Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome
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PROVIDER: PRJNA673126 | ENA |
REPOSITORIES: ENA
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Project description:Abstract: Quality control requires discrimination between functional and aberrant species to selectively target substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation and helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to selectively target and degrade an unmodified tRNA while leaving native tRNA intact. Inactivation of the distributive exoribonuclease activity of Rrp6 results in loss of substrate discrimination, leading to degradation of all RNAs. These data suggest that the activities of the Mtr4 helicase and Rrp6 exoribonuclease endow the TRAMP-RNA exosome complex with the ability to protect stable RNA while degrading defective RNA species. Rrp6 and its 3′-5′ exonuclease activity have previously been shown to contribute to quality control and processing of various types of nuclear RNAs. Our sequencing analysis further confirms that Rrp6 contributes to this process.
2021-03-18 | GSE160368 | GEO
Project description: Not available
| S-EPMC8040639 | biostudies-literature
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
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
Project description:Substrate Specificity of the TRAMP Nuclear Surveillance Complexes
| PRJNA559170 | ENA
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.
2012-11-14 | GSE42231 | GEO
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
Project description:The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g. heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate that using such a microarray we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy number variation detection and gene expression analysis. Keyword(s): MutationDetection_CGH Comparison of polymorphic vs non-polymorphic genomes when hybridrized to short probe microarrays
2010-05-18 | E-GEOD-19319 | biostudies-arrayexpress
Project description:Gene expression profile of Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) reveals murine targets for preclinical development of human prostate cancer therapy In this study, we have generated an open source TRAMP microarray dataset to identify differentially expressed genes from human prostate cancer that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. Affymetrix Mouse 430 2.0 chips were used for microarrays of total RNA from 9 TRAMP tumors and 9 normal prostates (ventral and dorsolateral lobes). Keywords: prostate cancer, TRAMP
2008-12-31 | GSE10525 | GEO
Project description:The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g. heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate that using such a microarray we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy number variation detection and gene expression analysis. Keyword(s): MutationDetection_CGH
2009-12-10 | GSE19319 | GEO