Identification and characterization of a novel C2B splice variant of synaptotagmin I

Abstract

We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-b, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-a. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-b exon in the nervous system. Synaptotagmin I C2B-b showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-a, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-b with a higher affinity than synaptotagmin I C2B-a. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.

Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans

“…There are nine exons, including two alternate forms of exon 6. The alternative sixth exons were first identified in C. elegans (Craxton, 2001), but were subsequently noted in Aplysia as well (Nakhost et al, 2004). Exon 6A is 134 bp and encodes 45 residues, while exon 6B is 140 bp and encodes 47 residues.…”

“…Alternative splicing of synaptotagmin 1 pre-mRNA gives rise to two distinct protein isoforms in C. elegans, as well as some other invertebrates (Craxton, 2001;Nakhost et al, 2004). Two alternative exons, designated 6A and 6B, encode the third, fourth, and fifth β-strands in the C2B domain, and alternative splicing of these exons creates structural diversity within this domain.…”

“…For example, alternative splicing of synaptotagmin 1 in Aplysia, mice, and humans specifies a change in the juxtamembrane region (reviewed in Craxton, 2004); this splicing appears to alter the release properties of synaptotagmin (Nakhost et al, 2003). In addition, the C2B domain of synaptotagmin 1 undergoes alternative splicing in Caenorhabditis and Aplysia, and RNA editing in Anopheles and Drosophila (Hoopengardner et al, 2003;Nakhost et al, 2004). In C. elegans and Aplysia, there are two alternative forms of the sixth exon, designated exons 6A and 6B.…”

A-to-I RNA editing alters less-conserved residues of highly conserved coding regions: Implications for dual functions in evolution

“…For example, four residues (Gly, Ser, Thr, and Ala) are also located at the equivalent position of edited Thr350 in squid sytI (Nakhost et al 2004). Based on the structural similarity of Ser and Thr and Ala and Gly, switching these amino acids should not result in a major structural change while substitution between Ser/Thr and Ala/Gly could potentially result in a major functional change.…”

“…For example, RNA editing generates a Ser/Gly substitution close to the fruit fly ADAR active site, which could result in less active ADAR than the genomeencoded, unedited isoform (Keegan et al 2005). A Thr/Gly mutation at the equivalent position of sytI at the edited site shows greater resistance to proteolysis in the absence of calcium (Nakhost et al 2004). Therefore, species-specific expansion at this site in DNA and RNA seems to have adaptive functional significance.…”