Cross-regulation between transcription and pre-mRNA splicing

Abstract/Project Summary The ubiquitous need for transcription and pre-mRNA processing in eukaryotes requires an understanding of the mechanisms by which they occur simultaneously and regulate each other. Our long-term goals are to determine the mechanisms that govern co-transcriptional intron removal and how co-transcriptional splicing contributes to gene expression regulation. With previous support from this grant, my lab has pioneered strategies for purifying and sequencing nascent RNA – the transient intermediates in transcription and RNA processing – to quantify pre-mRNA splicing relative to the position of elongating RNA Polymerase II (Pol II) on the gene. Most introns are removed as soon as they emerge from Pol II, indicating that Pol II and the spliceosome are timed to act together and physically close to one another. Knowing this, our overall objectives are to (i) elucidate the co-transcriptional mechanism of “all-or-none” RNA processing, in which individual nascent transcripts are either fully spliced and cleaved at the 3’ end (all) or fully unspliced and uncleaved (none). My lab discovered this recently by sequencing full-length nascent RNAs, and it is a major co-transcriptional regulatory mechanism for b-globin gene expression. (ii) Determine how the potential for splicing becomes limited as transcription proceeds and the nascent transcript gets longer. And (iii) Determine the fate and possible function of the unprocessed transcripts. The central hypothesis is that the potential for nascent RNA to bind positive- and negative-acting RNA binding proteins and/or to undergo intermolecular base-pairing and compaction increases as the nascent chain grows during transcription. We will test this hypothesis by pursuing three specific aims: 1) develop a method to determine the local base-pairing behavior of nascent RNA, using DMS chemistry and nascent RNA purification. The results of this aim will tell us the degree to which nascent RNA becomes transiently structured during transcription and if local structures correlate with positive or negative effects on splicing, 2) identify activator and/or repressor proteins that accumulate on the growing nascent chain during transcription, using biochemical purification and in vivo labeling approaches. 3) determine the nuclear response to unprocessed “none” transcripts. Are they delayed in processing? Or will they be degraded? For this aim, we have established the A-count method, which sequences the entire transcript from 5’ end to 3’ end, including the whole length of the polyA tail, which will be counted by nucleotide type and number. Our preliminary results show that the nuclear polyA binding protein PABPN1 is regulated by phosphorylation during mitosis, suggesting differential fates for nuclear retained mRNAs with introns. PABPN1 is a protein mutated in Oculopharyngeal muscular dystrophy (OPMD). The proposed research is significant because these mechanisms operating at the level of pre-mRNA processing can change protein product amounts by at least 10-fold, showing that RNA processing is as important for gene expression as regulation by transcription factors.