Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.

• Klíčová slova
• Prp8, RES complex, chromatin immunoprecipitation, cotranscriptional splicing, nineteen complex, spliceosome assembly,
• MeSH
• introny MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• malý jaderný ribonukleoprotein U2 metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sestřih RNA * MeSH
• spliceozomy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U1 MeSH
• malý jaderný ribonukleoprotein U2 MeSH
• PRP45 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

• MeSH
• databáze nukleových kyselin MeSH
• mutace genetika   MeSH
• prekurzory RNA genetika   MeSH
• průběh práce * MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• prekurzory RNA MeSH