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

项目摘要/摘要 在美国，大约10%的新诊断癌症是血液系统恶性肿瘤，包括 血癌和相关疾病的谱系。慢性淋巴细胞白血病(CLL)和髓系白血病 肿瘤、体细胞热点突变频繁发生在五种RNA剪接因子(SFS)中：SF3B1、SRSF2、 U2AF1、ZRSR2和编码U1短链RNA的基因。这些突变导致mRNAs的异常剪接 促进白血病发生。最近，一类新的被称为环状RNA(CircRNA)的RNA被发现 在多种类型的液体肿瘤中均有异常表达。与通过正常剪接形成的mRNAs不同，mRNAs 线性RNA，CircRNA是通过后剪接产生的，导致RNA环化。因为. 检测和注释方法的进展，CircRNA现在已知具有功能和临床 意义，暗示了癌症生物学中的一个新角色。然而，到目前为止，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。长期目标是研究它们的功能和 CircRNAs的治疗潜力。此外，SF突变(SF3B1、SRSF2、U2AF1、ZRSR2和 在CircRNAs的异常表达中，编码U1SnRNA的基因仍不清楚。因此，总体上， 目的是将SF突变与CircRNA的异常表达联系起来。为此，我假设 SF3B1和其他SFS，如SRSF2、U2AF1、ZRSR2和编码U1单链RNA的基因上调CircRNA 富含促进白血病发生的物质。这项研究的基本原理是将SF突变与异常联系起来 CircRNAs的表达将定义一个新的调节轴，释放新的治疗机会 治疗白血病。来自CLL患者B细胞和细胞系的初步数据显示，突变的SF3B1促进 CircRNAs在蛋白质转运和细胞等生物重要分子途径中的异常表达 周期调节。为了确定对细胞生存至关重要的CircRNAs，我验证了新出现的CRISPR CasRX 技术作为筛选CircRNA的工具。我还制作了一个试验性工作流的原型，用于验证 这些CircRNA的功能。此外，为了进一步确定SF突变在CircRNA生物发生中的作用， 具有SF3B1、SRSF2、U2AF1、ZRSR2和编码U1SnRNA基因的体细胞突变的细胞系 已经建立了。最后，我设计了基于细胞和基于微型基因的报告系统来研究 反向剪接的机制。使用这组工具，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(Aim 1)，并确定 反向剪接机制(目标2)。我预计这项提案的发现将定义一个新的监管轴心 SF突变和CircRNAs异常表达与白血病的发生有关。

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.