A. Rachid Rahmouni

The eukaryotic THO complex coordinates the assembly of so-called messenger RNA–ribonucleoprotein particles (mRNPs), a process that involves cotranscriptional coating of nascent mRNAs with proteins. Once formed, mRNPs undergo a quality control step that marks them either for active transport to the cytoplasm, or Rrp6/RNA exosome-mediated degradation in the nucleus. However, the mechanism behind the quality control of nascent mRNPs is still unclear. We investigated the cotranscriptional quality control of mRNPs in budding yeast by expressing the bacterial Rho helicase, which globally perturbs yeast mRNP formation. We examined the genome-wide binding profiles of the THO complex subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation of the mRNP biogenesis, and found that Tho2 plays two roles. In addition to its function as a subunit of the THO complex, upon perturbation of mRNP biogenesis Tho2 targets Rrp6 to chromatin via its carboxy-terminal domain. Interestingly, other THO subunits are not enriched on chromatin upon perturbation of mRNP biogenesis and are not necessary for localizing Rrp6 at its target loci. Our study highlights the potential role of Tho2 in cotranscriptional mRNP quality control, which is independent of other THO subunits. Considering that both the THO complex and the RNA exosome are evolutionarily highly conserved, our findings are likely relevant for mRNP surveillance in mammals.

The conserved 3′‐5′ exoribonuclease EXOSC10/Rrp6 processes and degrades RNA, regulates gene expression and participates in DNA double‐strand break repair and control of telomere maintenance via degradation of the telomerase RNA component. EXOSC10/Rrp6 is part of the multimeric nuclear RNA exosome and interacts with numerous proteins. Previous clinical, genetic, biochemical and genomic studies revealed the protein's essential functions in cell division and differentiation, its RNA substrates and its relevance to autoimmune disorders and oncology. However, little is known about the regulatory mechanisms that control the transcription, translation and stability of EXOSC10/Rrp6 during cell growth, development and disease and how these mechanisms evolved from yeast to human. Herein, we provide an overview of the RNA‐ and protein expression profiles of EXOSC10/Rrp6 during cell division, development and nutritional stress, and we summarize interaction networks and post‐translational modifications across species. Additionally, we discuss how known and predicted protein interactions and post‐translational modifications influence the stability of EXOSC10/Rrp6. Finally, we explore the idea that different EXOSC10/Rrp6 alleles, which potentially alter cellular protein levels or affect protein function, might influence human development and disease progression. In this review we interpret information from the literature together with genomic data from knowledgebases to inspire future work on the regulation of this essential protein's stability in normal and malignant cells.

In eukaryotic cells, aberrant mRNPs with processing and packaging defects are targeted co-transcriptionally by a surveillance system that triggers their nuclear retention and ultimately the degradation of their mRNA component by the 3′–5′ activity of the exosome-associated exonuclease Rrp6. This mRNP quality control process is stimulated by the NNS complex (Nrd1-Nab3-Sen1), which otherwise mediates termination, processing, and decay of ncRNAs. The process involves also the exosome co-activator TRAMP complex (Trf4-Air2-Mtr4). Here, we describe a genome-wide approach to visualize the dynamic movement and coordination of these quality control components over the yeast chromosomes upon perturbation of mRNP biogenesis. The method provides valuable information on how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events during perturbation of mRNP biogenesis. The overview shows also that the gathering of the quality control components over affected mRNA genes takes place at the expense of their commitment to be recruited at ncRNA genomic features, provoking termination and processing defects of ncRNAs.

Stress granules and P bodies are cytoplasmic structures assembled in response to various stress factors and represent sites of temporary storage or decay of mRNAs. Depending on the source of stress, the formation of these structures may be driven by distinct mechanisms, but several stresses have been shown to stabilize mRNAs via inhibition of deadenylation. A recent study identified yeast gene deletion mutants with constitutive stress granules and elevated P bodies; however, the mechanisms which trigger its formation remain poorly understood. Here, we investigate the possibility of accumulating mRNA with mammalian codon bias, which we termed the model RNA, in these mutants. We found that the model RNA accumulates in dcp2 and xrn1 mutants and in four mutants with constitutive stress granules overlapping with P bodies. However, in eight other mutants with constitutive stress granules, the model RNA is downregulated, or its steady state levels vary. We further suggest that the accumulation of the model RNA is linked to its protection from the main mRNA surveillance path. However, there is no obvious targeting of the model RNA to stress granules or P bodies. Thus, accumulation of the model RNA and formation of constitutive stress granules occur independently and only some paths inducing formation of constitutive stress granules will stabilize mRNA as well.

Stress granules and P bodies are cytoplasmic structures assembled in response to various stress factors and represent sites of temporary storage or decay of mRNAs. Depending on the source of stress, the formation of these structures may be driven by distinct mechanisms, but several stresses were shown to stabilize mRNAs via inhibition of deadenylation. A recent study identified yeast gene deletion mutants with constitutive stress granules and elevated P bodies; however, the mechanisms which trigger its formation remain poorly understood. Here, we investigate the possibility of accumulating mRNA with mammalian codon bias, which we termed the model RNA, in these mutants. We found that the model RNA accumulates in dcp2 and xrn1 mutants and in four mutants with constitutive stress granules overlapping with P bodies. However, in eight other mutants with constitutive stress granules the model RNA is downregulated, or its steady state levels vary. We further suggest that the accumulation of the model RNA is linked to its protection from the main mRNA surveillance path. However, there is no obvious targeting of the model RNA to stress granules or P bodies. Thus, accumulation of the model RNA and formation of constitutive stress granules occur independently and only some paths inducing formation of constitutive stress granules will stabilize mRNA as well.

Eukaryotic cells have evolved a nuclear quality control (QC) system to monitor the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent ribonucleoprotein particles (mRNPs). Aberrant mRNPs that fail to pass the QC steps are retained in the nucleus and eliminated by the exonuclease activity of Rrp6. It is still unclear how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events that may arise at each step of transcript elongation and mRNP formation. To dissect the QC mechanism, we previously implemented a powerful assay based on global perturbation of mRNP biogenesis in yeast by the bacterial Rho helicase. By monitoring model genes, we have shown that the QC process is coordinated by Nrd1, a component of the NNS complex (Nrd1-Nab3-Sen1) involved in termination, processing and decay of ncRNAs which is recruited by the CTD of RNAP II. Here, we have extended our investigations by analyzing the QC behaviour over the whole yeast genome. We performed high-throughput RNA sequencing (RNA-seq) to survey a large collection of mRNPs whose biogenesis is affected by Rho action and which can be rescued upon Rrp6 depletion. This genome-wide perspective was extended by generating high-resolution binding landscapes (ChIP-seq) of QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis. Our results show that perturbation of mRNP biogenesis redistributes the QC components over the genome with a significant hijacking of Nrd1 and Nab3 from genomic loci producing ncRNAs to Rho-affected protein-coding genes, triggering termination and processing defects of ncRNAs.

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.

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.