Activation of the Endonuclease that Defines mRNA 3′ Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex

Hill, C.P.; Boreikaitė, Vytautė; Kumar, Aman; Casañal, Ana; Kubík, Peter; Degliesposti, Gianluca; Maslen, Sarah; Mariani, Angelica; Loeffelholz, Ottilie von; Girbig, Mathias; Skehel, Mark; Passmore, Lori A.

doi:10.1016/j.molcel.2018.12.023

Cited by 81 publications

(181 citation statements)

References 92 publications

(124 reference statements)

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“…cDNAs encoding full length Gallus gallus FANCI and FANCD2 were synthesized (GeneArt) and cloned into pACEBac1. The individual genes were amplified by PCR and cloned into pBIG1a vector using a modified version of the biGBac system, as previously described 42 , 43 . The combined vector was transformed into EMBacY E. coli competent cells for bacmid generation.…”

Section: Methodsmentioning

confidence: 99%

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

Alcón

Shakeel

Chen

et al. 2020

Nat Struct Mol Biol

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120

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Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryoEM to determine the structure of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.

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Section: Methodsmentioning

confidence: 99%

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

Alcón

Shakeel

Chen

et al. 2020

Nat Struct Mol Biol

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120

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“…For subsequent protein preparations, A-G-B-L-100-C-E-F, A-G-B-L-100, G-B-L-100 and C-E-F complexes were prepared using a modified BiGBac system as described previously 29,30 . The individual genes were PCR amplified for cloning into pBIG vectors from pACEBac1, pIDC or pIDS vectors.…”

Section: Cloning Expression and Purificationmentioning

confidence: 99%

Structure of the Fanconi anaemia monoubiquitin ligase complex

Shakeel

Rajendra

Alcón

et al. 2019

Nature

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113

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The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress 1,2. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer 1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCI-FANCD2 4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase 6,7. Monoubiquitinated FANCD2 then recruits enzymes to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryoEM) and mass spectrometry to determine its structure. The FA core complex is comprised of two central dimers of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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“…However, other CFIA subunits, Pcf11 and Clp1, as well as the fission yeast specific factor Ctf1, did not crosslink with poly(A)+ RNA. Recombinantly reconstituted CFIA and polymerase module were both found to interact with RNA (Hill et al, 2019). In our RIC experiment CFIA was less enriched on poly(A)+ RNA than those polymerase subunits that bound RNA ( Figure 5A), possibly because CFIA associates with RNA elements downstream of the cleavage site.…”

Section: Using Ric To Study the Topology Of Multi-protein Complexesmentioning

confidence: 83%

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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Activation of the Endonuclease that Defines mRNA 3′ Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex

Cited by 81 publications

References 92 publications

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

Structure of the Fanconi anaemia monoubiquitin ligase complex

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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