Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex

Clerici, Marcello; Faini, Marco; Muckenfuss, Lena M.; Aebersold, Ruedi; Jinek, Martin

doi:10.1038/s41594-017-0020-6

Cited by 107 publications

(153 citation statements)

References 28 publications

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“…Among the protein complexes contributing to 3' processing of mammalian mRNA precursors, CstF and CF I have previously been reconstituted from recombinant proteins (Takagaki and Manley 1994;Rüegsegger et al 1998;Yang et al 2011;Yang et al 2018). Of the central factor, CPSF, only the mPSF subcomplex, active in polyadenylation, has been reconstituted (Schönemann et al 2014;Clerici et al 2017;Clerici et al 2018;Sun et al 2018), but the subunit composition of CPSF is reasonably clear from biochemical experiments (Bienroth et al 1991;Shi et al 2009;Schönemann et al 2014). In contrast, CF II had never been purified to the point that its subunit composition could be judged.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Schaefer

Tüting

Schoenemann

et al. 2018

Preprint

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Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequencespecificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

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Section: Discussionmentioning

confidence: 99%

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Schaefer

Tüting

Schoenemann

et al. 2018

Preprint

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“…We and others recently reported the structures of a quaternary complex of human CPSF-160, CPSF-30, WDR33 and an AAUAAA PAS RNA (Figs. 1A, 1B) (18,19), the structure of a ternary complex of the yeast protein homologs (Cft1, Yth1 and Pfs2, without RNA) (20), as well as the structure of a binary complex of human CPSF-160 and WDR33 (21). The structures of the quaternary complexes revealed extensive and specific interactions between the AAUAAA PAS and WDR33 and CPSF-30 ( Fig.…”

Section: Introductionmentioning

confidence: 94%

“…A U3-A6 Hoogsteen base pair was observed in the PAS when bound to the CPSF-160-WDR33-CPSF-30 ternary complex (Fig. 1C) (18,19). The binding mode of the PAS suggests that other Hoogsteen base pairs, such as C3-G6, could be accommodated, while a wobble U3-G6 base pair would not fit.…”

Section: Variation Of the U3-a6 Hoogsteen Base Pairmentioning

confidence: 99%

“…Besides the U3-A6 Hoogsteen base pair, the structures show that A1 and A4 of the AAUAAA PAS are specifically recognized by CPSF-30, while A2 and A5 also establish favorable hydrogen-bonding interactions (18,19). To assess the binding affinity of other PAS hexamers that can also support 3¢-end processing, we selected from those identified in mammalian pre-mRNAs (7,8), changing each of these four positions at a time.…”

Section: Variation Of Other Positions Of the Aauaaa Pasmentioning

confidence: 99%

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Biophysical characterizations of the recognition of the AAUAAA polyadenylation signal

Hamilton

Sun

Tong

2018

Preprint

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Most eukaryotic messenger RNA precursors must undergo 3¢-end cleavage and polyadenylation for maturation. We and others recently reported the structure of the AAUAAA polyadenylation signal (PAS) in complex with the protein factors CPSF-30, WDR33 and CPSF-160, revealing the molecular mechanism for this recognition. Here we have characterized in detail the interactions between the PAS RNA and the protein factors using fluorescence polarization experiments. Our studies show that AAUAAA is recognized with ~1 nM affinity by the CPSF-160-WDR33-CPSF-30 ternary complex. Variations in the RNA sequence can greatly reduce the affinity. Similarly, mutations of residues that have van der Waals interactions with the bases of AAUAAA also lead to substantial reductions in affinity. Finally, our studies confirm that both CPSF-30 and WDR33 are required for binding the PAS RNA, and determine a ~7 nM affinity between CPSF-30 and the CPSF-160-WDR33 binary complex. Short title: Recognition of the AAUAAA polyadenylation signal

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“…Recent studies of the S. cerevisiae complex demonstrated that CPF is organised into three functionally different, stably associated modules: poly(A) polymerase, nuclease, and phosphatase (Casañal et al, 2017). Reconstitution and structural analysis of the mammalian polymerase module (CPSF160-WDR33-CPSF30-Fip1) have revealed that the zinc finger domains 2 and 3 of CPSF30, as well as the WD40 domain and the N-terminal region of WDR33, interact with the PAS element (Clerici et al, 2017(Clerici et al, , 2018Schönemann et al, 2014;Sun et al, 2018). Although RNA is not present in the cryo-EM structure of the S. cerevisiae polymerase module, its overall organisation is very similar to the mammalian module (Casañal et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Kilchert

Kecman

Priest

et al. 2019

Preprint

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Abbreviations: gene ontology (GO); messenger RNA (mRNA); mass spectrometry (MS); polyadenylation site (PAS); RNA-binding domain (RBD); RNA-binding protein (RBP);RNA interactome capture (RIC); ribonucleoprotein particle (RNP); ribosomal protein (RP); transcription elongation factor (TEF); wild-type (WT); whole cell extract (WCE); cleavage and polyadenylation factor (CPF); cleavage factors IA and IB (CFIA and CFIB) 2 Abstract Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNAbinding activity and provide key information about the RNA-binding properties of large multiprotein complexes, such as the mRNA 3' end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

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Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex

Cited by 107 publications

References 28 publications

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Biophysical characterizations of the recognition of the AAUAAA polyadenylation signal

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

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