Simultaneous generation of many RNA-seq libraries in a single reaction

Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches

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 directly interacts with SHARP to silence transcription through HDAC3

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

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.Gordon and Betty Moore Foundation (Grant GBMF775)Beckman InstituteNational Institutes of Health (U.S.) (Director’s Early Independence Award DP5OD012190)Rose Hills FoundationEdward Mallinckrodt, Jr. FoundationSontag FoundationSearle Scholars ProgramCalifornia Institute of Technolog