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P-TEFb is degraded by Siah1/2 in quiescent cells-Huang etc

Citation

Fujinaga, Koh (2022), P-TEFb is degraded by Siah1/2 in quiescent cells-Huang etc, Dryad, Dataset, https://doi.org/10.7272/Q6ZP44CR

Abstract

P-TEFb, composed of CycT1 and CDK9, regulates the elongation of transcription by RNA polymerase II. In proliferating cells, it is regulated by 7SK snRNA in the 7SK snRNP complex. In resting cells, P-TEFb is absent, because CycT1 is dephosphorylated, released from CDK9 and rapidly degraded. In this study, we identified the mechanism of this degradation. We mapped the ubiquitination and degradation of free CycT1 to its N-terminal region from positions 1 to 280. This region is ubiquitinated at six lysines, where E3 ligases Siah1 and Siah2 bind and degrade these sequences. Importantly, the inhibition of Siah1/2 rescued the expression of free CycT1 in proliferating as well as resting primary cells. We conclude that Siah1/2 are the E3 ligases that bind and degrade the dissociated CycT1 in resting, terminally differentiated, anergic and/or exhausted cells.

Methods

All WBs were visualized by enhanced chemiluminescence (ECL) (Perkin Elmer) produced by HRP-conjugated secondary antibodies, and chemiluminescent signals were directly captured by LI-COR image analyzer. All unedited WB images were exported as TIFF files and directly pasted into Keynote file. WB bands used in the figures for the manuscript were indicated. The size of protein marker and brief description of how WBs were obtained were also included.

qPCR data were directed exported as excel file, and bar graph was generated in the same file.

Flow cytometry data were analyzed by Flowjo, and exported as one PDF file.

Usage Notes

This dataset is for read-only and not for further manipulations, and it includes all raw data and indicated figures for the paper already published. Please do not reuse this dataset for any other publications or purposes without permissions for the original authors. 

Funding

National Institute of Allergy and Infectious Diseases, Award: R01 AI049104

Nora Eccles Treadwell Foundation

National Institute of Allergy and Infectious Diseases, Award: P50AI150476