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Unknown,Transcriptomics,Genomics,Proteomics

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RNA-biotin based pull down assay with polyA site (PAS) RNA substrates in Hela Nuclear Extract followed by RNA-seq to discover trans-acting RNA molecules involved in mRNA 3 processing

ABSTRACT: We aimed to discover trans-acting RNA molecules involved in mRNA 3 processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3 processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3 processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used two polyA site (PAS) RNA substrates, SV40 late (SVL) and adenovirus L3 pre-mRNAs, and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3 end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3 processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. we extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing. See related experiments: E-MTAB-5384; E-MTAB-5389

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: chengguo yao

PROVIDER: E-MTAB-5383 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-biotin based pull down assay with Hela Nuclear Extract followed by RNA-seq

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HeLa mRNA interactome

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