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

Pharmacological inhibition of protein kinases that are responsible for the phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II during transcription by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) leads to severe inhibition of mRNA synthesis and activates p53. Transcription of the p53 effectors that are induced under these conditions, such as p21 or PUMA, must bypass the requirement for CTD phosphorylation by the positive elongation factor P-TEFb. Here, we have downregulated SNW1/SKIP, a splicing factor and a transcriptional co-regulator, which was found to interact with P-TEFb and synergistically affect Tat-dependent transcription elongation of HIV 1. Using the colon cancer derived cell line HCT116, we have found that both doxorubicin- and DRB-induced expression of p21 or PUMA is insensitive to SNW1 downregulation by siRNA. This suggests that transcription of stress response genes, unlike, e.g., the SNW1-sensitive mitosis-specific genes, can proceed uncoupled from regulators that normally function under physiological conditions.

• MeSH
• buněčné jádro metabolismus   MeSH
• down regulace genetika   MeSH
• fyziologický stres genetika   MeSH
• HCT116 buňky MeSH
• HeLa buňky MeSH
• koaktivátory jaderných receptorů genetika   metabolismus   MeSH
• lidé MeSH
• lidské chromozomy metabolismus   MeSH
• mitóza MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• koaktivátory jaderných receptorů MeSH
• nádorový supresorový protein p53 MeSH
• SNW1 protein, human MeSH Prohlížeč