Novel Spliceosomal Defects in Myelodysplastic Syndromes

 描述（由申请人提供）：骨髓增生异常综合征（MDS）的特征是发育异常的无效造血、血细胞减少和白血病进展。新的技术进步使得对遗传性体细胞缺陷的分析得以改进。在新发现的病变中，几个剪接体因子基因经常被发现发生突变。这些包括SF 3B 1、ZRSR 2、SRSF 2和U2 AF 1中的常见突变，以及PRPF 8、DDX 41等中不太常见的突变。这一发现导致了这样的假设，即靶基因剪接模式的改变在MDS的建立或进展中起着重要作用。该建议的重点是我们最近发现的LUC 7 L2基因失活突变和由于涉及LUC 7 L2基因座的7 q34缺失而导致的频繁单倍不足。临床分析显示LUC 7 L2的缺失/低表达与不良结局相关。最近发表的数据显示LUC 7 L2可以逆转del 7 q人iPS细胞中的缺陷性分化。初步数据显示，具有LUC 7 L2单倍不足的工程化人iPS细胞具有与del 7 q类似的分化缺陷。LUC 7 L2被认为在剪接期间调节5'剪接位点选择。RNA-Seq结果表明，由于LUC 7 L2的缺陷，多个基因改变了剪接模式。我们的建议是基于这样的假设，即突变和/或单倍不足的剪接体蛋白，如LUC 7 L2导致特定类型的错误剪接的特定或不同的组合TSG和最终，剪接体缺陷可能影响特定的基因或途径的其他分子缺陷的表型后果。该提案的目标是了解LUC 7 L2缺陷与MDS发展和进展相关的后果。将在培养物中研究原代细胞和工程化shRNA敲低细胞和LUC 7 L2敲除iPS细胞中单倍不足的影响，包括对增殖、分化和凋亡的影响。我们将使用深度RNA测序进行剪接分析，以表征剪接功能障碍及其对mRNA表达的影响，以确定下游机制和靶基因。为了鉴定直接剪接靶标，将在体内和体外用LUC 7 L2敲低和突变体敲入细胞进行RNA剪接测定。RNA CLIP-Seq和直接RNA结合分析将用于确定LUC 7 L2作用的体内RNA底物。最后，我们将研究候选下游基因的作用，其剪接和表达在LUC 7 L2缺陷细胞和患者样本中发生改变。初步数据表明，LUC 7 L2水平调节包括SMAD 5在内的几个下游基因的剪接。使用工程化细胞系和/或患者样本，我们将通过使用反义吗啉代寡核苷酸诱导或抑制可变剪接位点来测试错误剪接事件的作用。虽然体外测试将检查生长和分化，但体内实验将在含有LUC 7 L2中单倍不足的敲低、敲除或原代MDS细胞的NSG小鼠异种移植物中进行。

 DESCRIPTION (provided by applicant): Myelodysplastic syndromes (MDS) are characterized by dysplastic ineffective hematopoiesis, cytopenias and leukemic evolution. New technological advances have allowed for improved analysis of genetic somatic defects. Among newly identified lesions, several spliceosomal factor genes were frequently found to be mutated. These included common mutations in SF3B1, ZRSR2, SRSF2 and U2AF1 as well as less prevalent mutations in PRPF8, DDX41 and others. This discovery has led to the hypothesis that alterations in the pattern of splicing of target genes plays a major role in the establishment or progression of MDS. This proposal focuses on our recent identification of inactivating mutations in the LUC7L2 gene and frequent haploinsufficiency due to deletions at 7q34 involving the LUC7L2 locus. Clinical analysis shows that deletion/low expression of LUC7L2 is associated with poor outcome. Recently published data show that LUC7L2 can reverse defective differentiation in del7q human iPS cells. Preliminary data shows that engineered human iPS cells with LUC7L2 haploinsufficiency have defective differentiation similar to del7q. LUC7L2 is thought to regulate 5' splice site choice during splicing. RNA-Seq results suggest that multiple genes have altered splicing patterns due to defects in LUC7L2. Our proposal is based on the hypothesis that mutations and/or haploinsufficiency of spliceosomal proteins such as LUC7L2 leads to specific types of missplicing of specific or distinct combinations of TSG and ultimately, that spliceosomal defects may phenocopy consequences of other molecular defects affecting specific genes or pathways. The goals of the proposal are to understand the consequences of LUC7L2 deficiency in relation to the development and progression of MDS. The effects of haploinsufficiency in primary cells and in engineered shRNA knockdown cells and LUC7L2 knock out iPS cells will be investigated in culture including effects on proliferation, differentiaion and apoptosis. We will perform splicing analysis using deep RNA sequencing to characterize splicing dysfunction and its consequences on mRNA expression to determine downstream mechanisms and target genes. To identify direct splicing targets, RNA splicing assays will be performed in vivo and in vitro with LUC7L2 knocked down and mutant knock-in cells. RNA CLIP-Seq and direct RNA binding analyses will be used to define the in vivo RNA substrates of LUC7L2 action. Finally, we will examine the roles of candidate downstream genes whose splicing and expression is altered in LUC7L2 defective cells and patient samples. Preliminary data suggests that LUC7L2 levels regulate the splicing of several downstream genes including SMAD5. Using engineered cell lines and/or patient samples, we will test the roles of the missplicing events by inducing or inhibiting alternative splice sites using antisense morpholino oligonucleotides. While in vitro testing will examine growth and differentiation, in vivo experiments will be carried out in NSG mouse xenografts containing knockdown, knock out or primary MDS cells haploinsufficient in LUC7L2.