Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts

Abstract

The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery.

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

“…In our previous studies, we monitored the effect of Rho activity by measuring the steady-state levels of a set of widely-studied yeast mRNAs (PMA1, PGK1, FBA1 and ACT1) and we found that PMA1 was the most affected with a 60% to 70% reduction, which led us to use it as a model transcript for further characterization of the QC system. 15,16 As shown by Northern blot analysis and RT-qPCR quantifications in Fig. 1, a large drop in the level of PMA1 mRNA is observed under Rho inducing conditions and the absence of the exonuclease Rrp6p in the rrp6D strain restores the PMA1 mRNA to nearly normal level.…”

“…Our previous analyses by the Rho-based approach of a subset of commonly-studied yeast transcripts such as PMA1 and PGK1 substantiated the essential role of Rrp6p in the elimination of aberrant mRNPs and revealed the implication of Nrd1p-Nab3p complex in the activation of the QC process. [15][16][17] However, in the course of those studies we noticed that the HXK1 transcript, which is also strongly affected by Rho action, was not rescued from degradation upon alteration of the Nrd1p-Nab3p complex or removal of Rrp6p and its activating cofactors. In the present work, we show that the elimination of Rho-induced aberrant HXK1 mRNPs is in fact mediated by an alternative nuclear QC pathway that relies on the degradation activity of the 5 0 -3 0 exonuclease Rat1p.…”

“…Upon its recruitment, Nrd1p forms a heterodimer with Nab3p which also binds to specific RNA sequences via its own RRM and thus strengthen the interaction of the complex with the nascent transcript. 16 The requirement for Nrd1p recruitment to target Rho-induced aberrant mRNPs is clearly seen for PMA1 in Fig. 2A where most of the transcript is recovered under Rho inducing conditions upon deletion at the chromosomal locus of the CID domain (nrd1D6-150 strain) or the domain interacting with Nab3p (nrd1D151-214 strain) from the Nrd1p protein.…”

“…16,17 with the following minor modifications to the reported protocol: noninduced cells were grown in selective medium containing 5 mg/ ml of Doxycycline (final concentration) and the eluted DNA (50 mL) was purified using the Thermo Scientific PCR cleanup kit as recommended by the manufacturer. Anti-RNA polymerase II (Pol II) antibodies (8WG16) were obtained from Covance and the anti-Myc antibodies (sc-40 X) were bought from Santa Cruz Biotechnology.…”

“…Moreover, the results revealed that the QC process is coordinated by Nrd1p (a component of the early termination complex, Nrd1p-Nab3p) whose increased co-transcriptional recruitment promotes the attachment of Rrp6p to the transcription complex along with its cofactors Rrp47p and Mpp6p as well as the co-activator TRAMP complex. 16,17 A limited microarray analysis measuring differential steady-state mRNA levels in the absence and the presence of Rho activity (unpublished results) revealed HXK1 (hexokinase isoenzyme 1) as a transcript that was also highly sensitive to Rho action. In the present work, we investigated the mechanism underlying Rho-mediated HXK1 decay.…”