Expression of Bacterial Rho Factor in Yeast Identifies New Factors Involved in the Functional Interplay between Transcription and mRNP Biogenesis

Abstract

In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events.The expression of protein-encoding genes in eukaryotic cells is a multistep process in which the genetic message is transcribed from DNA into a pre-mRNA molecule that undergoes 5Ј-end capping, splicing, 3Ј-end cleavage, and polyadenylation before being assembled into a ribonucleoprotein particle (mRNP) and exported to the cytoplasm for translation. Genetic and biochemical studies, using the yeast Saccharomyces cerevisiae as a model organism in particular, suggest that the various mRNA processing and packaging events known as mRNP biogenesis are coupled physically and functionally to transcript elongation. The current view is that the nascent transcript emerging from the transcription elongation complex is coated sequentially with a variety of proteins that ensure its integrity and mediate its maturation and transport from the site of transcription to the nuclear pore for export (2,29,68). The cotranscriptional assembly of export-competent mRNPs seems to be facilitated by the C-terminal domain of the largest subunit of RNA polymerase II (RNAP II), which serves as a platform for the sequential recruitment of various factors. Among the general factors that are important for the production of export-competent mRNPs in yeast, the four-subunit THO complex (Hpr1p, Mft1p, Tho2p, and Thp1p) associates with RNAP II at early steps of transcription elongation and facilitates the loading onto the nascent transcript of two mRNP export factors, Yra1p and Sub2p (1,7,71). The hexameric complex formed by THO, Yra1p, and Sub2p, termed the transcription and export complex (TREX) (64), recruits the heterodimeric export receptor Mex6...

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

“…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.…”

“…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.…”

“…14 In previous studies, the steady-state levels of a set of mRNA transcripts were shown to be affected by Rho activity with the greatest effect observed for PMA1 mRNA. 15 Further analyses, using PMA1 as a model transcript, substantiated the essential role of Rrp6p in the nuclear mRNP QC. 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.…”

“…The 18S rRNA is used as an internal control since we have previously shown that Rho activity had no detectable effect on the steady-state levels of Pol I and Pol III dependent transcripts. 15 In the course of these studies, we also conducted a partial microarray analysis to study the effect of Rho on a wider set of transcripts (unpublished results). The analysis was done on single samples as a custom service by Eurogenetec (Belgium) on spotted cDNA arrays.…”

“…15 Growth of the strains harboring the Rho expression plasmid was performed at 25 C in YNB medium (YNB with ammonium sulfate) supplemented with SC-TRP (Synthetic Complete Supplement Mixture from MP Biomedicals) and glucose as carbon source (final concentration 2%). For the comparative experiments, glucose was replaced by galactose at the same final concentration (2%).…”