RBBP6 activates the pre-mRNA 3′ end processing machinery in humans

Abstract

3′ end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′ end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3′ endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.

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

“…However, ATP hydrolysis, a reaction similar to the kinase activity of hClp1, might facilitate some aspect of the processing reaction that is not assayed in our experiments, in which nonhydrolyzable analogs were functional. In parallel work, Passmore and colleagues ( Boreikaite et al 2022 ) have reconstituted 3′ cleavage of human pre-mRNA under conditions not requiring the presence of ATP. It remains to be determined which features of the reaction are responsible for the difference in ATP dependence.…”

Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3’ end biogenesis

“…To perform the coupled reaction steps of cleavage and polyadenylation, the 3’ end processing machinery likely undergoes a sequence of conformational and compositional rearrangements as polyadenylation site recognition by the mPSF module of CPSF and activation by RBBP6 triggers CstF- dependent cleavage by the mCF, after which the nascent 3’ end needs to be made accessible to PAP for subsequent poly(A) synthesis (Boreikaite et al, 2022; Schmidt et al, 2022). Based on our structural and biochemical findings, we propose a model in which hFip1 acts as a coordinator of the two steps of 3’ end processing.…”

“…RBBP6 associates with mCF and is essential for pre-mRNA cleavage (Di Giammartino et al, 2014;Boreikaite et al, 2022;Schmidt et al, 2022). Within mPSF, the CPSF160-WDR33 subcomplex forms a rigid scaffold (Clerici et al, 2017) that interacts with CPSF30 (Clerici et al, 2018;Sun et al, 2018;Zhang et al, 2019) and the CPSF100 subunit of mCF (Zhang et al, 2019).…”

“…The PAS is specifically recognized by CPSF (Chan et al, 2014; Schönemann et al, 2014; Clerici et al, 2018; Sun et al, 2018), which consists of two functional modules: the mammalian polyadenylation specificity factor (mPSF) comprising subunits CPSF160, WDR33, CPSF30 and hFip1 (for h uman factor interacting with p oly(A) polymerase 1) (Bienroth et al, 1991; Murthy and Manley, 1992; Kaufmann et al, 2004; Shi et al, 2009), and the mammalian cleavage factor (mCF) containing the endonuclease CPSF73 (Mandel et al, 2006), CPSF100 as well as Symplekin (Sullivan et al, 2009). RBBP6 associates with mCF and is essential for pre-mRNA cleavage (Di Giammartino et al, 2014; Boreikaite et al, 2022; Schmidt et al, 2022). Within mPSF, the CPSF160-WDR33 subcomplex forms a rigid scaffold (Clerici et al, 2017) that interacts with CPSF30 (Clerici et al, 2018; Sun et al, 2018; Zhang et al, 2019) and the CPSF100 subunit of mCF (Zhang et al, 2019).…”

The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

“…Transcription termination is generally accompanied by the 3′-end processing of transcripts. Such processing of genes producing polyadenylated mRNA is achieved by multimeric cleavage and polyadenylation-specific factors (CPSFs), consisting of CPSF1 (CPSF160), CPSF2 (CPSF100), CPSF3 (CPSF73), CPSF4 (CPSF30), hFip1 and WDR33, and multimeric cleavage stimulation factors (CstFs), which produce poly(A) tail transcripts 6 – 8 . In contrast, the 3′-end processing of replication-dependent histone (RDH) and snRNA genes, which produce non-polyadenylated mRNAs, is achieved by different sets of 3′-end processing factors 2 , 9 .…”