Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome
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ABSTRACT:
PROVIDER: PRJNA673126 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12931405_1.fastq.gz | Fastqsanger.gz | |||
| SRR12931405_2.fastq.gz | Fastqsanger.gz | |||
| SRR12931406_1.fastq.gz | Fastqsanger.gz | |||
| SRR12931406_2.fastq.gz | Fastqsanger.gz | |||
| SRR12931407_1.fastq.gz | Fastqsanger.gz |
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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:Quality control requires discrimination between functional and aberrant species to selectively target aberrant substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation followed by 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 protect stable RNA from degradation while selectively targeting and degrading less stable RNA. Substrate discrimination is lost when the distributive exoribonuclease activity of Rrp6 is inactivated, leading to degradation of stable and unstable RNA species. These data support a proofreading mechanism in which deadenylation by Rrp6 competes with Mtr4-dependent degradation to protect stable RNA while selectively targeting and degrading unstable RNA.
| 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: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: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. 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:TRAMP is a eukaryotic protein complex with RNA helicase and polyadenylase activity that marks and delivers a variety of RNA substrates to the nuclear exosome for degradation. Prior work has characterized binding of Trf4 and Air2 to the helicase core of Mtr4 and the regulation of both enzymatic activities in TRAMP. Air2 contains a conserved sequence that is predicted to bind the arch domain of Mtr4, though this interface is dispensable for TRAMP formation. To understand the interactions of the Saccharomyces cerevisiae TRAMP complex and its interactions with RNA, we have applied hydrogen-deuterium exchange mass spectrometry. We demonstrate that Air2 simultaneously binds the helicase core and the fist of Mtr4 in solution, constraining the arch to a conformation ready to bind RNA. We characterize the previously unknown interactions of Trf4 and Air2 with RNA and show how interactions with substrates are altered in TRAMP, supporting a mechanistic model for the enhancement of Mtr4 activity in TRAMP.
2024-12-11 | PXD053773 | Pride
Project description:In this dataset we show transcriptional profiles derived from adenocarcinoma or incipient neuroendocrine prostate cancer (NEPC) derived from TRAMP or SPARC-deficient TRAMP mice respectively. Genetic ablation of SPARC rendered prostate cancer cells to be prone to NEPC differentiation.
2024-07-01 | GSE242811 | 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 Comparison of polymorphic vs non-polymorphic genomes when hybridrized to short probe microarrays
2010-05-18 | E-GEOD-19319 | biostudies-arrayexpress