GSE159993
Genome-wide analysis of PTBP1-regulated retained introns in mESCs
Intron retention (IR) is an alternative splicing event where mRNAs containing unspliced introns are exported to the cytoplasm and then either translated, giving rise to new protein isoforms, or degraded via the nonsense-mediated decay pathway. However, unspliced introns are also seen in nuclear RNA. Some of these are spliced at a low rate but do eventually become fully spliced and their respective mRNAs exported, while others stay retained and are recognized by nuclear decay machineries. IR has been shown to be regulated during granulocyte and neuronal differentiation and in some cancers, and a subset of the nuclear retained introns, called detained introns, have been implicated in growth control pathways. Despite many findings on IR and its biological significance, it is still not clear whether an incompletely spliced intron observed is a true retained intron that is exported as an mRNA or is a product of slow splicing that remains in the nucleus. A better understanding is needed of the molecular events that reduce intron excision rate and determine the release of an RNA for export. We have found that some genes in mouse embryonic stem cells (mESCs) produce RNAs that are highly associated with the high molecular-weight nuclear pellet (chromatin), rather than the soluble nucleoplasm or cytoplasm. This chromatin-associated RNA is polyadenylated, usually contains one or more incompletely spliced introns, and is often extensively bound by polypyrimidine tract-binding protein 1 (PTBP1). We hypothesize that PTBP1 inhibits splicing of these introns, and this causes sequestration of the transcript on chromatin. We are studying the role of PTBP1 in inhibiting intron excision and anchoring RNAs in the nuclear and chromatin compartments of mESCs. Overall design: E14 mESCs were treated with eGFP (negative control), PTBP1 and PTBP1+PTBP2 siRNAs for 48 hours. The cells were fractionated, and total RNA from each subcellular compartment was extracted. PolyA+ RNA was isolated for RNA sequencing and intron retention analyses. Three biological replicates from each condition were used.