Diversification of human plasmacytoid predendritic cells in response to a single stimulus.

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.

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.

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.

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.

Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure.

Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3' single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.

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.

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.

Bases of antisense lncRNA-associated regulation of gene expression in fission yeast.

Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene ctt1 revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to ctt1 induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on ctt1 induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast.

Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

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.

Correction to: Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

Following publication of the original article [1], the authors reported that one of the authors' names is spelled incorrectly.

Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

BACKGROUND: The PIWI/piRNA pathway is a conserved machinery important for germ cell development and fertility. This piRNA-guided molecular machinery is best known for repressing derepressed transposable elements (TE) during epigenomic reprogramming. The extent to which piRNAs are involved in modulating transcripts beyond TEs still need to be clarified, and it may be a stage-dependent event. We chose chicken germline as a study model because of the significantly lower TE complexity in the chicken genome compared to mammalian species. RESULTS: We generated high-confidence piRNA candidates in various stages across chicken germline development by 3'-end-methylation-enriched small RNA sequencing and in-house bioinformatics analysis. We observed a significant developmental stage-dependent loss of TE association and a shifting of the ping-pong cycle signatures. Moreover, the stage-dependent reciprocal abundance of LINE retrotransposons, CR1-C, and its associated piRNAs implicated the developmental stage-dependent role of piRNA machinery. The stage dependency of piRNA expression and its potential functions can be better addressed by analyzing the piRNA precursors/clusters. Interestingly, the new piRNA clusters identified from embryonic chicken testes revealed evolutionary conservation between chickens and mammals, which was previously thought to not exist. CONCLUSIONS: In this report, we provided an original chicken RNA resource and proposed an analytical methodology that can be used to investigate stage-dependent changes in piRNA compositions and their potential roles in TE regulation and beyond, and also revealed possible conserved functions of piRNAs in developing germ cells.

LncRNAs, lost in translation or licence to regulate?

Over the last decade, advances in transcriptomics have revealed that the pervasive transcription of eukaryotic genomes produces plethora of long noncoding RNAs (lncRNAs), which are now recognized as major regulators of multiple cellular processes. Although they have been thought to lack any protein-coding potential, recent ribosome-profiling data indicate that lncRNAs can interact with the translation machinery, leading to the production of functional peptides in some cases. In this perspective, we have explored the idea that translation can be part of the fate of cytoplasmic lncRNAs, raising the possibility for them to work as bifunctional RNAs, endowed with dual coding and regulatory functions.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.

History, Discovery, and Classification of lncRNAs.

The RNA World Hypothesis suggests that prebiotic life revolved around RNA instead of DNA and proteins. Although modern cells have changed significantly in 4 billion years, RNA has maintained its central role in cell biology. Since the discovery of DNA at the end of the nineteenth century, RNA has been extensively studied. Many discoveries such as housekeeping RNAs (rRNA, tRNA, etc.) supported the messenger RNA model that is the pillar of the central dogma of molecular biology, which was first devised in the late 1950s. Thirty years later, the first regulatory non-coding RNAs (ncRNAs) were initially identified in bacteria and then in most eukaryotic organisms. A few long ncRNAs (lncRNAs) such as H19 and Xist were characterized in the pre-genomic era but remained exceptions until the early 2000s. Indeed, when the sequence of the human genome was published in 2001, studies showed that only about 1.2% encodes proteins, the rest being deemed "non-coding." It was later shown that the genome is pervasively transcribed into many ncRNAs, but their functionality remained controversial. Since then, regulatory lncRNAs have been characterized in many species and were shown to be involved in processes such as development and pathologies, revealing a new layer of regulation in eukaryotic cells. This newly found focus on lncRNAs, together with the advent of high-throughput sequencing, was accompanied by the rapid discovery of many novel transcripts which were further characterized and classified according to specific transcript traits.In this review, we will discuss the many discoveries that led to the study of lncRNAs, from Friedrich Miescher's "nuclein" in 1869 to the elucidation of the human genome and transcriptome in the early 2000s. We will then focus on the biological relevance during lncRNA evolution and describe their basic features as genes and transcripts. Finally, we will present a non-exhaustive catalogue of lncRNA classes, thus illustrating the vast complexity of eukaryotic transcriptomes.

Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer.

BACKGROUND: Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known. METHODS: We used RT-PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome. RESULTS: MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway. CONCLUSIONS: Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target.