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.

Comparison of the yeast and human nuclear exosome complexes

2012 ◽  
Vol 40 (4) ◽  
pp. 850-855 ◽  
Author(s):  
Katherine E. Sloan ◽  
Claudia Schneider ◽  
Nicholas J. Watkins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Processing ◽  
Eukaryotic Cells ◽  
Multiprotein Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Surveillance ◽  
Mature Transcript ◽  
Nuclear Exosome ◽  
And Function ◽  
Rna Maturation

Most RNAs in eukaryotic cells are produced as precursors that undergo processing at the 3′ and/or 5′ end to generate the mature transcript. In addition, many transcripts are degraded not only as part of normal recycling, but also when recognized as aberrant by the RNA surveillance machinery. The exosome, a conserved multiprotein complex containing two nucleases, is involved in both the 3′ processing and the turnover of many RNAs in the cell. A series of factors, including the TRAMP (Trf4–Air2–Mtr4 polyadenylation) complex, Mpp6 and Rrp47, help to define the targets to be processed and/or degraded and assist in exosome function. The majority of the data on the exosome and RNA maturation/decay have been derived from work performed in the yeast Saccharomyces cerevisiae. In the present paper, we provide an overview of the exosome and its role in RNA processing/degradation and discuss important new insights into exosome composition and function in human cells.

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 The CTEXT complex in Saccharomyces cerevisiae plays a crucial role in degrading distinct sets of aberrant mRNAs by the nuclear exosome

2021 ◽  
Author(s):  
Upasana Saha ◽  
Rajlaxmi Gaine ◽  
Sunirmal Paira ◽  
Satarupa Das ◽  
Biswadip Das
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Impairment ◽  
Rna Helicase ◽  
Vital Role ◽  
Genomic Deletions ◽  
The Core ◽  
Rrm Domain ◽  
Dead Box ◽  
Nuclear Exosome ◽  
Exosome Complex

AbstractIn Saccharomyces cerevisiae, DRN (Decay of RNA in the Nucleus) requiring Cbc1/2p, Tif4631p, and Upf3p promotes the exosomal degradation of aberrantly long 3′-extended-, export-defective transcripts and a small group of normal (special) mRNAs. In this study, using a systematic proteomic analysis we show that each of the known components interacts with one another and they exist as a separate complex, which was dubbed CTEXT (CBC-Tif4631p-dependent EXosome Targeting). We also identified a DEAD-box RNA helicase Dbp2p as an additional novel component of CTEXT during this analysis which was further bolstered by the finding that genomic deletions of Dbp2p led to the stabilization of all the signature nuclear messages. Interestingly, the RRM domain of Tif4631p located at the extreme N-termini of this polypeptide was found to play a vital role in in mediating the interaction of the CTEXT with the core exosome complex. These inferences were substantiated by the finding that deletion of this domain led to the functional impairment of the CTEXT complex. Thus, the CTEXT constitutes an independent complex that assists the nuclear exosome in degrading the select classes of nuclear transcripts in Saccharomyces cerevisiae.

scholarly journals 5-Fluorouracil Enhances Exosome-Dependent Accumulation of Polyadenylated rRNAs

2004 ◽  
Vol 24 (24) ◽  
pp. 10766-10776 ◽  
Author(s):  
Feng Fang ◽  
Jason Hoskins ◽  
J. Scott Butler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
General Result ◽  
Rna Processing ◽  
Ribosome Biogenesis ◽  
Dna Microarrays ◽  
Rrna Processing ◽  
Genome Wide ◽  
Genes Encoding ◽  
Nuclear Exosome

ABSTRACT The antimetabolite 5-fluorouracil (5FU) is a widely used chemotherapeutic for the treatment of solid tumors. Although 5FU slows DNA synthesis by inhibiting the ability of thymidylate synthetase to produce dTMP, the drug also has significant effects on RNA metabolism. Recent genome-wide assays for 5FU-induced haploinsufficiency in Saccharomyces cerevisiae identified genes encoding components of the RNA processing exosome as potential targets of the drug. In this report, we used DNA microarrays to analyze the effect of 5FU on the yeast transcriptome and found that the drug causes the accumulation of polyadenylated fragments of the 27S rRNA precursor and that defects in the nuclear exoribonuclease Rrp6p enhance this effect. The size distribution of these RNAs and their sensitivity to Rrp6p suggest that they are normally degraded by the nuclear exosome and a 5′-3′ exoribonuclease. Consistent with this hypothesis, 5FU inhibits the growth of RRP6 mutants with defects in the degradation function of the enzyme and it interferes with the degradation of an rRNA precursor. The detection of poly(A)+ pre-RNAs in strains defective in various steps in ribosome biogenesis suggests that the production of poly(A)+ pre-rRNAs may be a general result of defects in rRNA processing. These findings suggest that 5FU inhibits an exosome-dependent surveillance pathway that degrades polyadenylated precursor rRNAs.

Mechanisms of Nuclear Transport in the cell: RNA exosome in Saccharomyces cerevisiae

Bionatura ◽  
2020 ◽  
Vol 5 (4) ◽  
pp. 1423-1426
Author(s):  
Bruna Rech ◽  
Fernando A. Gonzales-Zubiate
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Activity ◽  
Rna Processing ◽  
Cellular Localization ◽  
Nuclear Transport ◽  
Single Units ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Transcripts ◽  
Non Coding Rnas ◽  
Rna Exosome

Ribonucleases (RNases) functions in the cell include precise maturation of non- coding RNAs and degradation of specific RNA transcripts that are no longer necessary. RNAses are present in the cell as single units or assembled as multimeric complexes; one of these complexes is the RNA exosome, a highly conserved complex essential for RNA processing and degradation. In the yeast Saccharomyces cerevisiae, the RNA exosome comprises eleven subunits, two with catalytic activity: Rrp6 and Rrp44, where the Rrp6 subunit is exclusively nuclear. Despite the RNA exosome has been intensively investigated since its discovery in 1997, only a few studies were accomplished concerning its nuclear transport. This review describes recent research about cellular localization and transport of this essential complex.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

1992 ◽  
Vol 12 (6) ◽  
pp. 2561-2569 ◽  
Author(s):  
L L Stohl ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Processing ◽  
Mitochondrial Rna ◽  
Rna Sequence ◽  
Rnase Mrp ◽  
Processing Activity ◽  
Yeast Mitochondrial Dna ◽  
Human And Mouse

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

A synthetic intron in a naturally intronless yeast pre-tRNA is spliced efficiently in vivo

1989 ◽  
Vol 9 (1) ◽  
pp. 329-331
Author(s):  
M Winey ◽  
I Edelman ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Single Copy ◽  
Single Copy Gene ◽  
Copy Gene

Saccharomyces cerevisiae glutamine tRNA(CAG) is encoded by an intronless, single-copy gene, SUP60. We have imposed a requirement for splicing in the biosynthesis of this tRNA by inserting a synthetic intron in the SUP60 gene. Genetic analysis demonstrated that the interrupted gene produces a functional, mature tRNA product in vivo.

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

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