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The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis. Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5′ splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5′ splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5′ splice site that is suppressed in the presence of m6A and upstream of a 5′ splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.

Xist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA.One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

RNAN6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5’-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5’-splice site, and downstream of an enhanced 5’-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.

Background: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5’ end of the transcript, and also further 3’, in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist–mediated gene silencing. Methods: We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5’ m6A region in interspecific XX mouse embryonic stem cells (mESCs). Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5’ m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. Results: We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing, and that the 5’ m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5’ m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. Conclusions: Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5’ Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.

The biology of non-coding RNA (ncRNA) and the regulation of mammalian gene expression is a rapidly expanding field. In this review, we consider how recent advances in technology, enabling the precise mapping of modifications to RNA transcripts, has provided new opportunities to dissect post-transcriptional gene regulation. With this has come the realisation that in the absence of translation, the modification of ncRNAs may play a fundamental role in their regulation, protein interactome and subsequent downstream effector functions. We focus upon modification of RNA by N6-methyladenosine (m6A); its readers, writers and erasers, before considering the differing role of m6A modified lncRNAs MALAT1 and Xist.This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing., Xist RNA is the master regulator of X chromosome inactivation. Here the authors describe a systematic analysis of Xist-mediated allelic silencing in mouse ESC models and define the contribution of different pathways that regulate gene silencing.

Long noncoding RNAs (lncRNAs) are pervasively transcribed in the human genome. Recent studies suggest that the involvement of lncRNAs in human diseases could be far more prevalent than previously appreciated. Here we have identified a lncRNA termed Lnc_bc060912 whose expression is increased in human lung and other tumors. Lnc_bc060912 is 1.2 kb in length and is composed of two exons. The expression of Lnc_bc060912 was repressed by p53. Lnc_bc060912 suppressed cell apoptosis. Using a recently developed method for RNA-pulldown with formaldehyde cross-linking, we found that Lnc_bc060912 interacted with the two DNA damage repair proteins PARP1 and NPM1. Together, these results suggest that Lnc_bc060912, via PARP1 and NPM1, affects cell apoptosis and may play important roles in tumorigenesis and cancer progression.

Noncoding RNAs are increasingly being recognized as important players in eukaryote biology. However, despite major efforts in mapping the Caenorhabditis elegans transcriptome over the last couple of years, nonpolyadenylated and intermediate-size noncoding RNAs (is-ncRNAs) are still incompletely explored. We have combined an enzymatic approach with full-length RNA-Seq of is-ncRNAs in C. elegans. A total of 473 novel is-ncRNAs has been identified, of which a substantial fraction was associated with transcription factor binding sites and developmentally regulated expression patterns. Analysis of sequence and secondary structure permitted classification of more than 200 is-ncRNAs into several known RNA classes, while another 33 is-ncRNAs were identified as belonging to two previously uncharacterized groups of is-ncRNAs. Three of the unclassified is-ncRNAs contain the 5′ Alu domain common to SRP RNAs and specifically bound with the SRP9/14 heterodimer in vitro. One of these (inc394) showed 65% sequence identity with the human, neuron-specific BC200 RNA. Structure-based clustering analysis and in vitro binding experiments supported the notion that the nematode stem-bulge RNAs (sbRNAs) are homologs (or functional analogs) of the Y RNAs. Moreover, analysis of the differential libraries showed that some mature snoRNAs undergo secondary 5′ cap modification after processing of the primary transcript, thus suggesting the existence of a wider range of functional RNAs arising from processed and modified fragments of primary transcripts.

The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological processes has recently developed rapidly. The large amounts of non-coding RNAs scale consistent with developmental complexity in eukaryotes, indicating that most of these transcripts may have functions in the regulation of biological processes and disorder in the organisms. In particular, Understanding of the overall biological significance of lncRNAs in cancers still remains limited. Here, we found a nuclear-retained lncRNA, termed Lnc_ASNR (apoptosis suppressing-noncoding RNA), which serves as a repressor of apoptosis. Lnc_ASNR was discovered in a set of microarray data derived from four kinds of tumor and adjacent normal tissue samples and displayed significant up-regulation in the tumor tissues. Using an RNA-pull down assay, we found that Lnc_ASNR interacted with the protein ARE/poly (U)-binding/degradation factor 1(AUF1), which is reported to promote rapid degradation of the Bcl-2 mRNA, an inhibitor of apoptosis. Lnc_ASNR binds to AUFI in nucleus, decreasing the cytoplasmic proportion of AUF1 which targets the B-cell lymphoma-2 (Bcl-2) mRNA. Taken together, the overall effect of Lnc_ASNR expression is thus a decrease in cell apoptosis indicating that Lnc_ASNR may play a vital role in tumorigenesis and carcinogenesis.

BACKGROUND: A network of DNA damage response (DDR) mechanisms functions coordinately to maintain genome integrity and prevent disease. The Nucleotide Excision Repair (NER) pathway is known to function in the response to UV-induced DNA damage. Although numbers of coding genes and miRNAs have been identified and reported to participate in UV-induced DNA damage response (UV-DDR), the precise role of non-coding RNAs (ncRNAs) in UV-DDR remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used high-throughput RNA-sequencing (RNA-Seq) to discover intermediate-size (70-500 nt) ncRNAs (is-ncRNAs) in C. elegans, using the strains of L4 larvae of wild-type (N2), UV-irradiated (N2/UV100) and NER-deficient mutant (xpa-1), and 450 novel non-coding transcripts were initially identified. A customized microarray assay was then applied to examine the expression profiles of both novel transcripts and known is-ncRNAs, and 57 UV-DDR-related is-ncRNA candidates showed expression variations at different levels between UV irradiated strains and non- irradiated strains. The top ranked is-ncRNA candidates with expression differences were further validated by qRT-PCR analysis, of them, 8 novel is-ncRNAs were significantly up-regulated after UV irradiation. Knockdown of two novel is-ncRNAs, ncRNA317 and ncRNA415, by RNA interference, resulted in higher UV sensitivity and significantly decreased expression of NER-related genes in C. elegans. CONCLUSIONS/SIGNIFICANCE: The discovery of above two novel is-ncRNAs in this study indicated the functional roles of is-ncRNAs in the regulation of UV-DDR network, and aided our understanding of the significance of ncRNA involvement in the UV-induced DNA damage response.

In recent years, large numbers of non-coding RNAs (ncRNAs) have been identified in C. elegans but their functions are still not well studied. In C. elegans, CEP-1 is the sole homolog of the p53 family of genes. In order to obtain transcription profiles of ncRNAs regulated by CEP-1 under normal and UV stressed conditions, we applied the ‘not-so-random’ hexamers priming strategy to RNA sequencing in C. elegans, This NSR-seq strategy efficiently depleted rRNA transcripts from the samples and showed high technical replicability. We identified more than 1,000 ncRNAs whose apparent expression was repressed by CEP-1, while around 200 were activated. Around 40% of the CEP-1 activated ncRNAs promoters contain a putative CEP-1-binding site. CEP-1 regulated ncRNAs were frequently clustered and concentrated on the X chromosome. These results indicate that numerous ncRNAs are involved in CEP-1 transcriptional network and that these are especially enriched on the X chromosome in C. elegans.