tél : 02.38.25.55.85 - fax : 02.38.63.15.17
The co-transcriptional biogenesis of export-competent messenger ribonucleoprotein particles (mRNPs) in yeast is under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from inappropriate or inefficient processing and packaging reactions are detected by the QC system and retained in the nucleus with ensuing elimination of their mRNA component by a mechanism that requires the catalytic activity of Rrp6p, a 3’-5’ exonuclease associated with the RNA exosome. In previous studies, we implemented a new experimental approach in which the production of aberrant mRNPs is massively increased upon perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor expressed in S. cerevisiae. The analyses of a subset of transcripts such as PMA1 led us to substantiate the essential role of Rrp6p in the nuclear mRNP QC and to reveal a functional coordination of the process by Nrd1p. Here, we extended those results by showing that, in contrast to PMA1, Rho-induced aberrant HXK1 mRNPs are targeted for destruction by an Nrd1p- and Rrp6p-independent alternative QC pathway that relies on the 5’-3’ exonuclease activity of Rat1p. We show that the degradation of aberrant HXK1 mRNPs by Rat1p occurs co-transcriptionally following decapping by Dcp2p and leads to premature transcription termination. We discuss the possibility that this alternative QC pathway might be linked to the well-known specific features of the HXK1 gene transcription such as its localization at the nuclear periphery and gene loop formation.
The structure and dynamics of Escherichia coli transcription elongation complex are now well documented. However, most of the studies have been conducted in vitro and frequently under artificial conditions that facilitate the biochemical characterization of the complex. Thus, little is known about relevance of these results for the regulatory aspects of transcription elongation inside the cell. Here, we describe the use of a single-strand-specific probe chloroacetaldehyde for in situ footprinting of E. coli elongation complex temporarily halted by a protein roadblock. The method provides valuable information on the dynamic features of transcriptionally engaged RNA polymerase within the cellular environment
In eukaryotic cells, the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from faulty events are detected by the QC apparatus and retained in the nucleus with ensuing elimination of their mRNA component by the RNA degradation machinery. A decade of biochemical and genetic experiments in yeast allowed the identification of the nuclear degradation machinery including the core exosome and its two associated catalytic subunits Rrp6p and Rrp44p, its cofactors Rrp47p and Mpp6p as well as the activator complex TRAMP. Similarly, studies of the THO-Sub2 complex of the mRNP assembly and export apparatus have provided valuable information on the nuclear retention and degradation of a particular class of aberrant mRNPs. However, a unifying mechanism of action underlying the QC process remains elusive. Here, we review the implementation of a new experimental approach whereby the production of aberrant mRNPs is massively increased upon heterologous expression of the bacterial Rho helicase in yeast. Using this methodology, we have shown that the QC process is coordinated by Nrd1p (a component of the early termination complex) whose increased co-transcriptional recruitment promotes the attachment of the 3’-5’ exonuclease Rrp6p along with the co-factors Rrp47p and Mpp6p. Interestingly, we established that Rrp6p functions independently from the core exosome, yet is stimulated by two forms of the TRAMP complex that include Trf4p or Trf5p and Air2p but not Air1p. The results suggest that specific substrates could be primed for decay via various QC pathways owing to the versatility of the mRNA degradation apparatus. In this context, the bacterial Rho helicase provides a valuable tool to decipher the QC molecular process in yeast and possibly the homologous process in mammalian cells.
The co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent mRNPs are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p which is recruited co-transcriptionally in association with Nrd1p following Rho action. Here, we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p and Mpp6p as well as the TRAMP components Trf4p, Trf5p and Air2p contribute significantly by stimulating the degradation process upon their co-transcriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs.
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.
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.
Transcriptional pausing by RNA polymerase has been the subject of extensive investigations in vitro, yet little is known about its occurrence and significance in vivo. The transient nature of the pausing events makes them difficult to observe inside the cell, whereas their studies in vitro by classical biochemical methods are usually conducted under non-physiological conditions that increase the pause duration. Here, we have used an Escherichia coli system in which several RNA polymerase molecules transcribing in tandem traverse a pausing sequence while approaching a protein roadblock. The in vivo DNA footprinting and RNA 3’ end mapping of the elongation complexes are consistent with a dynamic view of the pausing event, during which RNA polymerase first loses its lateral stability and slides backward, and is subsequently rescued from extended backtracking and stabilized at the pause site by a nascent RNA hairpin. Our results show also that the folding of the hairpin provides an assisting force that promotes forward translocation of a trailing polymerase under a strained configuration by balancing the opposing force created by a steric clash with a leading elongation complex. (c) 2005 Elsevier Ltd. All rights reserved.
Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre- and post-translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low K-m). The addition of an extra nucleotide destabilizes the repressor-operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high K(m)s) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.
The GreA and GreB proteins of Escherichia coil show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock, When RNAP is stalled within an (ATC/TAG), sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3’ terminus and its re-synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced ’cleavage-and-restart’ process to become catalytically competent. Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.
Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses ren amongst others, Recently, several proteins have been shown to assist Dorsal in the repression of ten, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of ten but not in the activation of twist.
DSP1 is an HMG-box containing protein of Drosophila melanogaster which was first identified as a co-repressor of the Dorsal protein. Recently, the analysis of the structure of the gene has led vs to propose that DSP1 is the Drosophila equivalent of the ubiquitous vertebrate HMG 1/2 proteins. In the present paper, the patterns of expression of DSP1 protein and RNA in adult flies and during development are reported. in the adults DSP1 protein is located in nurse cells of ovaries and in brain. During eggs development uniform expression of DSP1 protein persists until the end of germband retraction.
Chargé de recherche , Biologie de l’ARN et ARN thérapeutiques