Studies with recombinant U7 snRNP demonstrate that CPSF73 is both an endonuclease and a 5′–3′ exonuclease

Abstract

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3 ′ ′ ′ ′ ′ end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonuclease, consistent with the activity of other members of the β-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.

Reconstitution of 3’-processing of mammalian pre-mRNA reveals a central role of RBBP6

“…5C). Thus, the cleavage module lacking CstF64 is fully competent for pre-mRNA cleavage, as also observed in histone 3' processing (Sun et al, 2020;Yang et al, 2020). In XL-MS experiments employing the amine-reactive cross-linker DSBU (Muller et al, 2010) in the context of a CM3-containing cleavage reaction, the dominant partners of CstF64 were the other two CstF subunits, but cross-links to the CM subunit symplekin were also reproducibly observed (Suppl.…”

“…The second subcomplex of CPSF has been termed ‘mammalian cleavage factor’ (mCF) (Sun et al, 2020; Zhang et al, 2020), but we will use the term ‘cleavage module’ (Schönemann et al, 2014) to avoid confusion with cleavage factors CF I and CF II. The cleavage module is composed of the endonuclease CPSF73, the related but catalytically inactive protein CPSF100, symplekin, and perhaps CstF64 (Mandel et al, 2006; Sullivan et al, 2009; Sun et al, 2020; Yang et al, 2020; Zhang et al, 2020). CF I consists of a dimer of CF I-25 and two copies of CF I-59 or CF I-68 (Kim et al, 2010; Ruegsegger et al, 1998; Yang et al, 2011) and recognizes UGUA motifs upstream of the polyadenylation signal (Brown and Gilmartin, 2003; Venkataraman et al, 2005; Yang et al, 2011; Yang et al, 2010).…”

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

“…Previous PARE studies have used xrn1 single mutants or depletion, and we reasoned that adding rat1-1 would expand the degradome studies to nuclear events. Other 5′ exonucleases (e.g., Dxo1, Rai1, Rrp17, Ysh1) have been found to play minor roles in RNA degradation and do not interfere with PARE (41)(42)(43)(44). We also included xrn1Δ rrp44-endo − and xrn1Δ rat1-1 rrp44-endo − strains that additionally lack the endonuclease activity of the Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway RNA exosome.…”

β-CASP proteins removing RNA polymerase from DNA: when a torpedo is needed to shoot a sitting duck

“…Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage by CPSF73 guided by the U7 snRNP that binds downstream of the cleavage site. Following the cleavage, the downstream cleavage product (DCP) is rapidly degraded by the 5′–3′ exonuclease activity of CPSF73 and is functionally linked to the release of Pol II from histone genes ( 84 , 85 ). Analogously, the processing of snRNAs utilizes the Integrator complex subunit 11 (IntS11; member of the β-CASP family) for the endonucleolytic cleavage of the nascent transcript (Figure 2C ) ( 86 ) and the degradation of DCP was demonstrated to be independent of, or only modestly affected by Xrn2 ( 76 , 87 ).…”

“…Analogously, the processing of snRNAs utilizes the Integrator complex subunit 11 (IntS11; member of the β-CASP family) for the endonucleolytic cleavage of the nascent transcript (Figure 2C ) ( 86 ) and the degradation of DCP was demonstrated to be independent of, or only modestly affected by Xrn2 ( 76 , 87 ). The exonuclelytic activity of CPSF73/IntS11 was previously proposed as one of the possible ways to terminate Pol II ( 84 , 85 , 88–90 ). Moreover, IntS11 is involved in promoter-proximal premature termination of hundreds of protein-coding genes, releasing paused/stalled RNAPs ( 90 ).…”