Abstract
It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.
Original language | English (US) |
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Pages (from-to) | 3412-3423.e5 |
Journal | Molecular cell |
Volume | 82 |
Issue number | 18 |
DOIs | |
State | Published - Sep 15 2022 |
Funding
We are grateful to M. Morgan for critical reading of the manuscript. We thank the Shilatifard lab members for helpful discussions, B. Monroe for illustrations, and E. Rendleman for initial next-generation sequencing (NGS) support early in our studies. We thank M. Kanemaki, A. Holland, I. Cheeseman, and D. Foltz for providing reagents. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. Proteomic analysis was performed by the Northwestern Proteomics Core Facility, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, an instrumentation award (S10OD025194) from the NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569. The study is supported by funding from the National Cancer Institute R35CA197569 to A.S. Y.A. and A.S. conceived and designed the experiments. Y.A. performed experiments and data analyses. A.P.S. and S.G. performed cloning, ChIP, and NGS work. S.H.A.S. repeated MS experiments. B.-K.C. Y.A.G. and N.L.K. performed proteomics experiments and data processing, and Y.A. analyzed proteomics data. Y.A. and A.S. interpreted results and wrote the manuscript, with input from all authors. The authors declare no competing interests. We are grateful to M. Morgan for critical reading of the manuscript. We thank the Shilatifard lab members for helpful discussions, B. Monroe for illustrations, and E. Rendleman for initial next-generation sequencing (NGS) support early in our studies. We thank M. Kanemaki, A. Holland, I. Cheeseman, and D. Foltz for providing reagents. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship . Proteomic analysis was performed by the Northwestern Proteomics Core Facility, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center , an instrumentation award ( S10OD025194 ) from the NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569 . The study is supported by funding from the National Cancer Institute R35CA197569 to A.S.
Keywords
- NELF
- PAF1
- RNA polymerase II
- SPT6
- chromatin
- elongation
- gene expression
- pause/release
- promoter-proximal pausing
- transcription
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology