Control of Rrp6 stability in yeast sporulation

International audienceMeeting Abstract P-04.5-00

Control of Rrp6 stability in yeast sporulation

International audienceMeeting Abstract P-04.5-00

Roles of Rrp6/EXOSC10-targeted lncRNAs in anti-cancer drug toxicity and cell wall architecture

International audienc

Deciphering the Dynamic Landscape of Transcription-Associated mRNP Quality Control Components Over the Whole Yeast Genome.

International audienceIn 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

Recombinant yeast and human cells as screening tools to search for antibacterial agents targeting the transcription termination factor Rho

International audienceThe 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 exosome cofactor Rrp47 is critical for the stability and normal expression of its associated exoribonuclease Rrp6 in Saccharomyces cerevisiae

Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs