Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing.

The lncRNA silences gene expression by recruiting the protein SPEN through its 5'-proximal Repeat A domain. How Repeat A recruits SPEN, how SPEN enacts silencing, and why Repeat A is required for biogenesis in addition to silencing remain unclear. We find that sequences in Repeat A critical for SPEN recruitment, silencing, and biogenesis directly bind SR-rich splicing factors and not SPEN.

Isogenic comparison of Airn and Xist reveals core principles of Polycomb recruitment by lncRNAs.

The mechanisms and biological roles of Polycomb repressive complex (PRC) recruitment by long noncoding RNAs (lncRNAs) remain unclear. To gain insight, we expressed two lncRNAs that recruit PRCs to multi-megabase domains, Airn and Xist, from an ectopic locus in mouse stem cells and compared effects. Unexpectedly, ectopic Airn recruited PRC1 and PRC2 to chromatin with a potency resembling Xist yet did not repress genes.

SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells.

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs.

Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn.

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression.

Ectopically expressed lncRNA deposits Polycomb with a potency that rivals .

We report that when expressed at similar levels from an isogenic locus, the lncRNA induces Polycomb deposition with a potency that rivals . However, when subject to the same degree of promoter activation, is more abundant and more potent than . Our data definitively demonstrate that the lncRNA is functional and suggest that achieved extreme potency in part by evolving mechanisms to promote its own abundance.

The control of polycomb repressive complexes by long noncoding RNAs.

The polycomb repressive complexes 1 and 2 (PRCs; PRC1 and PRC2) are conserved histone-modifying enzymes that often function cooperatively to repress gene expression. The PRCs are regulated by long noncoding RNAs (lncRNAs) in complex ways. On the one hand, specific lncRNAs cause the PRCs to engage with chromatin and repress gene expression over genomic regions that can span megabases.

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity.

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear.

Inhibition of cytoplasmic cap methylation identifies 5' TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5' ends.

Cap homeostasis is the cyclical process of decapping and recapping that maintains the translation and stability of a subset of the transcriptome. Previous work showed levels of some recapping targets decline following transient expression of an inactive form of RNMT (ΔN-RNMT), likely due to degradation of mRNAs with improperly methylated caps. The current study examined transcriptome-wide changes following inhibition of cytoplasmic cap methylation.

RETRACTED: A 5' fragment of Xist can sequester RNA produced from adjacent genes on chromatin.

Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin.

A recap of RNA recapping.

The N7-methylguanosine cap is a hallmark of the 5' end of eukaryotic mRNAs and is required for gene expression. Loss of the cap was believed to lead irreversibly to decay. However, nearly a decade ago, it was discovered that mammalian cells contain enzymes in the cytoplasm that are capable of restoring caps onto uncapped RNAs.

RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome.

The -methylguanosine cap is added in the nucleus early in gene transcription and is a defining feature of eukaryotic mRNAs. Mammalian cells also possess cytoplasmic machinery for restoring the cap at uncapped or partially degraded RNA 5' ends. Central to both pathways is capping enzyme (CE) (RNA guanylyltransferase and 5'-phosphatase (RNGTT)), a bifunctional, nuclear and cytoplasmic enzyme.

RNA Cap Methyltransferase Activity Assay.

Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps.

RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs.

Cap homeostasis is a cyclical process of decapping and recapping that impacts a portion of the mRNA transcriptome. The metastable uncapped forms of recapping targets redistribute from polysomes to non-translating mRNPs, and recapping is all that is needed for their return to the translating pool. Previous work identified a cytoplasmic capping metabolon consisting of capping enzyme (CE) and a 5'-monophosphate kinase bound to adjacent domains of Nck1.