Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites inC. elegans

Abstract

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3 ′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3 ′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3 ′ splice site (furthest from the 5 ′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3 ′ splice site (closer to the 5 ′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3 ′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3 ′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3 ′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. [Supplemental material is available for this article.]Alternative splicing is a highly regulated process by which a cell can produce multiple messenger RNAs, potentially encoding multiple proteins, from a common precursor transcript. The de novo assembly of a spliceosome on each intron of a pre-mRNA transcript requires cis-elements within the intron that are recognized as signals marking the beginning, end, and branchpoint (Ares and Weiser 1995). A cassette exon is a form of alternative splicing in which an exon is either included or skipped in the mature mRNA. Conserved enhancer or silencer elements within the exon or the surrounding introns interact with an array of constitutive and tissue-specific trans-factors that promote or inhibit assembly of a functional spliceosome (Wang and Burge 2008). The use of alternative 3 ′ or 5 ′ splice sites modifies gene expression by including or skipping coding sequences at the ends of exons. Many examples of adjacent alternative 3 ′ splice sites, defined as being separated by 18 nucleotides (nt) or less, have been observed. In many species, a form of alternative 3 ′ splice site usage has been identified in which the alternative splicing acceptors are only 3 nt apart. Except for the rare example of AC dinucleotides observed for substrates of the minor spliceosome, introns end with AG dinucleotides. Alternative 3 ′ splice sites separated by only 3 nt are referred to as NAGNAGs, as the end of the intron consists of two AG splice acceptors separated by 3 nt. A...

SNRP-27, the C. elegans homolog of the tri-snRNP 27K protein, has a role in 5′ splice site positioning in the spliceosome

“…The upstream 5 ′ splice site is strongly preferred in the snrp-27(+) background and shows splicing to both alternative 3 ′ splice sites at relatively equal levels. Both 3 ′ splice sites have relatively strong consensus sequences, so from previous work in our laboratory we might expect their alternative usage to not be regulated in a tissue-specific manner (Ragle et al 2015). In contrast, the downstream 5 ′ splice site is strongly preferred in the snrp-27(az26) background, and it splices exclusively to the downstream 3 ′ splice site.…”

Prp8 impacts cryptic but not alternative splicing frequency

“…Sequences trimmed to 80 bases were mapped to the C. elegans genome using STAR (29). We used a custom script to de novo identify all alternative 5′ SS and 3′ SS events separated by ≦ 50 nt (30) and AltEventFinder (31) to identify cassette exon alternative splicing events. We used MISO (23) to do pairwise comparisons of these libraries for all alternative splicing events.…”

Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans

“…The nematode is a powerful model to explore networks involved in alternative splicing regulation and the physiological impact of their perturbation (Barberan-Soler et al 2009, 2011Zahler 2012). Recent efforts have been made to systematically identify all possible splice variants of the complete Caenorhabditis elegans genome using transcriptome sequencing (RNA-seq) (Hillier et al 2009;Ramani et al 2011;Gerstein et al 2014;Kuroyanagi et al 2014;Ragle et al 2015). Most of these analyses reported previously unannotated splice junctions, indicating that saturation has not yet been reached.…”