Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA742220 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14935652.fastq.gz | Fastqsanger.gz | |||
| SRR14935653.fastq.gz | Fastqsanger.gz | |||
| SRR14935654.fastq.gz | Fastqsanger.gz | |||
| SRR14935655.fastq.gz | Fastqsanger.gz | |||
| SRR14935656.fastq.gz | Fastqsanger.gz |
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Project description:Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3 end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways.
2022-01-20 | GSE179106 | GEO
Project description:Turnover of nucleoplasmic transcripts by the multi-subunit RNA exosome is mediated by two adaptors; the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely been based on long-term factor depletion strategies. Here, we rapidly deplete a NEXT, a PAXT and a core-exosome component, uncovering direct consequences of their losses. Global proteome analyses displayed only minor changes, yet exposed the immediate co-depletion of exosome and adaptor subunits, reflecting possible sub-complex compositions. Parallel high-resolution 3’end sequencing of newly synthesized RNA confirmed earlier established long-term depletion phenotypes. Still, rapid NEXT depletion displayed a narrower phenotype, illustrated by its restricted effect on snoRNA-hosting introns, whereas long-term NEXT depletion data had suggested a more general role of the complex in mediating intron turnover. While this validates snoRNA-hosting introns as primary NEXT targets, our data suggest that nuclear 5’-3’ RNA decay is altered after prolonged NEXT depletion, obscuring interpretation. Further analysis of snoRNA-hosting introns also uncovered an unusual mode of core exosome-independent RNA decay. Due to its non-processive nature, we speculate that this reflects a hitherto undisclosed exosome-independent activity of the EXOSC10 ribonuclease.
2021-12-23 | PXD026774 | Pride
Project description:Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3' end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways.
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Project description:The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.
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Project description:Purpose: Studying the dynamics of the transcriptional response to acute depletion of various critical mRNA processing factors Methods: We used 4tU labeling and high-throughput polyA RNA-seq in yeast following auxin inducible degradation of key proteins. Results: We captured the detailed dynamics of cells’ adaptation to rapid degradation of Xrn1 and observed a significant accumulation of mRNA, followed by a delayed global reduction in nascent transcription and a gradual return to baseline mRNA levels. We present evidence that this transcriptional response is linked to cell cycle progression, and that it is not unique to Xrn1 depletion; rather, it is induced earlier when upstream factors in the 5'-3’ degradation pathway are perturbed. Conclusions: Our data suggest that the RNA feedback mechanism is cell-cycle-linked and monitors the accumulation of inputs to the 5’-3’ exonucleolytic pathway rather than its outputs.
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