title: Analysis of Subcellular RNA Fractions Revealed a Transcription-Independent Effect of Tumor Necrosis Factor Alpha on Splicing, Mediated by Spt5.
abstract: The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Gil
authors.initials: G
authors.lastname: Diamant
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Eisenbaum
authors.affiliation: Department of Biological Services, Bioinformatics Unit, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Dena
authors.initials: D
authors.lastname: Leshkowitz
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel rivka.dikstein@weizmann.ac.il.
authors.forename: Rivka
authors.initials: R
authors.lastname: Dikstein
mesh_terms: D004257:DNA Polymerase II / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D013347:Subcellular Fractions / Q000502:physiology; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D016328:NF-kappa B; D009687:Nuclear Proteins; C112224:SUPT5H protein, human; D035602:Transcriptional Elongation Factors; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D004257:DNA Polymerase II