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| Status |
Public on Jun 26, 2024 |
| Title |
mRNAseq: Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occu- pancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings revise our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.
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| Overall design |
For POLR3A KD Hek293T cells are transfected with either control siRNA or POLR3A siRNA, and cells are collected 2 days after knockdown. For Pol III small molecule inhibition, Hek293T cells are treated with either DMSO (vehicle, 0.1%) or ML60218 (25uM) and cells are collected 4 hours post-treatment. RNA is collected using E.Z.N.A.® Total RNA Kit I. For RNA sequencing, the RNAseq libraries were prepared with the Kapa Hyper Stranded mRNA library kit (Roche) and sequenced on two 10B lanes for 151 cycles from both ends of the fragments on a NovaSeq X Plus with V1.0 sequencing kits, Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina).
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| Contributor(s) |
Cheng R, Zhou S, Rajendra K, Lizarazo S, Smith D, Van Bortle K |
| Citation(s) |
38895345 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R00 HG010362 |
The development and application of tools to characterize the level and function of RNA polymerase III transcription dynamics during cellular differentiation |
UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN |
Kevin Van Bortle |
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| Submission date |
May 28, 2024 |
| Last update date |
Jun 26, 2024 |
| Contact name |
Ruiying Cheng |
| E-mail(s) |
[email protected], [email protected]
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| Phone |
2178983908
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| Organization name |
The University of Illinois Urbana-Champaign
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| Department |
School of Molecular & Cellular Biology
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| Lab |
Van Bortle Lab
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| Street address |
B520 Chemical & Life Sciences Laboratory
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| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
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| Platforms (1) |
| GPL34284 |
Illumina NovaSeq X Plus (Homo sapiens) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA1117407 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE268457_counts.csv.gz |
10.9 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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