Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

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.

An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

“…We recently addressed the role of RNA cleavage and degradation in termination by employing gene editing to bring Xrn2 or CPSF73 under inducible control [14]. Xrn2 was tagged with an auxin-inducible degron (AID) and could be eliminated within minutes whereas CPSF73 was combined with an E.coli DHFR degron allowing its depletion over the course of several hours.…”

“…Mammalian native elongating transcript sequencing (mNET-seq) was used to establish the effects of Xrn2 loss on transcription [9,14]. This method provides a picture of Pol II location over the genome at single nucleotide resolution and, unlike run-on based methods, detects polymerases irrespective of their ability to transcribe.…”

“…It is important to note that, on most genes, mNET-seq and RNA-seq signals eventually drop to near-background levels even when Xrn2 is absent [14]. Analysis of individual genes suggests that this is still the case when inactive Xrn2 is expressed in the absence of Xrn2-AID suggesting that delayed termination even occurs when degradation from the 5ʹ end is blocked.…”

“…Interestingly, exosome depletion stabilises some read-through RNA in the absence of Xrn2, which may derive from such processes [14]. The existence of auxiliary termination mechanisms would be reminiscent of fail-safe termination in budding yeast where, for example, compromising Rat1 leads to termination via the Nrd1 pathway [17].…”

“…Experiments using in vitro systems have been used to show that PAS-dependent termination can occur without RNA cleavage [13], but it is unclear whether this mechanism applies in cells where multiple processes are coordinated. We found that catalytically inactive CPSF73 does not restore transcriptional termination, when CPSF73 is lost, suggesting a strict requirement for cleavage [14]. Alternatively, inactive CPSF73 may not fully recapitulate the function of the native protein, for example in forming stable or precise interactions with other factors.…”