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Genome-wide identification of polycomb-associated RNAs by RIP-seq.

ABSTRACT: Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.

SUBMITTER: Zhao J

PROVIDER: S-EPMC3021903 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Genome-wide identification of polycomb-associated RNAs by RIP-seq.

Zhao Jing J Ohsumi Toshiro K TK Kung Johnny T JT Ogawa Yuya Y Grau Daniel J DJ Sarma Kavitha K Song Ji Joon JJ Kingston Robert E RE Borowsky Mark M Lee Jeannie T JT

Molecular cell 20101201 6

Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number o ...[more]

PMID: 21172659

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Project description:BackgroundSmall non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm.ResultsHere, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm.ConclusionsUsing an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.

Dataset Information

Genome-wide identification of polycomb-associated RNAs by RIP-seq.

Publications

Genome-wide identification of polycomb-associated RNAs by RIP-seq.

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