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.

摘要/项目摘要 真核生物中普遍存在的转录和前信使核糖核酸加工的需要需要理解 它们同时发生并相互调节的机制。我们的长期目标是 确定控制共转录内含子去除的机制以及共转录剪接如何 有助于基因表达调控。在之前这笔赠款的支持下，我的实验室开创了 转录和RNA的瞬时中间产物新生RNA的纯化和测序策略 加工--相对于延长的RNA聚合酶II(POL II)的位置来量化前mRNA剪接 基因。大多数内含子一旦从Pol II出现就被移除，这表明Pol II和剪接体 都被安排在一起行动，而且身体上彼此接近。认识到这一点，我们的总体目标是：(一) 阐明“全有或全无”RNA加工的共转录机制，在该机制中，个体新生 转录本要么在3‘端完全剪接和切割(ALL)，要么完全未剪接和未切割(NONE)。我的实验室 最近通过对全长的新生RNA进行测序发现了这一点，它是一种主要的共转录调控 B-珠蛋白基因表达的机制。(2)确定剪接潜力如何变得有限，因为 转录继续进行，新生的抄本变得更长。和(三)决定命运和可能的功能 未处理的成绩单。中心假设是，新生的RNA结合正性和 负性作用的RNA结合蛋白和/或经历分子间碱基配对和紧凑增加 随着新生的链在转录过程中增长。我们将通过追求三个具体目标来检验这一假设：1) 开发一种方法来确定新生RNA的局部碱基配对行为，使用DMS化学和 新生的RNA提纯。这个目标的结果将告诉我们，新生的RNA在多大程度上 转录过程中的瞬时结构以及局部结构是否与对 剪接，2)识别在生长的新生链上积累的激活蛋白和/或抑制蛋白 转录，使用生化纯化和体内标记方法。3)确定核反应 到未经处理的“无”成绩单。他们在处理过程中有没有延误？或者它们会被降级吗？为此，我们 已经建立了A-Count方法，该方法将整个转录本从5‘端到3’端进行测序，包括 Polya尾巴的全长，将按核苷酸类型和数量计算。我们的初步结果 证明核聚合体结合蛋白PABPN1在有丝分裂过程中受到磷酸化的调节， 提示核内含子保留的mRNAs的不同命运。PABPN1是一种突变的蛋白质 眼咽肌营养不良症(OPMD)。拟议的研究具有重要意义，因为这些机制 在前信使核糖核酸加工的水平上操作可以使蛋白质产品的数量至少增加10倍，这表明 RNA加工对基因表达的影响与转录因子的调控同等重要。

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