A lifelong duty: how Xist maintains the inactive X chromosome.

Female eutherians transcriptionally silence one X chromosome to balance gene dosage between the sexes. X-chromosome inactivation (XCI) is initiated by the lncRNA Xist, which assembles many proteins within the inactive X chromosome (Xi) to trigger gene silencing and heterochromatin formation. It is well established that gene silencing on the Xi is maintained through repressive epigenetic processes, including histone deacetylation and DNA methylation. Recent studies revealed a new mechanism where RNA-binding proteins that interact directly with the RNA contribute to the maintenance of Xist localization and gene silencing. In addition, a surprising plasticity of the Xi was uncovered with many genes becoming upregulated upon experimental deletion of Xist. Intriguingly, immune cells normally lose Xist from the Xi, suggesting that thisXist dependence is utilized in vivo to dynamically regulate gene expression from the Xi. These new studies expose fundamental regulatory mechanisms for the chromatin association of RNAs, highlight the need for studying the maintenance of XCI and Xist localization in a gene- and cell-type-specific manner, and are likely to have clinical impact.

Publisher Correction: A protein assembly mediates Xist localization and gene silencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A protein assembly mediates Xist localization and gene silencing.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3-5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.

Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

The "lnc" between 3D chromatin structure and X chromosome inactivation.

The long non-coding RNA Xist directs a remarkable instance of developmentally regulated, epigenetic change known as X Chromosome Inactivation (XCI). By spreading in cis across the X chromosome from which it is expressed, Xist RNA facilitates the creation of a heritably silent, heterochromatic nuclear territory that displays a three-dimensional structure distinct from that of the active X chromosome. How Xist RNA attaches to and propagates across a chromosome and its influence over the three-dimensional (3D) structure of the inactive X are aspects of XCI that have remained largely unclear. Here, we discuss studies that have made significant contributions towards answering these open questions.

The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins--SHARP, SAF-A and LBR--are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.

The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome.

Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

Pre-mRNA splicing during transcription in the mammalian system.

Splicing of RNA polymerase II transcripts is a crucial step in gene expression and a key generator of mRNA diversity. Splicing and transcription have generally been studied in isolation, although in vivo pre-mRNA splicing occurs in concert with transcription. The two processes appear to be functionally connected because a number of variables that regulate transcription have been identified as also influencing splicing. However, the mechanisms that couple the two processes are largely unknown. This review highlights the observations that implicate splicing as occurring during transcription and describes the evidence supporting functional interactions between the two processes. I discuss postulated models of how splicing couples to transcription and consider the potential impact that such coupling might have on exon recognition. WIREs RNA 2011 2 700-717 DOI: 10.1002/wrna.86 For further resources related to this article, please visit the WIREs website.

Co-transcriptional splicing of constitutive and alternative exons.

In metazoan organisms, pre-mRNA splicing is thought to occur during transcription, and it is postulated that these two processes are functionally coupled via still-unknown mechanisms. Current evidence supports co-transcriptional spliceosomal assembly, but there is little quantitative information on how much splicing is completed during RNA synthesis. Here we isolate nascent chromatin-associated RNA from free, nucleoplasmic RNA already released from the DNA template. Using a quantitative RT-PCR assay, we show that the majority of introns separating constitutive exons are already excised from the human c-Src and fibronectin pre-mRNAs that are still in the process of synthesis, and that these introns are removed in a general 5'-to-3' order. Introns flanking alternative exons in these transcripts are also removed during synthesis, but show differences in excision efficiency between cell lines with different regulatory conditions. Our data suggest that skipping of an exon can induce a lag in splicing compared to intron removal under conditions of exon inclusion. Nevertheless, excision of the long intron encompassing the skipped exon is still completed prior to transcript release into the nucleoplasm. Thus, we demonstrate that the decision to include or skip an alternative exon is made during transcription and not post-transcriptionally.