scholarly journals Mapping domains of ARS2 critical for its RNA decay capacity

2020 ◽  
Vol 48 (12) ◽  
pp. 6943-6953 ◽  
Author(s):  
Mireille Melko ◽  
Kinga Winczura ◽  
Jérôme Olivier Rouvière ◽  
Michaela Oborská-Oplová ◽  
Pia K Andersen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Rna Helicase ◽  
Rna Decay ◽  
Essential Properties ◽  
Nuclear Rna ◽  
Cap Binding Complex ◽  
Terminal Amino ◽  
Targeting Ability ◽  
Destructive Processes

Abstract ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5′cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.

scholarly journals Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Elizabeth V Wasmuth ◽  
John C Zinder ◽  
Dimitrios Zattas ◽  
Mom Das ◽  
Christopher D Lima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Central Region ◽  
Rna Processing ◽  
Rna Helicase ◽  
Rna Decay ◽  
Central Channel ◽  
Nuclear Rna ◽  
Nuclear Exosome

Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model.

scholarly journals The human ZC3H3 and RBM26/27 proteins are critical for PAXT-mediated nuclear RNA decay

2020 ◽  
Vol 48 (5) ◽  
pp. 2518-2530 ◽  
Author(s):  
Toomas Silla ◽  
Manfred Schmid ◽  
Yuhui Dou ◽  
William Garland ◽  
Miha Milek ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Rna Decay ◽  
Finger Protein ◽  
Nuclear Rna ◽  
Rna Exosome

Abstract Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

scholarly journals Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 31
Author(s):  
Ann Christina Bergmann ◽  
Cecilie Kyllesbech ◽  
Rimantas Slibinskas ◽  
Evaldas Ciplys ◽  
Peter Højrup ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Biological Function ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Therapeutic Target ◽  
Charged Amino Acids ◽  
Chaperone Protein ◽  
Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

scholarly journals TERMINAL AMINO ACIDS OF RHODOPSIN

1957 ◽  
Vol 229 (1) ◽  
pp. 477-487
Author(s):  
Genia Albrecht
Keyword(s):  
Amino Acids ◽  
Terminal Amino

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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