Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

Foucher, Anne-Emmanuelle; Touat‐Todeschini, Leila; Juarez-Martinez, Ariadna B.; Laroussi, Hamida; Karczewski, Claire; Acajjaoui, Samira; Soler-López, Montserrat; Cusack, Stephen; Mackereth, Cameron D.; Verdel, André; Kadlec, Jan

doi:10.1101/2022.01.19.476877

Cited by 2 publications

(2 citation statements)

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“…Red1 directly interacts with each submodule of the MTREC complex, including Pla1, and connects them to the Mtl1 helicase to deliver the associated RNA cargo to the exosome (ref. 29,30 , Supplementary Fig. 1A).…”

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confidence: 99%

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

et al. 2023

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The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.

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Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

et al. 2023

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“…As a proof of concept, we focused on human ZFC3H1, a 200 kDa protein that together with MTR4 forms the heterotetrameric poly(A) tail exosome targeting (PAXT) complex, which directs a subset of long polyadenylated poly(A) RNAs for exosomal degradation (Fig. 6A and ref( 31, 32 )). We designed binders against ZFC3H residues 594-620 (PLP 4 PEDPEQPPKPPF) which lie within a ∼100 residue disordered region (Fig.…”

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De novodesign of modular peptide binding proteins by superhelical matching

Bai

Chang

et al. 2022

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General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptide backbone polar groups. Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity, we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide; the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW-- in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein side chain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occurring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

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Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

Cited by 2 publications

References 56 publications

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

De novodesign of modular peptide binding proteins by superhelical matching

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