Increased large conductance calcium-activated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS

Background  

Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined.     

VAMP-1 has a highly variable C-terminus generated by alternative splicing.

VAMP-1 (synaptobrevin1) is one of the key proteins in the SNARE complex which is involved in regulated exocytosis. Recently, Isenmann et al. (1998, Mol. Biol. Cell 9, 1649-1660) showed the extreme C-terminal region of VAMP-1A and 1B to be involved in subcellular targeting of the isoforms. Four new splice variants (VAMP-1C to F) were identified in addition to the previously published variants VAMP-1A and VAMP-1B. Interestingly, the four new isoforms also have variable sequences only at the extreme C-terminus. This suggests that the C-terminal region has an important function for VAMP-1 and vesicle targeting. All six variants were a result of alternative splicing that linked exons 1-4 which encode the conserved region of VAMP-1 with one of the exons 5A to 5F that encodes the highly variable extreme C-terminus. Exon (5A-E) encode C-termini of two to five amino acid residues, whereas exon 5F encoded a long C-terminal amino acid extension. The splice variants were differentially expressed in human brain, kidney, and inflammatory cells.     

Redirecting splicing with bifunctional oligonucleotides

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5′ splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.     

Splice Variants of Na(V)1.7 Sodium Channels Have Distinct β Subunit-Dependent Biophysical Properties

Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na_V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na_V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na_V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na_V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.     

Alternative splicing of the <Emphasis Type="Italic">antitrypsin</Emphasis> gene in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Alternative splicing plays an important role in expanding protein diversity. In the present study, different splice variants of the antitrypsin gene (sw-AT) in the silkworm were identified by bioinformatics analyses using expressed sequence tags and genomic information. Four splice variants were obtained by RT-PCR with suitably designed primers, confirmed by sequencing, and designated as sw-AT-1, sw-AT-2, sw-AT-3, and sw-AT-4. The sw-AT gene contains 10 exons and nine introns. The splice variants differ in exon 9, with sw-AT-1, sw-AT-2, and sw-AT-3 using different versions of the exon, namely exon 9a, 9b, and 9c, respectively. In sw-AT-4, exon 9 consists of the combination of exons 9b and 9c. The expression patterns of the four isoforms in different tissues, at different developmental stages, and under different stress conditions (temperature, starvation, and mycotic infection) were characterized and quantified. The sw-AT isoforms showed tissue-specific expression patterns, with sw-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. The four isoforms were predominantly expressed in the fat body, body wall, and testes of larvae, and exhibited similar expression profiles during development of the fat body. Among the stress treatments, low temperature had the greatest effect on isoform expression, and expression was also upregulated with mycotic infection.     

SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase-9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 +/- 0.11 to 0.56 +/- 0.08 (P < 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.     

Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (IVS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases,and dramatically changes SUV39H2 functions

Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.