetal bovine serum: SH-SY5Y, 1:1 DMEM+Ham's F12 supplemented with gentamicin (50 μg/ml); Jurkat, RPMI supplemented with 10 mM HEPES and PEN-STREP. C63, HepG2 and HeLa cells were kindly provided by colleagues from other laboratories.
RNA extraction and retrotranscription
Total RNA from cell cultures was extracted using TRIzol (Invitrogen) according to manufacturer's instructions. Aliquots (5 μg) of DNase I-treated total RNA were reverse transcribed using SuperScript® III RNase H-reverse transcriptase (Invitrogen). Total RNA from human tissues was purchased from BD Clontech. Aliquots (1 μg) of total RNA from human tissues were reverse transcribed using QuantiTect® reverse transcription kit (Qiagen).
Non quantitative PCR
PCR experiments were performed using Ampli Taq Gold DNA Polymerase (Applied Biosystems), with buffer recommended by the manufacturer, in a final reaction volume of 10 μl. An Express (Thermo-Hybaid) thermal cycler was used for the analysis of the 5' half of SYBL1 gene, set as follows: 9' at 96°C followed by 35 cycles consisting of 30" melting at 95°C + 45" annealing at 58°C + 45" extension at 72°C; a final extension step at 72°C was then performed for 5'. The forward (F) and reverse (R) (MWG)-primer sequences were: SYN1F 5'-ATGGCGATTCTTTTTGCTGTTGTT-3'; SYN1R 5'-AGTGCTGTCTGTGCTCTTGAACCGT-3'. The 3' architecture of LD+ isoforms was investigated carrying out a seminested PCR in the Robocycler (Stratagene) with an annealing temperature of 55°C and steps of annealing and extension of 1'. In both PCRs the same forward primer, SYBL2/3F, was used: 5'-CGTACTCACATGGCAATTAT-3'. This forward primer (ASOa) is specific to VAMP7a; it was combined to the TEL3 reverse primer: 5'-AGTCACATGGATTGCTTTTA-3' in the first PCR round (28 cycles) and to the TBP5: 5'-CAAGATTTCTGCTGGTAG-3' in the second PCR round (35 cycles) as nested reverse primer.
All experiments were performed as follows: RNA for each sample was extracted and reverse transcribed in three replicates. In order to minimize variation depending on experimental error, real-time experiments were then performed in five replicates for each sample, resulting in 3x5 = 15 replicates. Real-time PCR experiments were performed using SYBR®-Green core reagent kit and Ampli Taq Gold DNA Polymerase (Applied Biosystem) in a final reaction volume of 25 μl. The thermal cycler (Rotor-Gene 3000 from Corbett Research) was set as follows: 9' at 95°C followed by 40 cycles consisting of 30" melting at 95°C + 30" annealing at 60°C + 35" extension at 72°C. The gene encoding human ribosomal protein S13 is the housekeeping control for normalization . For quantitative data analysis, the Rotor Gene software (version 6.0.34) was used according to Pflaffl  and Marino et al.. The following F and R primers (SIGMA-Genosys) were used:
Primers were designed and combined so that all amplicons had (or almost) equal length (250 to 254 bps) hence quantitative comparison could be extended to different amplicons from all isoforms. Primer combinations to amplify specific isoforms were: VAMP7a, VAMP7a-F + VAMP7a-R; VAMP7b, VAMP7a-F + VAMP7b-R; VAMP7c, VAMP7c-F + VAMP7a-R; VAMP7d, VAMP7d-F + VAMP7a-R; VAMP7h, VAMP7h-F + VAMP7h-R; VAMP7i, VAMP7i-F + VAMP7i-R; VAMP7j, VAMP7j-F + VAMP7j-R.
Plasmid pGEX expressing the cytoplasmic domain of VAMP7a fused to the N-terminal GST tag was a kind gift of Dr. Monica Cuccurese; the LD of VAMP7a/b (aa 1-118) and the novel C-terminal region of VAMP7b (aa 151-260) were cloned in a pET32(a) plasmid (Novagen) and expressed in E. coli BL21 cells following standard protocols.
