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.

TGFß-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma.

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor ß (TGFß), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFß signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFß target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFß induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis.

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.

Chromatin remodeling by Pol II primes efficient Pol III transcription.

The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is coupled with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here a mechanism where RNA Polymerase II (Pol II) transcription is required to prime and maintain nucleosome depletion at Pol III loci and contributes to efficient Pol III recruitment upon re-initiation of growth from stationary phase in Fission yeast. The Pcr1 transcription factor participates in the recruitment of Pol II, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

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.

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.

Long noncoding (lnc)RNAs modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, their ability to be transferred and control adjacent cells is unknown. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and antitumourigenic functions by controlling the immune response. In contrast to extracellular nonvesicular RNA, few studies have addressed the full RNA content within human fluids' EVs and have compared them with their tissue of origin. Here, we performed Total RNA-Sequencing on six Formalin-Fixed-Paraffin-Embedded (FFPE) prostate cancer (PCa) tumour tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. UEVs contain simplified transcriptome with intron-free cytoplasmic transcripts and enriched lnc/circular (circ)RNAs, strikingly common to an independent 20 patients' urinary cohort. Our full cellular and EVs transcriptome comparison within three PCa cell lines identified a set of overlapping 14 uEV-circRNAs characterized as essential for prostate cell proliferation in vitro and 28 uEV-lncRNAs belonging to the cancer-related lncRNA census (CLC2). In addition, we found 15 uEV-lncRNAs, predicted to encode 768 high-affinity neoantigens, and for which three of the encoded-ORF produced detectable unmodified peptides by mass spectrometry. Our dual analysis of EVs-lnc/circRNAs both in urines' and in vitro's EVs provides a fundamental resource for future uEV-lnc/circRNAs phenotypic characterization involved in PCa.

Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation.

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.

From Yeast to Mammals, the Nonsense-Mediated mRNA Decay as a Master Regulator of Long Non-Coding RNAs Functional Trajectory.

The Nonsense-Mediated mRNA Decay (NMD) has been classically viewed as a translation-dependent RNA surveillance pathway degrading aberrant mRNAs containing premature stop codons. However, it is now clear that mRNA quality control represents only one face of the multiple functions of NMD. Indeed, NMD also regulates the physiological expression of normal mRNAs, and more surprisingly, of long non-coding (lnc)RNAs. Here, we review the different mechanisms of NMD activation in yeast and mammals, and we discuss the molecular bases of the NMD sensitivity of lncRNAs, considering the functional roles of NMD and of translation in the metabolism of these transcripts. In this regard, we describe several examples of functional micropeptides produced from lncRNAs. We propose that translation and NMD provide potent means to regulate the expression of lncRNAs, which might be critical for the cell to respond to environmental changes.

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.

The Crohn's disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack.

Patients with Crohn's disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.

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.

Transient DNMT3L Expression Reinforces Chromatin Surveillance to Halt Senescence Progression in Mouse Embryonic Fibroblast.

Global heterochromatin reduction, which is one of the hallmarks of senescent cells, is associated with reduced transposable element repression and increased risk of chromatin instability. To ensure genomic integrity, the irreparable cells in a population exit permanently from the cell cycle, and this process is termed "senescence." However, senescence only blocks the expansion of unwanted cells, and the aberrant chromatin of senescent cells remains unstable. Serendipitously, we found that the transient ectopic expression of a repressive epigenetic modulator, DNA methyltransferase 3-like (DNMT3L) was sufficient to delay the premature senescence progression of late-passage mouse embryonic fibroblasts (MEFs) associated with a tightened global chromatin structure. DNMT3L induces more repressive H3K9 methylation on endogenous retroviruses and downregulates the derepressed transposons in aging MEFs. In addition, we found that a pulse of ectopic DNMT3L resulted in the reestablishment of H3K27me3 on polycomb repressive complex 2 (PRC2)-target genes that were derepressed in old MEFs. We demonstrated that ectopic DNMT3L interacted with PRC2 in MEFs. Our data also suggested that ectopic DNMT3L might guide PRC2 to redress deregulated chromatin regions in cells undergoing senescence. This study might lead to an epigenetic reinforcement strategy for overcoming aging-associated epimutation and senescence.

Subtelomeric Transcription and its Regulation.

The subtelomeres, highly heterogeneous repeated sequences neighboring telomeres, are transcribed into coding and noncoding RNAs in a variety of organisms. Telomereproximal subtelomeric regions produce non-coding transcripts i.e., ARRET, αARRET, subTERRA, and TERRA, which function in telomere maintenance. The role and molecular mechanisms of the majority of subtelomeric transcripts remain unknown. This review depicts the current knowledge and puts into perspective the results obtained in different models from yeasts to humans.

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Reference-free transcriptome exploration reveals novel RNAs for prostate cancer diagnosis.

The use of RNA-sequencing technologies held a promise of improved diagnostic tools based on comprehensive transcript sets. However, mining human transcriptome data for disease biomarkers in clinical specimens are restricted by the limited power of conventional reference-based protocols relying on unique and annotated transcripts. Here, we implemented a blind reference-free computational protocol, DE-kupl, to infer yet unreferenced RNA variations from total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into putative long noncoding (lnc)RNAs expressed in prostate cancer. Through filtering of 1,179 candidates, we defined 21 lncRNAs that were further validated by NanoString for robust tumor-specific expression in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling more than 90% of true-positive detections of cancer in an independent The Cancer Genome Atlas cohort. Remarkably, this clinical signature made of only nine unannotated lncRNAs largely outperformed PCA3, the only used prostate cancer lncRNA biomarker, in detection of high-risk tumors. This modular workflow is highly sensitive and can be applied to any pathology or clinical application.

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.

Bridging the gap between reference and real transcriptomes.

Genetic, transcriptional, and post-transcriptional variations shape the transcriptome of individual cells, rendering establishing an exhaustive set of reference RNAs a complicated matter. Current reference transcriptomes, which are based on carefully curated transcripts, are lagging behind the extensive RNA variation revealed by massively parallel sequencing. Much may be missed by ignoring this unreferenced RNA diversity. There is plentiful evidence for non-reference transcripts with important phenotypic effects. Although reference transcriptomes are inestimable for gene expression analysis, they may turn limiting in important medical applications. We discuss computational strategies for retrieving hidden transcript diversity.

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.

Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions.

The eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast, fly, and human. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at varying levels in yeast and found that Sir3 spreads into extended silent domains (ESDs), eventually reaching saturation at subtelomeres. We observed the spread of Sir3 into subtelomeric domains associated with specific histone marks in wild-type cells, and stopping at zones of histone mark transitions including H3K79 trimethylation levels. Our study shows that the conserved H3K79 methyltransferase Dot1 is essential in restricting Sir3 spread beyond ESDs, thus ensuring viability upon overexpression of Sir3. Last, our analyses of published data demonstrate how ESDs unveil uncharacterized discrete domains isolating structural and functional subtelomeric features from the rest of the genome. Our work offers a new approach on how to separate subtelomeres from the core chromosome.