High-throughput identification of RNA nuclear localization signals
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE98828 | GEO | 2018/01/10
SECONDARY ACCESSION(S): PRJNA386325
REPOSITORIES: GEO
Similar Datasets
Project description:Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. Here we investigate the localization mechanisms of Xist 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 initially accumulates on the periphery of actively transcribed regions and requires its silencing domain to spread across active regions. This suggests a model where Xist coats the entire X-chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in multiple cell contexts: 1) differentiated female cells (MLFs); 2) a time-course of Xist localization in male embryonic stem (ES) cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33); 3) a time-course of Xist localization in differentiating female ES cells (F1 2-1); and 4) wild-type (delXF6) and A-repeat deletion (delSXC9) Xist transgenes incorporated into the Hprt locus under the control of a tet-inducible promoter.
2013-05-30 | E-GEOD-46918 | biostudies-arrayexpress
Project description:Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. Here we investigate the localization mechanisms of Xist 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 initially accumulates on the periphery of actively transcribed regions and requires its silencing domain to spread across active regions. This suggests a model where Xist coats the entire X-chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.
2013-05-30 | GSE46918 | GEO
Project description:Pervasive transcription in the mammalian genome produces thousands of long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts. They preferentially locate to chromatin, at which some regulate chromatin structure, transcription and RNA processing. While several RNA sequences responsible for nuclear localization have been identified, such as repeats in the lncRNA Xist and Alu-like elements for long RNAs, how lncRNAs as a class are enriched on chromatin remains elusive. To screen for cis-elements that contribute to RNA-chromatin localization, we developed a high-throughput method named RNA elements for subcellular localization by sequencing (REL-seq), and discovered a U1 small nuclear ribonucleoprotein (snRNP)-recognition motif being critical for chromatin localization of reporter RNAs. Across the genome, chromatin-bound lncRNAs, which are enriched with 5’ splice sites and depleted of 3’ splice sites, exhibit high levels of U1 snRNA binding compared to cytoplasm-localized protein-coding mRNAs. Acute depletion of U1 snRNA, or U1 snRNP protein component SNRNP70, drastically reduces the chromatin association of hundreds of lncRNAs and unstable transcripts without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear-speckles and genome-wide localization of Malat1, a highly conserved and abundant lncRNA. Moreover, chromatin-bound U1 snRNP interacts with transcriptionally engaged RNA polymerase (Pol) II. Together, these results demonstrate that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a Pol II transcription-dependent manner. Our findings uncover a novel role of U1 snRNP beyond pre-mRNA processing and provide molecular insights into how lncRNAs are recruited to Pol II-transcribed genes and have a propensity for chromatin-associated functions.
2020-01-01 | GSE134287 | GEO
Project description:HNRNPU regulates lncRNAs nuclear localization
| PRJNA694053 | ENA
Project description:Gene regulation during the process of osteoblastogenesis has been well-described, yet the discovery of novel regulatory regions has been limited by how we currently predict the locations of functional cis-regulatory modules. Historically, the de novo identification of sequences critical for the control of gene expression relied primarily on sequence conservation in promoters. Queries for binding motifs were based on position weight matrices of known transcription factors and the identification of disease-causing, non-coding mutations near critical genes. However, we now must consider that regulatory elements also rely on 3-dimensional chromosomal interactions between far-distal regions, epigenetic chromosomal modifications, and RNA:DNA interactions. Traditionally, DNaseI-hypersensitivity assays have been used for the identification of regulatory regions via preferential digestion at chromatin depleted or displaced of nucleosomes, as a result of transcription factor occupancy. We probed DNase hypersensitivity on a genome-wide scale to determine whether osteogenic differentiation and/or bone-related gene regulation is marked by the presence of commonly utilized DNA motifs within active cis-regulatory modules. We thus sought to evaluate the gain or loss of motif representation within hypersensitive regions during osteoblastogenesis, from day-0 (growth-phase) to day-28 (mineralizing) MC3T3 cultures. We find that differentiation is marked by an increased enrichment of NFkB-p65, MEF2, and bHLH/E-box motifs within hypersensitive regions, while CTCF, NF1, TEAD, and AP1 motifs decrease. Furthermore, grouping hypersensitive regions based on genomic positioning (promoters, introns, exons, and far-distal regions) reveals significant differences in motif abundance in first introns versus other genomic positions. This finding suggests that the regulation conferred within first intron sequences may be somewhat distinct. Interestingly, the majority of motifs that were enriched, regardless of genomic position or differentiation time-point, were not completely matched to currently known transcription factor motifs (curated in the JASPAR database). Taken together, the changes in DNase-hypersensitive regions during osteoblastogenesis and the enrichment of distinct motifs within these regions indicate that osteoblasts utilize unique sets of motif rules for transcription factor binding or that regulatory control operates through undiscovered factors.