Oligoclonal antibodies and immunoblotting
Whenever possible, peptide sequences were designed to start N-terminally by a Cys residue (for conjugation to the carrier protein) already present in the VAMP7 isoform sequences:
MD2 (CITDDDFERSRAFNFLNE; residues 64-81 of VAMP7a and VAMP7b)
MD3 (Cys-RGERLELLIDKTENLVD; residues 150-166 of VAMP7a)
MD4 (CSSHVYEEPQAHYYH; residues 156-170 of VAMP7b)
MD5 (CDSSLSHTDRWYLPV; residues 219-233 of VAMP7b)
Peptide synthesis and conjugation, immunisation of rabbits and sera collection were performed by a company (Sigma-Genosys); we then purified the antibodies by affinity chromatography. Peptides were Cys-conjugated to the Sulfolink resin and chromatography was performed using binding and elution buffers from Pierce, following manufacturer's instructions. After elution, antibodies were concentrated by ultrafiltration using 10 kDa cut off filter tubes (Amicon). Strength and specificity of each antibody was checked by dot-blot analysis, using as targets progressive dilutions of (i) each specific peptide, (ii) the other three VAMP7 peptides and (iii) peptides from non-homologous proteins. For immunoblotting, protein samples were separated by standard SDS-PAGE and blotted onto nitrocellulose filters using Protean and Transblot cells (Bio-Rad). Primary antibodies were diluted 1:1000; secondary antibody (anti rabbit IgG-HRP, Santa Cruz Biotechnology) was diluted 1:20000. Enhanced chemiluminescence detection was performed using the Supersignal system (Pierce), following manufacturer's instructions; blot images were acquired using a Chemi-Doc image analysis system (Bio-Rad).
Subcellular localization experiments
The main isoform and splice variants of VAMP7 were tagged with a fluorescent protein (either red, RFP, or green, EGFP) cloning the isoform cds in pRFP-C3, pEGFP-C3 or pEGFP-N1 (Invitrogen). VAMP7b has C-terminal tag, whereas VAMP7d/h, VAMP7i and VAMP7j have N-terminal tags.
HeLa cells were transiently cotransfected using Lipofectamine™2000 protocol (Invitrogen) with pRFP-C3-VAMP7a and pEGFP-N1-VAMP7b or pEGFP-C3-VAMP7dh, pEGFP-C3-VAMP7i, pEGFP-C3-VAMP7j. Then, in vivo analysis was performed at 15/22/24/48h by using a TCS SP2 confocal scanning microscope (Leica, Heidelberg, Germany), sequential excitation with 488 nm, 543 nm laser beams, 63X 1.4 NA lens (low magnification: zoom 2-3; high magnification: zoom 5-8) and LAS AF Software. Images (size set to 512x512 pixels) were assembled by using ImageJ.
Melting temperatures of PCR primers were calculated using DNA Calculator http://www.sigma-genosys.com/calc/DNACalc.asp; possible occurrence of hairpins, self- and hetero-dimers was ruled out by Oligo analyzer 3.1 http://eu.idtdna.com/analyzer/applications/oligoanalyzer/default.aspx and the specificity was checked with MFEprimer http://biocompute.bmi.ac.cn/MFEprimer/, scanning both genomic and transcriptomic human databases. Translation Initiation Site (TIS) prediction was performed using StartScan http://bioinformatics.psb.ugent.be/webtools/startscan/; in silico translation of coding sequences resulting from likely alternative start codons was obtained using the Traslate tool at the ExPAsy server http://www.expasy.ch/tools/dna.html.
Antigenic regions of VAMP7 isoforms were predicted based on the antigenic index plot obtained using the Protean module of the Lasergene Package (DNASTAR); selection of peptide sequences was based on both their conservation/specifity and predicted solubility.
We thank Paul R. Pryor for critical reading and helpful suggestions, Thierry Galli and Simona Paladino for positive criticism and suggestions, Ciro Campanile for technical assistance, Elisabetta Caniato and Paolo Bolognese for help in some experiments. We thank Karen Gustafson for proofreading the manuscript. We acknowledge support from the University of Padua (PRAT2007 project CPDA077345/07) to FF and by PRIN2008 project 2008338WNL to OR. LA and FDR are recipients of PhD fellowships supported by: respectively, the Italian Ministry of University and Research (MIUR) and the contract LSHB-CT-2004-503243 from SAFE Network of Excellence European Commission Funded 6th Framework to MDE.
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