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

基本信息

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
项目类别：
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

项目摘要

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环是当新生的RNA链退火时在转录过程中形成的非B DNA结构模板DNA链形成RNA：DNA杂交。 Chedin实验室已经证明R-loops很普遍并构成在整个人类基因组中形成的结构。了解R-loops的功能生理和病理状况是该领域的重要目标，因为R环的错误调节具有越来越多的人类疾病暗示了我们。该领域的主要机制是剪接抑制作用会导致未剪切的新生转录物增加，然后更容易侵入后面的DNA 前进的RNA聚合酶。要发现剪接破坏与R环之间的连接，我将专注于SF3B1，这是SF3B复合体中的亚基，在剪接体的早期阶段起着至关重要的作用集会。重要的是，Pladienolide B（PladB）是一种直接抑制SF3B剪接的天然产物结合。这就是PLADB提供了一种以临时方式评估动态变化的工具。符合可用的文献，我最初的假设是PLADB治疗将导致R环的形成升高积累未填充成绩单的区域。但是，初步数据与这个想法不一致，相反，大多数涉及SF3B抑制的R环变化是由扰动引起的转录动力学。早期终止事件通过基因体导致定向R环损失。缺乏相比之下，基因末端的终止是“基因（狗）下游”的转录和定向R环的导致在狗区域上获得收益。这两个事件都集体影响一千多个基因。狗的转录已经观察到几种环境应力。这增加了剪接抑制是一个的可能性共享分子链路，驱动狗的转录。病毒感染后的狗转录已进一步联系大规模染色质在整个狗区域开放。这增加了R-loops的可能性，包括一个刚性A形式的RNA：DNA杂化，通过预防核小体引起染色质脱敏包装或沉积。那是我修订的假设是急性剪接抑制会影响转录伸长谱并导致共转录R环的基因组模式的变化。目标1意志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。我期望阐明剪接抑制与R环形成之间的暂时和位置关系高分辨率并确定SF3B在调节转录动力学中的新作用。 AIM 2将确定是否由狗转录驱动器在不同细胞下染色质体系结构的变化产生的R环变化压力。该项目将为共同剪接和 R环形成及其对在压力下的转录动态和染色质体系结构的影响状况。