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RNA-seq reveals that Air1 and Air2 control TRAMP substrate specificity for nuclear RNA surveillance

ABSTRACT: The Saccharomyces cerevisiae TRAMP4 and TRAMP5 complexes, which consist of the poly(A) polymerase Trf4 or Trf5, respectively, the zinc knuckle proteins Air1 or Air2, and the RNA helicase Mtr4, play a critical role in nuclear RNA surveillance. Although it is known to enhance the nuclease activity of the exosome, relatively little is known about the exact mechanism and specificity of TRAMP. To better define the specificities of the TRAMP complexes, we used phenotypic analysis and RNA deep-sequencing technology to measure differences in global RNA polyadenylation in air mutants, revealing specific requirements for each Air protein in the regulation of the levels of non-coding and coding RNAs. These findings reveal differential functions for Air proteins in eukaryotic RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE42231 | GEO | 2012/11/14

SECONDARY ACCESSION(S): PRJNA179372

REPOSITORIES: GEO

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RNA-seq reveals that Air1 and Air2 control TRAMP substrate specificity for nuclear RNA surveillance

Project description:The Saccharomyces cerevisiae TRAMP4 and TRAMP5 complexes, which consist of the poly(A) polymerase Trf4 or Trf5, respectively, the zinc knuckle proteins Air1 or Air2, and the RNA helicase Mtr4, play a critical role in nuclear RNA surveillance. Although it is known to enhance the nuclease activity of the exosome, relatively little is known about the exact mechanism and specificity of TRAMP. To better define the specificities of the TRAMP complexes, we used phenotypic analysis and RNA deep-sequencing technology to measure differences in global RNA polyadenylation in air mutants, revealing specific requirements for each Air protein in the regulation of the levels of non-coding and coding RNAs. These findings reveal differential functions for Air proteins in eukaryotic RNA metabolism and indicate that they control the substrate specificity of the RNA exosome. Poly(A)+ RNA from WT, rrp6-M-NM-^T, air1-M-NM-^T rrp6-M-NM-^T and air2-M-NM-^T rrp6-M-NM-^T was sequenced using ABI SOLiD platform, in duplicate.

2012-11-14 | E-GEOD-42231 | biostudies-arrayexpress

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

Project description:During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed coenrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

2020-07-02 | PXD017114 | Pride

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

Project description:During nuclear surveillance, the RNA exosome functions together with the TRAMP complexes, which include the RNA helicase Mtr4 together with a RNA binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions by TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. Comparison of the binding sites of TRAMP components with multiple nuclear RNA binding proteins revealed preferential colocalization of subsets of factors. On pre-mRNAs, ∆trf5 deletion caused reduced Mtr4 binding and RNA accumulation of Trf5 top targets.

2020-04-20 | GSE135526 | GEO

Genes identified by poly(A)+ transcriptome sequencing as overexpressed in trf5-Δ98-117

Project description:Purpose: To study the effects of disrupting the Trf5-Mtr4 interaction Methods: Analyzed TRF5 and trf5-Δ98-117 strains by transcriptome sequencing of poly(A)+ RNA Results: The set of most significantly affected genes included 71 that were overexpressed in trf5-Δ98-117 and only 5 that were down-regulated.The set of 71 overexpressed genes was predominated by known TRAMP substrates Conclusions: Disruption of the Trf5-Mtr4 interaction results in a snoRNA processing defect

2016-06-29 | GSE83827 | GEO

Genes identified by poly(A)+ transcriptome sequencing as overexpressed in trf5-Î?98-117

Project description:Purpose: To study the effects of disrupting the Trf5-Mtr4 interaction Methods: Analyzed TRF5 and trf5-Î?98-117 strains by transcriptome sequencing of poly(A)+ RNA Results: The set of most significantly affected genes included 71 that were overexpressed in trf5-Î?98-117 and only 5 that were down-regulated.The set of 71 overexpressed genes was predominated by known TRAMP substrates Conclusions: Disruption of the Trf5-Mtr4 interaction results in a snoRNA processing defect Duplicate RNA samples of trf4Î?, trf5Î? yeast strains expressing either TRF5 or trf5-Î?98-117 were enriched for poly(A)+ transcripts and converted to a sequencing library. Transcriptome sequencing was performed on a HiSeq 2500. Each sequenced library yielded between 10 and 14 million 50nt reads that were mapped to the annotated yeast genes using Bowtie. Significantly up- or down-regulated genes in the trf5-Î?98-117 mutant were identified via edgeR.

2016-06-29 | E-GEOD-83827 | biostudies-arrayexpress

The Nuclear Exosome is Active and Important during Budding Yeast Meiosis

Project description:We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). One sample each of Cbc2-associated RNA from wild-type and trf4-deleted cells at 6 hours of meiosis

2014-08-09 | E-GEOD-60221 | biostudies-arrayexpress

The Nuclear Exosome is Active and Important during Budding Yeast Meiosis

2014-08-09 | GSE60221 | GEO

Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast

Project description:In budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprograming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II (Pol II) and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV-crosslinking immediately following glucose withdrawal (0, 4, and 8 minutes). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. This indicates transcription termination followed by TRAMP-mediated RNA decay. Upregulated transcripts generally showed low or strongly reduced binding of Nab3 and Mtr4. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability.

2017-01-01 | GSE86483 | GEO

MPP6 stimulates both RRP6 and DIS3 to degrade a specified subset of MTR4-sensitive substrates in the human nucleus

Project description:Recent in vitro reconstitution analyses have proven that the physical interaction between the exosome core and MTR4 helicase, which promotes the exosome activity, is maintained by either MPP6 or RRP6. However, knowledge regarding the function of MPP6 with respect to in vivo exosome activity remains scarce. Here we demonstrate a facilitative function of MPP6 that composes a specific part of MTR4-dependent substrate decay by the human exosome. Using RNA polymerase II-transcribed poly(A)+ substrate accumulation as an indicator of a perturbed exosome, we found functional redundancy between RRP6 and MPP6 in the decay of these poly(A)+ transcripts. MTR4 binding to the exosome core via MPP6 was essential for MPP6 to exert its redundancy with RRP6. However, at least for the decay of our identified exosome substrates, MTR4 recruitment by MPP6 was not functionally equivalent to recruitment by RRP6. Genome-wide classification of substrates based on their sensitivity to each exosome component revealed that MPP6 deals with a specific range of substrates, and highlights the importance of MTR4 for their decay. Considering recent findings of competitive binding to the exosome between auxiliary complexes, our results suggest that the MPP6-incorporated MTR4-exosome complex is one of multiple alternative complexes rather than the prevailing one.

2022-06-19 | GSE184274 | GEO

Yeast exosome: immunoprecipitation 1

Project description:Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unstable transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis which are undetectable in wild-type cells, suggesting that the nuclear exosome remains functional, and we further show that the meiotic exosome complex still contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

2015-05-15 | PXD001223 | Pride

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