Effects of Transcription Elongation Rate and Xrn2 Exonuclease Activity on RNA Polymerase II Termination Suggest Widespread Kinetic Competition

Abstract

Summary The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5′PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

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

“…Although the role of Rat1 was recently re-affirmed [8], RNAi of Xrn2 did not reveal a general termination defect at the 3ʹ end of protein-coding genes [9]. However, termination was subsequently shown to be affected when a catalytically inactive version of Xrn2 is expressed in an RNAi background [10]. The inactive protein may block termination by trace amounts of Xrn2, remaining after RNAi, or prevent redundant 5ʹ-3ʹ exonucleases from accessing the nascent RNA in the Xrn2-depleted situation [11].…”

“…Perhaps these are examples of where Pol II does not pause and responds more acutely to Xrn2 loss or these genes may not employ alternative termination mechanisms that could act in the absence of Xrn2 (see below). Other possibilities would include slower 3ʹ end processing that would delay the production of an Xrn2 entry site or rapid Pol II elongation rates previously shown to extend transcription [10]. A more detailed analysis of primary sequence or chromatin features will be important to understand the basis of these different Xrn2 effects and whether they are variations on the same termination mechanism or represent distinct processes.…”

“…Indeed, recent work shows the sensitivity of many C.Elegans protein-coding genes to Xrn2-dependent termination is determined by promoter identity [27]. Intriguingly, Xrn2 is still recruited to transcription units that are unaffected by its loss, including human RDH and snRNA genes, raising the possibility that an additional feature or factor determines its involvement in termination [10,27]. …”

“…We have discussed how paused Pol II may constitute a frequent target for Xrn2 and data from purified systems shows that prone polymerases are more effectively terminated by 5ʹ-3ʹ exonucleases [15]. However, Xrn2 may also have to pursue elongating polymerases as modulating polymerase transcription rates affects the position of termination in cells and extended mNET-seq signal is often observed in its absence [10,14]. …”

Transcription Regulation in Archaea

“…Transcription factors involved in initiation and elongation have been characterized in all domains, while a transcription termination factor(s) has been characterized only in Bacteria and Eukarya (142)(143)(144)(145). By inference, from known termination factors that are employed in bacterial and eukaryotic systems, it is easily argued that protein factors are encoded in archaeal genomes that have the capacity to direct transcription termination in vivo.…”