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.

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Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01117-15.

ACKNOWLEDGMENTS

We are grateful to members of the Crown Institute for Genomics in the Nancy and Stephen Grand Israel National Center for Personalized Medicine for the RNA-Seq analysis.

R.D. is the incumbent of the Ruth and Leonard Simon Chair of Cancer Research.

R.D., G.D., and T.E. conceived and designed the study, analyzed the data and wrote the paper; G.D. and T.E. carried out most of the experiments; D.L analyzed the RNA-Seq data.

We declare that we have no competing financial interests.

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Funding

This work, including the efforts of Rivka Dikstein, was funded by a grant from the Pearl Welinsky Merolo Foundation Scientific Research Progress Fund to Rivka Dikstein.