Impact of RNA splicing factor mutations on circular RNA biogenesis in leukemia

项目总结/摘要 在美国，大约10%的新癌症诊断是血液恶性肿瘤，包括 一系列血癌和相关疾病。慢性淋巴细胞白血病（CLL）和髓系白血病 在肿瘤中，体细胞热点突变经常发生在五个RNA剪接因子（SF）中：SF 3B1，SRSF2， U2AF1、ZRSR2和编码U1 snRNA的基因。这些突变驱动mRNA的异常剪接， 促进白血病发生。最近，发现了一类称为环状RNA（circRNA）的新型RNA， 在多种类型的液体肿瘤中异常表达。与通过正常剪接形成的mRNA不同， 线性RNA，circRNA通过导致RNA环化的反向剪接产生。因为 随着检测和注释方法的进步，现在已知circRNA具有功能性和临床应用价值。 意义，表明在癌症生物学中的新作用。然而，迄今为止，它们在血液学中的确切作用 恶性肿瘤，即白血病，仍然没有定义。长期目标是研究功能和 CircRNA的治疗潜力。此外，SF突变（SF3B1、SRSF2、U2AF1、ZRSR2和 编码U1 snRNA的基因）在circRNA异常表达中的作用仍然未知。因此， 目的是将SF突变与异常circRNA表达联系起来。为此，我假设， SF3B1和其他SF如SRSF2、U2AF1、ZRSR2和编码U1 snRNA的基因上调circRNA 促进白血病发生。这项研究的基本原理是，将SF突变与异常 circRNA的表达将定义一个新的调控轴，为癌症的治疗提供新的机会。 治疗白血病来自CLL患者B细胞和细胞系的初步数据显示，突变型SF 3B1促进了细胞凋亡。 在生物学重要的分子途径如蛋白质转运和细胞中的异常表达 周期调节为了鉴定对细胞存活至关重要的circRNA，我验证了新出现的CRISPR CasRX。 技术作为筛选circRNA的工具。我还建立了一个实验性的工作流程原型， 这些circRNA的功能。此外，为了进一步确定SF突变对circRNA生物发生的作用， 具有SF 3B1、SRSF 2、U2 AF 1、ZRSR2和编码U1 snRNA的基因体细胞突变的细胞系 确立了习最后，我设计了基于细胞和基于小基因的报告系统，以研究 backsplicing的机制。使用这些工具，我提出了以下目标：识别和验证 SF突变相关circRNA在白血病中的功能影响（目的1），并确定 反向剪接的机制（目的2）。我希望这项提案的结果将定义一个新的监管轴心 将SF突变和circRNA异常表达与白血病发生联系起来。

PROJECT SUMMARY/ABSTRACT Approximately 10% of new cancer diagnoses in the United States are hematological malignancies that include a spectrum of blood cancers and related disorders. In chronic lymphocytic leukemia (CLL) and myeloid neoplasms, somatic hotspot mutations frequently occur in five RNA splicing factors (SFs): SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA. These mutations drive aberrant splicing of mRNAs to promote leukemogenesis. Recently, a novel class of RNAs called circular RNA (circRNA) was found to be aberrantly expressed in many types of liquid tumors. Unlike mRNAs that form through normal splicing to produce linear RNAs, circRNAs are produced through backsplicing that results in RNA circularization. Because of advances to detection and annotation methods, circRNAs are now known to possess functional and clinical significances, suggesting a novel role in cancer biology. However, to date, their precise role in hematological malignancies, namely leukemia, remains undefined. The long-term goal is to investigate the functions and therapeutic potentials of circRNAs. Moreover, the role of SF mutations (SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA) in the aberrant expression of circRNAs remains unknown. Thus, the overall objective is to link SF mutations to aberrant circRNA expressions. To this end, I hypothesize that mutations in SF3B1 and other SFs such as SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA upregulate circRNA abundance to promote leukemogenesis. The rationale for this research is that linking SF mutations to aberrant expressions of circRNAs would define a novel regulatory axis, unlocking new therapeutic opportunities for treating leukemia. Preliminary data from CLL patient B cells and cell lines showed that mutant SF3B1 promoted aberrant expression of circRNAs in biological important molecular pathways such as protein transport and cell cycle regulation. To identify circRNAs critical for cell survival, I have validated the emergent CRISPR CasRX technology as a tool for screening circRNAs. I have also prototyped an experimental workflow for validating the functions of these circRNAs. Additionally, to further determine the role of SF mutations on circRNA biogenesis, cell lines with somatic mutations for SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA have been established. Finally, I have engineered cell-based and minigene-based reporter systems to investigate the mechanisms of backsplicing. Using this collection of tools, I propose the following aims: to identify and validate the functional impact of SF mutation-associated circRNA in leukemia (Aim 1) and to determine the mechanism of backsplicing (Aim 2). I expect the findings from this proposal will define a novel regulatory axis linking SF mutations and circRNAs aberrant expressions to leukemogenesis.