Abstract
Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' → 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.
Highlights
Transcriptional termination can be defined as the cessation of RNA polymerization and dissolution of the ternary complex of RNA polymerase II (Pol II), DNA, and RNA
AAUAAA is recognized by the CPSF30 and WDR33 subunits of cleavage and polyadenylation specificity factor (CPSF), with endonuclease activity provided by CPSF73 (Mandel et al 2006; Shi et al 2009; Chan et al 2014; Schonemann et al 2014)
Selection markers were separated from the tag by a P2A sequence that was cleaved during translation (Kim et al 2011)
Summary
Transcriptional termination can be defined as the cessation of RNA polymerization and dissolution of the ternary complex of RNA polymerase II (Pol II), DNA, and RNA. Rat was shown to promote the recruitment of some polyadenylation factors to budding yeast genes and so may sometimes affect termination indirectly through impacting PAS function (Luo et al 2006). CPSF73 was identified as the nuclease over a decade ago (Mandel et al 2006), its function in termination is not fully characterized This issue has been tackled using in vitro systems competent for transcription and RNA processing, which revealed that a PAS can promote termination in the absence of cleavage (Zhang et al 2015). While highlighting the capacity of PAS recognition to affect Pol II activity, it is unknown whether this mechanism promotes termination in cells
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