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Splicing factors facilitate RNAi-directed silencing in fission yeast.

ABSTRACT: Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.

SUBMITTER: Bayne EH

PROVIDER: S-EPMC2585287 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Splicing factors facilitate RNAi-directed silencing in fission yeast.

Bayne Elizabeth H EH Portoso Manuela M Kagansky Alexander A Kos-Braun Isabelle C IC Urano Takeshi T Ekwall Karl K Alves Flavia F Rappsilber Juri J Allshire Robin C RC

Science (New York, N.Y.) 20081001 5901

Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect t ...[more]

PMID: 18948543

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Project description: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.

Dataset Information

Splicing factors facilitate RNAi-directed silencing in fission yeast.

Publications

Splicing factors facilitate RNAi-directed silencing in fission yeast.

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