Xrn2 substrate mapping identifies torpedo loading sites and extensive premature termination of RNA pol II transcription

Abstract

The exonuclease torpedo Xrn2 loads onto nascent RNA 5′-PO4ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and “travels” to 3′ ends of genes. Mapping of 5′-PO4ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3′ ends. We propose that processing of all mRNA 3′ ends comprises cleavage and limited 5′–3′ trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3′ ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3′ flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3′ flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5′-PO4ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

“…These observations are consistent with the published ChIP-seq data that demonstrated Pol II accumulation downstream of PAS observed in Xrn2 D235A catalytic mutant and recent analyses of nascent transcription (that employed bromouridine for metabolic labelling of RNA, Bru-seq) when Xrn2 D235A expression was induced in addition to the endogenous WT Xrn2 54,85,100 . Global accumulation of the 3’-reads observed in Xrn2 M is accompanied by a decrease in TT-seq signal over the gene-body region for many genes (Figure 4D, clusters 1 and 3).…”

“…We provide evidence that Xrn2 forms a stable complex with Pol II which supports the idea of Xrn2 travelling together with elongation complex. This aligns with the observations from other studies as Xrn2 was detected at gene promoters by ChIP 54,85 and it was implicated in the premature dislodgement of Pol II at metazoan and fission yeast promoters 54,85,171 .…”

“…These data confirm the importance of the catalytic activity of Xrn2 for its function in transcription termination. In agreement with our data, Xrn2 exonucleolytic activity has been recently demonstrated to play a role in the control of a widespread premature transcription termination in human cells 85 in addition to its function in Pol II dislodgement at the end of the genes.…”

“…These insights bring a new perspective on the accepted view that Xrn2 chases down still transcribing Pol II after cleavage, and rather suggest that Xrn2 functions as a part of Pol II complex in a collaboration with Spt5. In agreement with our findings, recent studies that employed transcriptome analyses mapping Xrn2 entry sites and PAS usage proposed that Xrn2 acts in close association with Pol II throughout the transcription cycle and engages with the 5’end of the nascent RNA immediately after it is cleaved by CPA 52,85 . We show that Spt5 activates Xrn2 independently of its phosphorylation by Cdk9.…”

“…To study the nucleolytic activity of Xrn2 we expressed WT and catalytic mutant (Xrn2 M , D237A), in which a conserved aspartic acid located in the catalytic pocket of the enzyme was replaced with alanine (Figure 1D) 18,42,54,85,100,101 . To obtain a high yield of stable protein, both constructs were expressed without the C-terminal part (1-885 aa of Xrn2) as previously reported 101 .…”