Cross-regulation between transcription and pre-mRNA splicing

转录和前 mRNA 剪接之间的交叉调节

• 批准号: 10735170
• 负责人: Karla M Neugebauer
• 金额: $ 50.41万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735170
• 关键词: Acute; Address; Architecture; Base Pairing; Behavior; Binding; Binding Proteins; Biochemical; Biological; Cells; Chemicals; Chemistry; Code; Coupling; DNA; Erythropoiesis; Eukaryota; Event; Excision; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Globin; Goals; Grant; Individual; Introns; Knowledge; Label; Learning; Length; Link; Mammalian Cell; Mammals; Mass Spectrum Analysis; Measures; Messenger RNA; Methods; Mitosis; Modeling; Mutate; Nuclear; Nuclear RNA; Nucleotides; Oculopharyngeal Muscular Dystrophy; Output; Phosphorylation; Point Mutation; Polymerase; Positioning Attribute; Predisposition; Proteins; RNA; RNA Folding; RNA Polymerase II; RNA Processing; RNA Splicing; RNA Stability; RNA purification; RNA-Binding Proteins; Regulation; Repressor Proteins; Research; Resolution; Role; Saccharomycetales; Site; Splice-Site Mutation; Spliceosomes; Structure; System; Tail; Testing; Therapeutic; Trans-Activators; Transact; Transcript; Transcription Elongation; Transcriptional Regulation; U1 Small Nuclear Ribonucleoprotein; Vaccines; Yeasts; beta Globin; exosome; gene panel; improved; in vivo; mRNA Precursor; novel; overexpression; posttranscriptional; recruit; response; species difference; transcription factor

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.

抽象/项目总结

项目成果

Long-read sequencing of nascent RNA reveals coupling among RNA processing events.

• DOI: 10.1101/gr.232025.117
• 发表时间: 2018-07
• 期刊: Genome research
• 影响因子: 7
• 作者: Herzel L;Straube K;Neugebauer KM
• 通讯作者: Neugebauer KM

Nascent RNA and the Coordination of Splicing with Transcription.

新生 RNA 以及剪接与转录的协调。

• DOI: 10.1101/cshperspect.a032227
• 发表时间: 2019
• 期刊: Cold Spring Harbor perspectives in biology
• 影响因子: 7.2
• 作者: Neugebauer,KarlaM

Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II.

• DOI: 10.1016/j.cell.2016.02.045
• 发表时间: 2016-04-07
• 期刊: Cell
• 影响因子: 64.5
• 作者: Oesterreich FC;Herzel L;Straube K;Hujer K;Howard J;Neugebauer KM
• 通讯作者: Neugebauer KM

Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA.

整个转录组的映射揭示了包括mRNA在内的二氢岛景观。

• DOI: 10.1371/journal.pbio.3001622
• 发表时间: 2022-05
• 期刊: PLOS BIOLOGY
• 影响因子: 9.8
• 作者: Draycott, Austin S.;Schaening-Burgos, Cassandra;Rojas-Duran, Maria F.;Wilson, Loren;Scharfen, Leonard;Neugebauer, Karla M.;Nachtergaele, Sigrid;Gilbert, Wendy, V
• 通讯作者: Gilbert, Wendy, V

Nuclear mechanisms of gene expression control: pre-mRNA splicing as a life or death decision.

• DOI: 10.1016/j.gde.2020.11.002
• 发表时间: 2021-04
• 期刊: Current opinion in genetics & development
• 影响因子: 4
• 作者: Gordon JM;Phizicky DV;Neugebauer KM
• 通讯作者: Neugebauer KM