Novel Spliceosomal Defects in Myelodysplastic Syndromes

骨髓增生异常综合征中的新型剪接体缺陷

• 批准号: 9335972
• 负责人: Jaroslaw P Maciejewski
• 金额: $ 60.83万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335972
• 关键词: 5' Splice Site; 5q31; Affect; Alleles; Alternative Splicing; Apoptosis; Biological Assay; CD34 gene; Cell Line; Cell model; Cells; Chromosome abnormality; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Defect; Development; Differentiation and Growth; Disease; Dysmyelopoietic Syndromes; Engineering; Event; Evolution; Failure; Functional disorder; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genomics; Goals; Hematopoiesis; Heterogeneity; Human; Human Engineering; In Vitro; Ineffective Hematopoiesis; Knock-in; Knock-out; Lesion; Link; Marrow; Mediating; Messenger RNA; Modeling; Molecular; Mus; Mutate; Mutation; Myeloproliferative disease; Oligonucleotides; Oncogenic; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pattern; Phenocopy; Phenotype; Play; Protein Isoforms; Proteins; Publishing; RNA; RNA Binding; RNA Helicase; RNA Processing; RNA Splicing; Regulation; Role; SRSF2 gene; Sampling; Site; Somatic Mutation; Specificity; Spliced Genes; Spliceosomes; System; Technology; Testing; Tumor Suppressor Genes; Xenograft procedure; base; clinical phenotype; crosslinking and immunoprecipitation sequencing; cytopenia; experimental study; genetic analysis; improved; in vitro testing; in vivo; induced pluripotent stem cell; knock-down; leukemia; leukemogenesis; loss of function mutation; mRNA Expression; mutant; new therapeutic target; novel; overexpression; public health relevance; small hairpin RNA; transcriptome sequencing

 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.

 描述（由申请人提供）：骨髓增生异常综合征（MDS）的特征是发育不良、无效造血、血细胞减少和白血病进化。在新发现的体细胞缺陷中，经常发现一些剪接体因子基因。这些包括 SF3B1、ZRSR2、SRSF2 和 U2AF1 中的常见突变以及 PRPF8 中不太常见的突变， DDX41 等发现导致了这样的假设：靶基因剪接模式的改变在 MDS 的建立或进展中发挥着重要作用。该提议的重点是我们最近发现的 LUC7L2 基因失活突变和频繁的单倍体不足。由于涉及 LUC7L2 基因座的 7q34 缺失，临床分析表明 LUC7L2 的缺失/低表达与不良结果相关。初步数据显示，具有 LUC7L2 单倍体不足的工程化人类 iPS 细胞具有与 del7q 类似的分化缺陷，RNA-Seq 结果表明多个基因具有调节 5' 剪接位点的选择。由于 LUC7L2 的缺陷而改变的剪接模式我们的建议是基于以下假设：突变和/或单倍体不足。 LUC7L2 等剪接体蛋白会导致特定类型的 TSG 特定或不同组合的错误剪接，最终，剪接体缺陷可能会影响特定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 LUC7L2 缺陷的后果。原代细胞和工程 shRNA 敲除细胞和 LUC7L2 敲除 iPS 细胞中单倍体不足的影响将与 MDS 的发生和进展相关。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。使用 LUC7L2 敲低和突变型敲入细胞进行体内和体外实验，并使用直接 RNA 结合分析来定义 LUC7L2 作用的体内 RNA 底物。 LUC7L2 缺陷细胞和患者样本中剪接和表达发生改变的候选下游基因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，我们将使用工程细胞系和/或患者样本来测试这些下游基因的剪接。通过使用反义吗啉寡核苷酸诱导或抑制选择性剪接位点来研究错误剪接事件的作用，而体外测试将检查生长和分化，而体内实验将在 NSG 中进行。小鼠异种移植物含有 LUC7L2 中敲低、敲除或单倍体不足的原代 MDS 细胞。