Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans

Regulation of alternative splicing is controlled by pre-mRNA sequences (cis-elements) and trans-acting protein factors that bind them. The combinatorial interactions of multiple protein factors with the cis-elements surrounding a given alternative splicing event lead to an integrated splicing decision. The mechanism of multifactorial splicing regulation is poorly understood. Using a splicing-sensitive DNA microarray, we assayed 352 Caenorhabditis elegans alternative cassette exons for changes in embryonic splicing patterns between wild-type and 12 different strains carrying mutations in a splicing factor. We identified many alternative splicing events that are regulated by multiple splicing factors. Many splicing factors have the ability to behave as splicing repressors for some alternative cassette exons and as splicing activators for others. Unexpectedly, we found that the ability of a given alternative splicing factor to behave as an enhancer or repressor of a specific splicing event can change during development. Our observations that splicing factors can change their effects on a substrate during development support a model in which combinatorial effects of multiple factors, both constitutive and developmentally regulated ones, contribute to the overall splicing decision

Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene

Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (∼18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 – now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation

A computational screen for mammalian pseudouridylation guide H/ACA RNAs

The box H/ACA RNA gene family is one of the largest non-protein-coding gene families in eukaryotes and archaea. Recently, we developed snoGPS, a computational screening program for H/ACA snoRNAs, and applied it to Saccharomyces cerevisiae. We report here results of extending our method to screen for H/ACA RNAs in multiple large genomes of related species, and apply it to the human, mouse, and rat genomes. Because of the 250-fold larger search space compared to S. cerevisiae, significant enhancements to our algorithms were required. Complementing extensive cloning experiments performed by others, our findings include the detection and experimental verification of seven new mammalian H/ACA RNAs and the prediction of 23 new H/ACA RNA pseudouridine guide assignments. These assignments include four for H/ACA RNAs previously classified as orphan H/ACA RNAs with no known targets. We also determined systematic syntenic conservation among human and mouse H/ACA RNAs. With this work, 82 of 97 ribosomal RNA pseudouridines and 18 of 32 spliceosomal RNA pseudouridines in mammals have been linked to H/ACA guide RNAs

Global analysis of alternative splicing uncovers developmental regulation of nonsense-mediated decay in C. elegans

Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a mechanism for post-transcriptional regulation of gene expression. We analyzed the global effects of mutations in seven genes of the C. elegans NMD pathway on AS isoform ratios. We find that mutations in two NMD factors, smg-6 and smg-7, have weaker global effects relative to mutations in other smg genes. We did an in-depth analysis of 12 pre-mRNA splicing factor genes that are subject to AS-NMD. For four of these, changes in the ratio of alternatively spliced isoforms during development are caused by developmentally regulated inhibition of NMD, and not by changes in alternative splicing. Using sucrose gradient analysis of mRNAs undergoing translation, we find several examples of NMD-dependent enrichment of premature termination codon (PTC) isoforms in the monosome fraction. In contrast, we present evidence of two genes for which the PTC-containing isoforms are found in polysomes and have a translational profile similar to non-PTC-containing transcripts from the same gene. We propose that NMD of certain alternatively spliced isoforms is regulated, and that some stabilized NMD targets may be translated

Coordinated tissue-specific regulation of adjacent alternative 3' splice sites in C. elegans

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