Novel Spliceosomal Defects in Myelodysplastic Syndromes

 描述(申请人提供)：骨髓增生异常综合征(MDS)的特征是发育不良的无效造血，细胞减少和白血病的演变。新的技术进步使得对遗传体细胞缺陷的分析得到了改进。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。这些突变包括常见的SF3B1、ZRSR2、SRSF2和U2AF1突变，以及PRPF8、DDX41等不太常见的突变。这一发现导致了一种假说，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。这项建议集中在我们最近发现的LUC7L2基因的失活突变和由于涉及LUC7L2基因的7q34缺失而导致的频繁的单倍体不足。临床分析表明，LUC7L2基因缺失/低表达与预后不良相关。最近发表的数据表明，LUC7L2可以逆转del7q人iPS细胞的分化缺陷。初步数据显示，具有LUC7L2单倍体缺陷的工程化人iPS细胞具有类似于del7q的分化缺陷。LUC7L2被认为在剪接过程中调节5‘剪接位点的选择。RNA-Seq结果表明，由于LUC7L2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，即剪接体蛋白(如LUC7L2)的突变和/或单倍体不足导致特定类型的TSG特定或不同组合的错接，最终剪接体缺陷可能是影响特定基因或途径的其他分子缺陷的表型复制后果。该提案的目标是了解LUC7L2缺陷与MDS的发展和进展有关的后果。单倍体缺陷在原代细胞、工程shRNA敲除细胞和LUC7L2敲除iPS细胞中的影响将在培养中进行研究，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。为了确定直接剪接靶点，将在体内和体外对LUC7L2被击倒的细胞和突变的敲入细胞进行RNA剪接实验。RNA Clip-Seq和直接RNA结合分析将用于定义LUC7L2作用的体内RNA底物。最后，我们将研究剪接和表达发生变化的候选下游基因在LUC7L2缺陷细胞和患者样本中的作用。初步数据表明，LUC7L2水平调节包括Smad5在内的几个下游基因的剪接。使用工程细胞系和/或患者样本，我们将通过使用反义吗啉寡核苷酸诱导或抑制替代剪接位点来测试错剪事件的作用。虽然体外测试将检查生长和分化，但体内实验将在含有LUC7L2基因敲除、敲除或原代MDS细胞单倍体缺陷的NSG小鼠移植瘤中进行。

 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.