Abstract
Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3’ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5’ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5’ss in dtd1+ intron 1 and of an upstream alternative 3’ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5’ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5’ ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3’ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.
Highlights
Nuclear pre-mRNA splicing, a key process in eukaryotic gene expression, is carried out by the spliceosome consisting of U1, U2, U4, U5 and U6 snRNAs and ~ 150 non-snRNP proteins
These compiled splice junction sequences were mapped onto two independently reported transcriptomes of exponentially growing wild-type fission yeast cultures [40, 16]. This analysis identified only 104 non-consecutive splice events as common to both reported transcriptome datasets and we inferred these as stringent and reproducible events (S3 Table). Most of these events were of very low abundance, only 17 exon skipped junctions had total generation sequence (NGS) read count more than 10 reaffirming that in wild-type cells exon skipping, even when it occurs is at low levels
To deduce any role for the splicing factor SpSlu7 in these events, we first interrogated our microarray data [36] from two biological replicates of stationary phase slu7+ O/E cells to identify exon skipped events detected by the custom microarray probes
Summary
Nuclear pre-mRNA splicing, a key process in eukaryotic gene expression, is carried out by the spliceosome consisting of U1, U2, U4, U5 and U6 snRNAs and ~ 150 non-snRNP proteins. This riboprotein machinery assembles on the pre-mRNA and catalyses the two invariant transesterification reactions by which introns are precisely excised and exons ligated [1]. The flexibility in splice-site choice, reflected as alternative splicing, is crucial for for diversifying the metazoan transcriptome, the proteome and for genome evolution [2, 3]. Functions for SR proteins in alternative splice-site choice are well described, roles for core spliceosomal proteins are less understood even in these organisms
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