Abstract
RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5′ cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 261-274.e5 |
| Journal | Molecular cell |
| Volume | 78 |
| Issue number | 2 |
| DOIs | |
| State | Published - Apr 16 2020 |
Funding
We thank the Shilatifard lab members, D. Reinberg, D. Taatjes, A. Stark, E. Bartom, K. Eagen, J. Lis, and J. Conaway for helpful discussions and P. Ozark for advice on the data analysis. We thank M. Kanemaki, A. Holland, I. Cheeseman, and D. Foltz for providing reagents. We thank N. Ethen for illustrations. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. E.R.S. was supported by the National Institutes of Health grant R50CA211428 . R.S. was supported by funding from the University of Miami Miller School of Medicine , Sylvester Comprehensive Cancer Center , and grants R01 GM078455 and DP1 CA228041 from the National Institutes of Health . Studies in the Shilatifard laboratory related to transcription elongation are funded by the National Institutes of Health grant R01CA214035 . We thank the Shilatifard lab members, D. Reinberg, D. Taatjes, A. Stark, E. Bartom, K. Eagen, J. Lis, and J. Conaway for helpful discussions and P. Ozark for advice on the data analysis. We thank M. Kanemaki, A. Holland, I. Cheeseman, and D. Foltz for providing reagents. We thank N. Ethen for illustrations. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. E.R.S. was supported by the National Institutes of Health grant R50CA211428. R.S. was supported by funding from the University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, and grants R01 GM078455 and DP1 CA228041 from the National Institutes of Health. Studies in the Shilatifard laboratory related to transcription elongation are funded by the National Institutes of Health grant R01CA214035. Y.A. E.R.S. R.S. and A.S. conceived and designed the experiments. Y.A. carried out most experiments and data analyses. A.R.W. developed the initial framework for PRO-seq data analysis. F.X.C. and A.P.S. assisted in ChIP-seq experiments. S.A.M. and E.J.R. carried out next-generation sequencing experiments. Y.A. E.R.S. R.S. and A.S. interpreted results and wrote the manuscript, with input from all authors. The authors declare no competing interests.
Keywords
- NELF
- PRO-cap
- PRO-seq
- RNA Polymerase II
- cap-binding complex
- m7G cap
- mRNA capping
- promoter-proximal pausing
- super elongation complex
- transcription elongation
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology