Endogenous RNAi pathway evolutionarily shapes the destiny of the antisense lncRNAs transcriptome

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The 5-Fluorouracil RNA Expression Viewer (5-FUR) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast

International audienceSaccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5‐Fluorocuracil (5‐FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA‐dependent processes. Since 5‐FU inhibits the activity of the 3′−5′‐exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5‐FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5‐Fluorouracil RNA (5‐FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1‐dependent‐, stable‐, cryptic‐, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2‐transformed or linear data can be displayed as filled diagrams, line graphs or color‐coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5‐FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org

The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast

International audience5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3'-5' exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU's cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets

The 5-Fluorouracil RNA Expression Viewer (5-FUR) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast

International audienceSaccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5‐Fluorocuracil (5‐FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA‐dependent processes. Since 5‐FU inhibits the activity of the 3′−5′‐exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5‐FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5‐Fluorouracil RNA (5‐FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1‐dependent‐, stable‐, cryptic‐, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2‐transformed or linear data can be displayed as filled diagrams, line graphs or color‐coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5‐FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org

Transcription-dependent spreading of the Dal80 yeast GATA factor across the body of highly expressed genes

GATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well defined in vitro, the in vivo selectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites, correlating with nitrogen- and/or Dal80-sensitive gene expression. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies requires active transcription. Consistently, Dal80 co-immunoprecipitated with the initiating and post-initiation forms of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway toward SARS-CoV-2

International audienceInvestigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and long non-coding RNAs (lncRNAs), including transcripts coding for antiviral factors, such as interferons (IFNs). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-κB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-κB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells

Transinteractome analysis reveals distinct niche requirements for isotype‐based plasma cell subsets in the bone marrow

International audienceBone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR +) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype

Chromatin remodelling by INO80 at promoter proximal pause sites promotes premature termination of mRNA synthesis

Abstract How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process