The Role of mRNA 3’ end Processing Factors in Regulating Global RNA Pol II Transcription and its Termination

Not only is mRNA 3’ processing essential for gene expression, it is crucial in gene regulation as well. The regulation of 3’ end processing is important in maintaining normal processes like neural activity, T-cell activity and stem cell differentiation and renewal, whereas its dysregulation can result in many diseases such as cancers. To study how 3’ processing factors regulate gene expression, comprehensive ChIP-seq (Chromatin Immunoprecipitation-Seq) analyses of subunits of the 3’ processing complex were performed. Unexpectedly, one subunit, CPSF100, showed minimal peaks at the 3’ ends of genes, with maximum signal at the promoters of a large subset of genes and another large subset of strong peaks at enhancers, both regulatory elements that can be dysregulated to precipitate aberrant gene expression patterns. To analyze the effect of CPSF100 and the CPSF complex on transcription and its termination at protein coding genes and enhancers, we performed RNA PolII ChIP-Seq, 4SU-Seq and PolyA Site (PAS)-Seq in HeLa cells where CPSF73 and CPSF100 were knocked down using an shRNA-mediated lentiviral delivery method. We found that knocking down CPSF factors led to bidirectional transcriptiontermination defects at enhancers and protein coding genes. In addition, it led to an aberrant response to transcription activation by reducing the ratio of PolII elongating on the gene body to the amount of PolII at the promoter. Finally, depletion of CPSF led to the formation of long and abundant noncoding RNAs from enhancers, promoters of genes, and at permissive intergenic regions. These findings advance the field of RNA 3’ end processing in several ways. We show that CPSF factors are important for facilitating productive elongation. In addition, the effect of depleting them on transcription termination far surpasses the effect of depleting the exonuclease that is thought to enable termination, suggesting that another exonuclease-independent model of termination may be the major mechanism. Finally, our findings show that CPSF is primarily responsible for mediating the cleavage and termination of eRNA transcripts, which was previously thought to occur via another factor. These findings open up new avenues of research and highlight the importance of the 3’end processing machinery in regulating transcription