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 Rhoaffected 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.

Selective DNA binding by transcription factors (TFs) is crucial for the correct regulation of DNA transcription. In healthy cells, promoters of active genes are hypomethylated. A single CpG methylation within a TF response element may change the binding preferences of the protein thus causing the dysregulation of transcription programs. Here we investigate a molecular mechanism driving the downregulation of the NDUFA13 gene, due to hypermethylation, which is associated with multiple cancers. Using bioinformatic analyses of breast cancer cell line MCF7, we identify a hypermethylated region containing the binding sites of two TFs dimers, CEBPB and E2F1-DP1, located 130 b.p. from the gene transcription start site. All-atom extended MD simulations of wild-type and methylated DNA alone and in complex with either one or both TFs dimers provide mechanistic insights into the cooperative asymmetric binding order of the two dimers; the CEBPB binding should occur first to facilitate the E2F1-DP1-DNA association. The CpG methylation within the E2F1-DP1 response element and the linker decreases the cooperativity effects and renders the E2F1-DP1 binding site less recognizable by the TF dimer. Taken together, the identified CpG methylation site may contribute to the downregulation of the NDUFA13 gene.

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 ncRNAs genomic features, provoking termination and processing defects of ncRNAs.

Selective DNA binding by transcription factors (TFs) is crucial for the correct regulation of DNA transcription. In healthy cells, promoters of active genes are hypomethylated. A single CpG methylation within a TF response element may change the binding preferences of the protein thus causing the dysregulation of transcription programs. Here we investigate a molecular mechanism driving the downregulation of NDUFA13 gene, due to hypermethylation, which is associated with multiple cancers. Using bioinformatic analyses of breast cancer cell line MCF7, we identify a hypermethylated region containing the binding sites of two TFs dimers, CEBPB and E2F1-DP1, located 130 b.p. from the gene transcription start site. All-atom extended MD simulations of wild-type and methylated DNA alone and in complex with either one or both TFs dimers provide mechanistic insights into the cooperative asymmetric binding order of the two dimers; the CEBPB binding should occur first to facilitate the E2F1-DP1-DNA association. The CpG methylation within the E2F1-DP1 response element and the linker decreases the cooperativity effects and renders the E2F1-DP1 binding site less recognizable by the TF dimer. Taken together, the identified CpG methylation site may contribute to the downregulation of NDUFA13 gene and has a potential as a biomarker for breast cancer.