A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

Pervasive translation of Xrn1-sensitive unstable long noncoding RNAs in yeast.

Despite being predicted to lack coding potential, cytoplasmic long noncoding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNA translation remain poorly studied. In yeast, cytoplasmic Xrn1-sensitive unstable transcripts (XUTs) are targeted by nonsense-mediated mRNA decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are pervasively translated, which impacts their decay. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired. Ribo-seq confirmed ribosomes binding to XUTs and identified ribosome-associated 5'-proximal small ORFs. Mechanistically, the NMD-sensitivity of XUTs mainly depends on the 3'-untranslated region length. Finally, we show that the peptide resulting from the translation of an NMD-sensitive XUT reporter exists in NMD-competent cells. Our work highlights the role of translation in the posttranscriptional metabolism of XUTs. We propose that XUT-derived peptides could be exposed to natural selection, while NMD restricts XUT levels.

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

Bone 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.

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

Saccharomyces 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.

HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions.

Epithelial-to-mesenchymal transition (EMT) describes the loss of epithelial traits and gain of mesenchymal traits by normal cells during development and by neoplastic cells during cancer metastasis. The long noncoding RNA HOTAIR triggers EMT, in part by serving as a scaffold for PRC2 and thus promoting repressive histone H3K27 methylation. In addition to PRC2, HOTAIR interacts with the LSD1 lysine demethylase, an epigenetic regulator of cell fate during development and differentiation, but little is known about the role of LSD1 in HOTAIR function during EMT. Here, we show that HOTAIR requires its LSD1-interacting domain, but not its PRC2-interacting domain, to promote the migration of epithelial cells. This activity is suppressed by LSD1 overexpression. LSD1-HOTAIR interactions induce partial reprogramming of the epithelial transcriptome altering LSD1 distribution at promoter and enhancer regions. Thus, we uncover an unexpected role of HOTAIR in EMT as an LSD1 decommissioning factor, counteracting its activity in the control of epithelial identity.

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.

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

5-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 RNA helicases Dbp2 and Mtr4 regulate the expression of Xrn1-sensitive long non-coding RNAs in yeast.

The expression of yeast long non-coding (lnc)RNAs is restricted by RNA surveillance machineries, including the cytoplasmic 5'-3' exonuclease Xrn1 which targets a conserved family of lncRNAs defined as XUTs, and that are mainly antisense to protein-coding genes. However, the co-factors involved in the degradation of these transcripts and the underlying molecular mechanisms remain largely unknown. Here, we show that two RNA helicases, Dbp2 and Mtr4, act as global regulators of XUTs expression. Using RNA-Seq, we found that most of them accumulate upon Dbp2 inactivation or Mtr4 depletion. Mutants of the cytoplasmic RNA helicases Ecm32, Ski2, Slh1, Dbp1, and Dhh1 did not recapitulate this global stabilization of XUTs, suggesting that XUTs decay is specifically controlled by Dbp2 and Mtr4. Notably, Dbp2 and Mtr4 affect XUTs independently of their configuration relative to their paired-sense mRNAs. Finally, we show that the effect of Dbp2 on XUTs depends on a cytoplasmic localization. Overall, our data indicate that Dbp2 and Mtr4 are global regulators of lncRNAs expression and contribute to shape the non-coding transcriptome together with RNA decay machineries.

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

Investigations 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.

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

Antisense long noncoding (aslnc)RNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNAi. Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive aslncRNAs levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for Xrn1-sensitive aslncRNAs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared with S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.