Uncovering novel roles for splicing factor SF3B1 in transcription dynamics, R-loop metabolism, and chromatin structure

揭示剪接因子 SF3B1 在转录动力学、R 环代谢和染色质结构中的新作用

• 批准号: 9910740
• 负责人: Daisy Castillo Guzman
• 金额: $ 3.79万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-26 至 2022-03-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910740
• 关键词: ATAC-seq; Acute; Acute Erythroblastic Leukemia; Acute Myelocytic Leukemia; Affect; Architecture; Binding; Cell Line; Cells; Cellular Stress; Chromatin; Chromatin Structure; Complex; DNA; DNA Structure; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Defect; Deposition; Disease; Dysmyelopoietic Syndromes; ENG gene; Elderly; Enzymes; Essential Genes; Event; Gene Expression; Genes; Genetic Transcription; Genomic Instability; Genomics; Goals; Hematological Disease; Hour; Human; Human Genome; Hybrids; Invaded; K-562; K562 Cells; Lead; Link; Literature; Measures; Mediating; Medical; Metabolism; Molecular; Mutagenesis; Mutation; Natural Products; Nucleosomes; Oxidative Stress; Pathologic; Pattern; Physiological; Play; Process; RNA; RNA Splicing; Regulation; Resolution; Role; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Stress; Structure; Testing; Time; Transcript; Transcription Elongation; U2 Small Nuclear Ribonucleoprotein; Virus Diseases; base; driver mutation; genome-wide; insight; novel; prevent; replication stress; response; ribonuclease H1; tool; transcriptome sequencing

Project Summary R-loops are non-B DNA structures that form during transcription when the nascent RNA strand anneals to the template DNA strand forming a RNA:DNA hybrid. The Chedin lab has demonstrated that R-loops are prevalent and conserved structures that form throughout the human genome. Understanding the function of R-loops under physiological and pathological conditions is an important goal in the field, because mis-regulation of R-loops has been implicated in a growing number of human disorders. A leading mechanism in the field is that splicing inhibition causes an increase in unspliced nascent transcripts that can then more readily invade the DNA behind the advancing RNA polymerase. To uncover the connections between splicing disruption and R-loops, I will focus on SF3B1, a subunit in the SF3b complex which plays a critical role in the early stages of spliceosome assembly. Importantly, Pladienolide B (PladB) is a natural product that directly inhibits splicing upon SF3b binding. Thus, PladB provides a tool for assessing dynamic changes in a temporal manner. In keeping with available literature, my initial hypothesis was that PladB treatment will lead to elevated R-loop formation over regions that accumulate unspliced transcripts. Preliminary data, however, is inconsistent with this idea and instead suggests that most R-loop changes that accompany SF3b inhibition are caused by perturbation of transcriptional dynamics. Early termination events cause directional R-loop losses through gene bodies. Lack of termination at gene ends, by contrast, cause “downstream of gene (DoG)” transcription and directional R-loop gains over DoG regions. Both events collectively affect over a thousand genes. DoG transcription has been observed in response to several environmental stresses. This raises the possibility that splicing inhibition is a shared molecular link that drives DoG transcription. DoG transcription upon viral infection has been further linked to large scale chromatin opening throughout the DoG region. This raises the possibility that R-loops, which include a rigid A-form-like RNA:DNA hybrid, cause chromatin decondensation by preventing nucleosome wrapping or deposition. Thus, my revised hypothesis is that acute splicing inhibition affects transcription elongation profiles and leads to shifts in the genomic patterns of co-transcriptional R-loops. Aim 1 will determine the global dynamic effects of acute splicing inhibition on splicing, R-loop and transcription patterns. I expect to clarify the temporal and positional relationships between splicing inhibition and R-loop formation at high-resolution and to identify a novel role for SF3b in regulating transcription dynamics. Aim 2 will determine if R-loops generated from DoG transcription drive changes in chromatin architecture under different cellular stresses. This project will provide key insights into the inter-relationship between co-transcriptional splicing and R-loop formation and their impact on transcriptional dynamics and chromatin architecture under stress conditions.

项目摘要 R环是在转录过程中当新生RNA链退火到DNA链时形成的非B DNA结构。 模板DNA链形成RNA：DNA杂交体。Chedin实验室已经证明了R环是普遍存在的 和整个人类基因组中形成的保守结构。了解R环的功能 生理和病理条件是该领域的重要目标，因为R环的错误调节 与越来越多的人类疾病有关该领域的主要机制是， 抑制导致未剪接的新生转录物增加，然后更容易侵入DNA， 不断发展的RNA聚合酶为了揭示剪接中断和R环之间的联系，我将 SF3B1是SF3b复合物中的一个亚基，在剪接体的早期阶段起着关键作用 组装件.重要的是，普拉地辛B（Plad B）是直接抑制SF 3 B剪接的天然产物 约束力因此，PladB提供了一个工具，用于评估动态变化的时间方式。符合 根据现有文献，我最初的假设是PladB治疗将导致R环形成增加， 积累未剪接转录本的区域。然而，初步数据与这一想法不一致， 相反，这表明大多数伴随SF3b抑制的R环变化是由SF3b的扰动引起的。 转录动力学早期终止事件导致通过基因体的定向R环损失。缺乏 相反，在基因末端终止引起“基因下游（DoG）”转录和定向R环 超过DOG地区。这两个事件共同影响了一千多个基因。DoG转录已被 这是对几种环境压力的反应。这就提出了剪接抑制是一种可能性， 驱动DoG转录的共有分子链。病毒感染后的DoG转录进一步与 大规模的染色质开放整个DoG区域。这就提出了R环的可能性， 包括刚性A型样RNA：DNA杂合体，通过阻止核小体 包裹或沉积。因此，我修正的假设是，急性剪接抑制影响转录 延伸概况，并导致共转录R环的基因组模式的转变。目标1将 确定急性剪接抑制对剪接、R环和转录模式的全局动态影响。我 希望澄清剪接抑制和R环形成之间的时间和位置关系， 高分辨率，并确定SF3b在调节转录动力学的新作用。目标2将确定是否 由DoG转录产生的R环在不同细胞分化下驱动染色质结构的变化。 压力该项目将提供关键的见解之间的相互关系共转录剪接和 R环形成及其对应激下转录动态和染色质结构的影响 条件