Many long non-coding RNAs (lncRNAs) affect gene expression1, but the mechanisms by which they act are still largely unknown2. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X-chromosome during development in female mammals3,4. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role3. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell5. Here we develop a method to purify a lncRNA and identify its direct interacting proteins using quantitative mass spectrometry. We identify 10 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-repressor6 that activates HDAC37, is not only essential for silencing, but is also required for the exclusion of RNA Polymerase II (PolII) from the inactive X. Both SMRT and HDAC3 are also required for silencing and PolII 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 PolII across the X-chromosome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Chronic social isolation causes severe psychological effects in humans, but their neural bases remains poorly understood. Two weeks (but not 24 hrs) of social isolation stress (SIS) caused multiple behavioral changes in mice, and induced brain-wide up-regulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS, via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function, and suggest potential new therapeutic applications for Nk3R antagonists.

Plunging into a domain of silence Female mammals have two X chromosomes. One must be silenced to “balance” gene dosage with male XY cells. The Xist long noncoding RNA coats the inactive X chromosome in female mammalian cells. Chen et al. show that the Xist RNA helps recruit the X chromosome to the internal rim of the cell nucleus, a region where gene expression is silenced. Xist is recruited to the domain through an interaction with the Lamin B receptor. This recruitment allows the Xist RNA to spread across the future inactive X chromosome, shutting down gene expression. Science , this issue p. 468

Single molecule methods have given researchers the ability to investigate the structural dynamics of biomolecules at unprecedented resolution and sensitivity. One of the preferred methods of studying single biomolecules is single-molecule fluorescence resonance energy transfer (smFRET). The popularity of smFRET stems from its ability to report on dynamic, either intra- or intermolecular interactions in real-time. For example, smFRET has been successfully used to characterize the role of dynamics in functional RNAs and their protein complexes, including ribozymes, the ribosome, and more recently the spliceosome. Being able to reliably extract quantitative kinetic and conformational parameters from smFRET experiments is crucial for the interpretation of their results. The need for efficient, unbiased analysis routines becomes more evident as the systems studied become more complex. In this article we focus on the practical utility of statistical algorithms, particularly hidden Markov models, to aid in the objective quantification of complex smFRET trajectories with three or more discrete states, and to extract kinetic information from the trajectories. Additionally, we present a method for systematically eliminating transitions associated with uncorrelated fluorophore behavior that may occur due to dye anisotropy and quenching effects. We also highlight the importance of data condensation through the use of various transition density plots to fully understand the underlying conformational dynamics and kinetic behavior of the biological macromolecule of interest under varying conditions. Finally, the application of these techniques to studies of pre-mRNA conformational changes during eukaryotic splicing is discussed.