N-terminal tagging of RNA Polymerase II shapes transcriptomes more than C-terminal alterations

RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains unclear. Here, we investigate how altering the CTD’s length and fusing it with protein tags affects transcriptional output on a genome-wid...

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Bibliographic Details
Published in:iScience Vol. 27; no. 6; p. 109914
Main Authors: Callan-Sidat, Adam, Zewdu, Emmanuel, Cavallaro, Massimo, Liu, Juntai, Hebenstreit, Daniel
Format: Journal Article
Language:English
Published: United States Elsevier Inc 21.06.2024
Elsevier
Subjects:
Biophysics
Molecular biology
Transcriptomics
Molecular biology
Transcriptomics
Biophysics
ISSN:2589-0042, 2589-0042
Online Access:Get full text
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Summary:RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains unclear. Here, we investigate how altering the CTD’s length and fusing it with protein tags affects transcriptional output on a genome-wide scale in mammalian cells at single-cell resolution. While transcription generally appears to occur in burst-like fashion, where RNA is predominantly made during short bursts of activity that are interspersed with periods of transcriptional silence, the CTD’s role in shaping these dynamics seems gene-dependent; global patterns of bursting appear mostly robust to CTD alterations. Introducing protein tags with defined structures to the N terminus cause transcriptome-wide effects, however. We find the type of tag to dominate characteristics of the resulting transcriptomes. This is possibly due to Pol II-interacting factors, including non-coding RNAs, whose expression correlates with the tags. Proteins involved in liquid-liquid phase separation appear prominently. [Display omitted] •ScRNA-seq datasets for cells expressing CTD-mutant RNA polymerase II (Pol II)•Transcriptional bursting is robust to Pol II CTD alterations•N-terminal- rather than CTD-modifications of Pol II shape transcriptomes•Altered expression of ncRNAs and LLPS-related factors due to N-terminal Pol II tags Molecular biology; Biophysics; Transcriptomics
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ISSN:2589-0042
2589-0042
DOI:10.1016/j.isci.2024.109914