2015-05-18 | GSE55046 | GEO
Project description:Gene regulation during the process of osteoblastogenesis has been well-described, yet the discovery of novel regulatory regions has been limited by how we currently predict the locations of functional cis-regulatory modules. Historically, the de novo identification of sequences critical for the control of gene expression relied primarily on sequence conservation in promoters. Queries for binding motifs were based on position weight matrices of known transcription factors and the identification of disease-causing, non-coding mutations near critical genes. However, we now must consider that regulatory elements also rely on 3-dimensional chromosomal interactions between far-distal regions, epigenetic chromosomal modifications, and RNA:DNA interactions. Traditionally, DNaseI-hypersensitivity assays have been used for the identification of regulatory regions via preferential digestion at chromatin depleted or displaced of nucleosomes, as a result of transcription factor occupancy. We probed DNase hypersensitivity on a genome-wide scale to determine whether osteogenic differentiation and/or bone-related gene regulation is marked by the presence of commonly utilized DNA motifs within active cis-regulatory modules. We thus sought to evaluate the gain or loss of motif representation within hypersensitive regions during osteoblastogenesis, from day-0 (growth-phase) to day-28 (mineralizing) MC3T3 cultures. We find that differentiation is marked by an increased enrichment of NFkB-p65, MEF2, and bHLH/E-box motifs within hypersensitive regions, while CTCF, NF1, TEAD, and AP1 motifs decrease. Furthermore, grouping hypersensitive regions based on genomic positioning (promoters, introns, exons, and far-distal regions) reveals significant differences in motif abundance in first introns versus other genomic positions. This finding suggests that the regulation conferred within first intron sequences may be somewhat distinct. Interestingly, the majority of motifs that were enriched, regardless of genomic position or differentiation time-point, were not completely matched to currently known transcription factor motifs (curated in the JASPAR database). Taken together, the changes in DNase-hypersensitive regions during osteoblastogenesis and the enrichment of distinct motifs within these regions indicate that osteoblasts utilize unique sets of motif rules for transcription factor binding or that regulatory control operates through undiscovered factors.
2017-12-31 | GSE55045 | GEO
Project description:Gene regulation during the process of osteoblastogenesis has been well-described, yet the discovery of novel regulatory regions has been limited by how we currently predict the locations of functional cis-regulatory modules. Historically, the de novo identification of sequences critical for the control of gene expression relied primarily on sequence conservation in promoters. Queries for binding motifs were based on position weight matrices of known transcription factors and the identification of disease-causing, non-coding mutations near critical genes. However, we now must consider that regulatory elements also rely on 3-dimensional chromosomal interactions between far-distal regions, epigenetic chromosomal modifications, and RNA:DNA interactions. Traditionally, DNaseI-hypersensitivity assays have been used for the identification of regulatory regions via preferential digestion at chromatin depleted or displaced of nucleosomes, as a result of transcription factor occupancy. We probed DNase hypersensitivity on a genome-wide scale to determine whether osteogenic differentiation and/or bone-related gene regulation is marked by the presence of commonly utilized DNA motifs within active cis-regulatory modules. We thus sought to evaluate the gain or loss of motif representation within hypersensitive regions during osteoblastogenesis, from day-0 (growth-phase) to day-28 (mineralizing) MC3T3 cultures. We find that differentiation is marked by an increased enrichment of NFkB-p65, MEF2, and bHLH/E-box motifs within hypersensitive regions, while CTCF, NF1, TEAD, and AP1 motifs decrease. Furthermore, grouping hypersensitive regions based on genomic positioning (promoters, introns, exons, and far-distal regions) reveals significant differences in motif abundance in first introns versus other genomic positions. This finding suggests that the regulation conferred within first intron sequences may be somewhat distinct. Interestingly, the majority of motifs that were enriched, regardless of genomic position or differentiation time-point, were not completely matched to currently known transcription factor motifs (curated in the JASPAR database). Taken together, the changes in DNase-hypersensitive regions during osteoblastogenesis and the enrichment of distinct motifs within these regions indicate that osteoblasts utilize unique sets of motif rules for transcription factor binding or that regulatory control operates through undiscovered factors. Genome-wide DNase hypersensitivity mapping of osteoblast cultures was performed by adapting the DNase-seq protocol from Song et al. (Song and Crawford, 2010) with slight modifications. Growth-phase (day 0), matrix-deposition stage (day 9), or mineralization stage (day 28) MC3T3-E1 clone-4 cultures were subjected to DNase-seq library preparation. Libraries of purified DNA were generated using custom adapters described in Song et al. High-throughput sequencing was performed by Illumina Genome Analyzer II with 36 base reads and on an Illumina Hiseq-1000 with 100 base reads. Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina). Two independent biological repeats of DNase-seq libraries were prepared for each time point. Each biological repeat is represented by two technical repeats.
2015-05-18 | E-GEOD-55046 | biostudies-arrayexpress
Project description:Xist orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the 3-dimensional structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here we show that Xist directly interacts with the Lamin B Receptor (LBR), an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing. We show that this interaction recruits the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the accessibility of DNA thereby enabling Xist, and its silencing proteins, to spread across the X to silence transcription. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in male mouse ES cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33) containing 1) wild-type Xist (WT), 2) A-repeat deletion Xist (dA), 3) LBR binding site deletion Xist (dLBS), 4) dLBS-Xist rescued with LMNB1 (LMNB1Res), 5) LBR CRISPRi knock down (LBRKD), or 6) SHARP CRISPRi knock down (SHARPKD).
2016-07-24 | E-GEOD-80510 | biostudies-arrayexpress
Project description:XIST is a long non-coding RNA (lncRNA) that mediates transcriptional silencing of X chromosome genes. Here we show that XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues, a reversible base modification whose function in lncRNAs is unknown. We show that m6A formation in XIST, as well as cellular mRNAs, is mediated by RBM15 and its paralog RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in methylation of adenosines in adjacent m6A consensus motifs. Furthermore, knockdown of RBM15 and RBM15B, or knockdown of the m6A methyltransferase METTL3 impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTHDC1 preferentially recognizes m6A in XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. Three to four biological HEK293T replicates were used to perform iCLIP of endogenous YTH proteins, RBM15, and RBM15B. Crosslinking induced truncations were identified using CIMS-CITS pipeline.
2016-09-07 | E-GEOD-78030 | biostudies-arrayexpress
Project description:Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes (~1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: specificity to the X and chromosome-wide gene silencing, and suggest a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets.
2022-01-21 | GSE192573 | GEO