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RIP-PEN-seq for 15.5K in HEK293T cells

ABSTRACT: Here, we developed a RNA ImmunoPrecipitation coupled with sequencing of Paired-Ends of NcRNAs (RIP-PEN-seq) method to identify the full-length sequences of ncRNAs bound by 15.5K protein and discover a novel class of backward K-turn RNAs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE160970 | GEO | 2023/04/10

REPOSITORIES: GEO

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Genome-wide identification of Polycomb-associated RNAs by RIP-seq

Project description:Large-scale analyses have revealed that 60-90% of the mammalian genome is transcriptionally active. Because <2% of sequences have protein-coding potential, why so much cellular energy is expended on RNA synthesis is a major question in the post-genomic era. The hypothesis that RNA may serve as recruiting platforms for chromatin modifiers has gained ground with discoveries linking long ncRNAs, such as RepA, Xist, and Tsix, to locus-specific targeting of Polycomb proteins to the X-chromosome. Long ncRNAs have also been associated with Polycomb proteins at human HOX loci. Here, we ask if RNA targeting may be a general feature of regulation for mouse Polycomb repressive complex 2 (PRC2). We develop a method to capture the 'PRC2 transcriptome' by combining RNA immunoprecipitation (RIP) with deep sequencing (seq). RIP-seq of mouse embryonic stem cells identifies >3000 RNAs in the PRC2 transcriptome. Approximately 14% of RNAs originate from previously described PRC2-binding sites and many are promoter-associated. The transcriptome includes a large number of unannotated noncoding and antisense RNAs, with the X-chromosome exhibiting a high density of PRC2 RNAs. Imprinted genes and other disease genes, including those involved in cancer, are also well-represented. Functional validation of select candidates confirms RNA-PRC2 interactions in vivo and recruitment of Polycomb proteins in cis. Thus, RNA cofactors may be one general mechanism, among others, for targeting mammalian PRC2. Given PRC2's essential roles during stem cell renewal, development, and cancer, the PRC2 transcriptome described herein will provide a valuable resource for regenerative medicine and cancer biology. Identification and characterization of RNAs associated with PRC2 complex in mouse embryoinc stem cells

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