Analysis of co-transcriptional RNA processing by RNA-ChIP assay

Abstract

It was initially assumed that RNA biogenesis and processing were two independent processes with transcripts undergoing splicing only after being completely synthesized and released from the DNA template. However, transcription and splicing are tightly linked and increasing evidence shows that nascent transcripts can undergo splicing in the vicinity of chromatin while still attached to the RNA polymerase II (RNAPII) transcriptional machinery. These co-transcriptionally spliced RNA molecules are very labile due to dynamic processing and represent a minor subpopulation among total cellular RNA species. Thus, it is difficult to isolate these RNAs in order to study the dynamics and mechanisms of co-transcriptional RNA splicing. To overcome this problem, the RNA-chromatin immunoprecipitation (ChIP) assay, adapted from classical ChIP, allows to co-purify and isolate nascent RNAs after immunoprecipitation of RNAPII. Thanks to this technique, we have shown that co-transcriptional RNA splicing occurs with distinct efficiencies for different genes and different exons of a given transcript and can represent a rate-limiting step in the biological response of messenger RNA synthesis to extracellular stimuli and drug treatments.