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Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs
Claire Torchet1, Cecile Bousquet-Antonelli, Laura Milligan
1Wellcome Trust Centre for Cell Biology, University of Edinburgh, EH9 3JR Edinburgh, Scotland, UK.
Abstract:
Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination. Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p. The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype. Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation. Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation. The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media.
Insights
Mutations in RNA14 and RNA15 disrupt mRNA processing. The exosome and RNA helicase Dob1p/Mtr4p stabilize read-through transcripts, while Rrp6p stabilizes functional mRNAs, with degradation favored on glucose.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Processing
Background:
- RNA processing, including 3' cleavage and polyadenylation, is crucial for gene expression.
- The exosome complex and associated factors like Rrp41p and Dob1p/Mtr4p play key roles in RNA degradation and processing.
- Nuclear-specific exosome components, such as Rrp6p, have distinct functions in RNA metabolism.
Purpose of the Study:
- To investigate the roles of RNA14, RNA15, Rrp41p, Dob1p/Mtr4p, and Rrp6p in pre-mRNA processing and stability.
- To elucidate the mechanisms regulating the balance between degradation and processing of pre-mRNAs.
- To understand how cellular conditions, like glucose availability, influence these regulatory pathways.
Main Methods:
- Analysis of yeast strains with specific mutations (rna14.1, rna15.2).
- Depletion of key proteins (Rrp41p, Dob1p/Mtr4p, Rrp6p) using conditional expression systems.
- Assessment of pre-mRNA stability, polyadenylation status, and functionality for translation.
- Investigation of the impact of media composition (glucose) on RNA processing outcomes.
Main Results:
- Strains with rna14.1 and rna15.2 mutations exhibit defects in pre-mRNA 3' cleavage, polyadenylation, and transcription termination.
- Depletion of Rrp41p or Dob1p/Mtr4p stabilizes long, read-through transcripts in these mutant strains.
- Absence of Rrp6p in rna14.1 strains leads to stabilization of short, polyadenylated, and translatable pre-mRNAs.
- These Rrp6p-dependent mRNAs are products of exosome processing followed by uncoupled polyadenylation and their production is suppressed by Rrp41p depletion.
- Degradation of 3'-unprocessed pre-mRNAs is favored under glucose-rich conditions.
Conclusions:
- RNA14 and RNA15 are essential for proper pre-mRNA 3' end formation and transcription termination.
- The exosome complex (Rrp41p) and RNA helicase (Dob1p/Mtr4p) are involved in degrading aberrant transcripts.
- Rrp6p plays a critical role in processing and stabilizing functional mRNAs, potentially through a pathway distinct from complete degradation.
- A regulatory balance exists between RNA degradation and processing to functional mRNA, influenced by environmental cues like glucose availability.