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环是非B DNA结构，可将晶状RNA链退火至 模板DNA链形成RNA：Chedin实验室的DNA杂交。 以及整个人类基因组形成的保守结构。 生理环境和病理状况是现场的重要目标 与越来越多的人类混乱有关。 抑制作用会导致未剪切的新生转录物增加，然后更多地侵入后面的DNA RNA聚合酶的前进。 专注于SF3B1，这是SF3B复合体中的亚基，在剪接的早期阶段起着至关重要的作用 组件。 因此，PLADB提供了一种以时间方式评估动态变化的工具。 可用的文献，我的最初假设是。 但是 相反，表明大多数tacompany sf3b抑制是由扰动引起的大多数R环变化 转录动力学。 相比之下，基因末端的终止是“基因（狗）下游”的转录和定向R-L-L-loop 狗区域的收益均已影响一千个基因。 观察到响应磨光应力。 在病毒感染时驱动狗转录的共享分子链接已进一步联系 大规模染色质开放狗区域。 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位 包装或沉积。 伸长曲线并导致共转录R-L环的基因组模式的变化 确定急性剪接的全局动态效应对剪接，R环和转录模式的效果 期望阐明剪接抑制与R环形成之间的时间和位置关系 高分辨率并确定SF3B在调节转录动力学中的新作用。 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 强调这个项目。 R环形成以及对转录动力学和染色质体系结构的影响 状况。