1. Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.
- Author
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Ni Z, Ahmed N, Nabeel-Shah S, Guo X, Pu S, Song J, Marcon E, Burke GL, Tong AHY, Chan K, Ha KCH, Blencowe BJ, Moffat J, and Greenblatt JF
- Subjects
- Humans, Cyclin-Dependent Kinases metabolism, Cyclin-Dependent Kinases genetics, Genome, Human, HEK293 Cells, Polyadenylation, RNA Cleavage, RNA Polymerase II metabolism, CRISPR-Cas Systems, Genes, Reporter, mRNA Cleavage and Polyadenylation Factors metabolism, mRNA Cleavage and Polyadenylation Factors genetics, RNA Precursors metabolism, RNA Precursors genetics
- Abstract
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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