SPT5 stabilization of promoter-proximal RNA polymerase II

Yuki Aoi; Yoh hei Takahashi; Avani P. Shah; Marta Iwanaszko; Emily J. Rendleman; Nabiha H. Khan; Byoung Kyu Cho; Young Ah Goo; Sheetal Ganesan; Neil L. Kelleher; Ali Shilatifard

doi:10.1016/j.molcel.2021.08.006

SPT5 stabilization of promoter-proximal RNA polymerase II

Yuki Aoi, Yoh hei Takahashi, Avani P. Shah, Marta Iwanaszko, Emily J. Rendleman, Nabiha H. Khan, Byoung Kyu Cho, Young Ah Goo, Sheetal Ganesan, Neil L. Kelleher, Ali Shilatifard^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

42 Scopus citations

Abstract

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

Original language	English (US)
Pages (from-to)	4413-4424.e5
Journal	Molecular cell
Volume	81
Issue number	21
DOIs	https://doi.org/10.1016/j.molcel.2021.08.006
State	Published - Nov 4 2021

Funding

We are grateful to Dr. Edwin Smith for discussions and critical reading of the manuscript. We thank the Shilatifard lab members for helpful discussions and support and B. Monroe for illustrations. We thank M. Kanemaki, A. Holland, I. Cheeseman, D. Foltz, and B. Zheng for providing reagents. We thank K. Eagan for advice on TT-seq. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. Proteomics 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 National Cancer Institute grant R01CA214035 to A.S.

Keywords

CDK9
Cullin 3
NELF
RNA polymerase II
SPT5
VCP
auxin-inducible degron
degradation
elongation
transcription

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1016/j.molcel.2021.08.006

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title = "SPT5 stabilization of promoter-proximal RNA polymerase II",

abstract = "Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.",

keywords = "CDK9, Cullin 3, NELF, RNA polymerase II, SPT5, VCP, auxin-inducible degron, degradation, elongation, transcription",

author = "Yuki Aoi and Takahashi, {Yoh hei} and Shah, {Avani P.} and Marta Iwanaszko and Rendleman, {Emily J.} and Khan, {Nabiha H.} and Cho, {Byoung Kyu} and Goo, {Young Ah} and Sheetal Ganesan and Kelleher, {Neil L.} and Ali Shilatifard",

note = "Funding Information: We are grateful to Dr. Edwin Smith for discussions and critical reading of the manuscript. We thank the Shilatifard lab members for helpful discussions and support and B. Monroe for illustrations. We thank M. Kanemaki, A. Holland, I. Cheeseman, D. Foltz, and B. Zheng for providing reagents. We thank K. Eagan for advice on TT-seq. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. Proteomics 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 National Cancer Institute grant R01CA214035 to A.S. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",

year = "2021",

month = nov,

day = "4",

doi = "10.1016/j.molcel.2021.08.006",

language = "English (US)",

volume = "81",

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AU - Aoi, Yuki

AU - Takahashi, Yoh hei

AU - Shah, Avani P.

AU - Iwanaszko, Marta

AU - Rendleman, Emily J.

AU - Khan, Nabiha H.

AU - Cho, Byoung Kyu

AU - Goo, Young Ah

AU - Ganesan, Sheetal

AU - Kelleher, Neil L.

AU - Shilatifard, Ali

N1 - Funding Information: We are grateful to Dr. Edwin Smith for discussions and critical reading of the manuscript. We thank the Shilatifard lab members for helpful discussions and support and B. Monroe for illustrations. We thank M. Kanemaki, A. Holland, I. Cheeseman, D. Foltz, and B. Zheng for providing reagents. We thank K. Eagan for advice on TT-seq. Y.A. was supported by the JSPS Research Fellowship for Young Scientists and the Uehara Memorial Foundation Research Fellowship. Proteomics 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 National Cancer Institute grant R01CA214035 to A.S. Publisher Copyright: © 2021 Elsevier Inc.

PY - 2021/11/4

Y1 - 2021/11/4

N2 - Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

AB - Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

KW - CDK9

KW - Cullin 3

KW - NELF

KW - RNA polymerase II

KW - SPT5

KW - VCP

KW - auxin-inducible degron

KW - degradation

KW - elongation

KW - transcription

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U2 - 10.1016/j.molcel.2021.08.006

DO - 10.1016/j.molcel.2021.08.006

M3 - Article

C2 - 34480849

AN - SCOPUS:85119985326

SN - 1097-2765

VL - 81

SP - 4413-4424.e5

JO - Molecular cell

JF - Molecular cell

IS - 21

ER -

SPT5 stabilization of promoter-proximal RNA polymerase II

Abstract

Funding

Keywords

ASJC Scopus subject areas

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