The Role of DDX41 in the Spliceosome and Myeloid Neoplasia

DDX41 在剪接体和骨髓瘤形成中的作用

基本信息

批准号：
9332094
负责人：
James Matthew Hiznay
金额：
$ 3.83万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9332094
关键词：
Acute Myelocytic Leukemia Alleles Biochemical Biological Assay Blood Blood Cells Bone Marrow Catalytic Domain Cell Differentiation process Cell Line Cells Complex Disease Dysmyelopoietic Syndromes Dysplasia Event Evolution Family Fellowship Gene Expression Gene Mutation Generations Genes Genomics Germ-Line Mutation Goals Grouping Hematology Heterozygote High-Throughput Nucleotide Sequencing Immunoprecipitation In Vitro Investigation Knock-in Knowledge Laboratories Lead Lesion Malignant - descriptor Malignant Neoplasms Mammalian Cell Methods Missense Mutation Modeling Molecular Molecular Biology Mutation Myelogenous Myeloid Cells Neoplasms Nonsense Mutation Nuclear Nuclear Extract Patients Pattern Pharmaceutical Preparations Phenotype Play Polymerase Chain Reaction Process Proteins Proteomics RNA RNA Binding RNA Helicase RNA Sequences RNA Splicing Recording of previous events Recurrence Reverse Transcription Ribonucleoproteins Role Scientist Site Small Nuclear RNA Somatic Mutation Spliceosome Assembly Pathway Spliceosomes System Training Translational Research Tumor Markers Tumor Suppressor Genes Validation Work aspartylglutamate base carcinogenesis carcinogenicity crosslink crosslinking and immunoprecipitation sequencing disease phenotype exome sequencing experimental study glutamylalanine helicase in vivo inhibitor/antagonist insight knock-down lenalidomide leukemia treatment leukemogenesis mRNA Precursor member mutant novel outcome forecast polypeptide prevent protein crosslink protein function public health relevance small molecule success survival outcome transcriptome sequencing tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Myelodysplastic syndrome (MDS) is a grouping of disease phenotypes characterized as hematological pre-malignancies. MDS treatment focuses on preventing the further transformation of diseased cells into acute myeloid leukemia (AML). A recent unique finding of myeloid pre-malignancies and malignancies is the mutation of components of the spliceosome, the nuclear complex that catalyzes pre-mRNA splicing during gene expression. Whole exome sequencing of MDS patients has identified novel mutations of the gene DDX41 in the germline and somatically. The germline mutation results in a frameshift in the protein, most likely leading to a nonfunctional polypeptide, while the somatic mutation is missense. Patients present primarily as compound heterozygotes, and these mutations confer a more advanced stage of disease and a decreased patient survival outcome. The gene DDX41 encodes a member of the DEAD-box RNA helicase family. Although proteomics has identified DDX41 as a component of the spliceosome catalytic core, no information is available on the exact role the protein plays in splicing or on its interaction partners in the spliceosome. We hypothesize that DDX41 is a bona fide tumor suppressor gene (TSG) and that mutations of it result in the aberrant splicing of other TSGs, resulting in myeloid tumorigenesis. In this project, we will investigate the roles of DDX41 in the spliceosome as well as in myeloid neoplasia. We will determine the RNA interaction sequences of wild type and mutant DDX41 in vivo using a form of crosslinking-immunoprecipitation to purify RNAs bound to DDX41 followed by high-throughput sequencing to analyze these RNAs. We will investigate the effects of DDX41 mutations on the biochemical steps of splicing using DDX41-deficient nuclear extract created from mammalian cells in an in vitro system. We will analyze alterations in splicing as well as cellular phenotype conferred by DDX41 mutations in mammalian cells using in vivo splicing assays as well as RNA- seq. Since MDS patients with DDX41 mutations respond very well to lenalidomide treatment, we will study the impact of this drug on DDX41 both in vivo and in vitro to determine a potential mechanism of action. Successful completion of this application will result in new information on the role a spliceosome core component plays in gene expression as well as disease. Furthermore, the opportunities provided by this fellowship application will enhance the training of a young scientist in the process of collaborative translational research.

描述（由适用提供）：骨髓增生综合征（MDS）是疾病表型的分组，其特征在于血液学前的疗法。 MDS治疗的重点是防止解散细胞进一步转化为急性髓样白血病（AML）。最近对髓样前和恶性肿瘤的独特发现是剪接组成分的突变，剪接体的成分是催化基因表达过程中mRNA剪接的核复合物。 MDS患者的整个表达测序已经确定了种系中基因DDX41的新突变。种系突变会导致蛋白质的移率，最有可能导致非功能性多肽，而体细胞突变是错义的。患者呈现为复合杂合子的初级，并且这些突变会导致疾病的更晚期阶段和患者的生存结果降低。基因DDX41编码Dead-Box RNA解旋酶家族的成员。尽管蛋白质组学已将DDX41识别为剪接催化核的组成部分，但尚无有关蛋白质在剪接或其相互作用伙伴在剪接体中所起的确切作用的信息。我们假设DDX41是一种真正的肿瘤抑制基因（TSG），并且其突变导致其他TSG的异常剪接，从而导致髓样肿瘤发生。在这个项目中，我们将研究DDX41在剪接体以及髓样肿瘤中的作用。我们将使用一种交联免疫沉淀的形式在体内确定野生型和突变型DDX41的RNA相互作用序列，以纯化与DDX41结合的RNA，然后进行高通量测序以分析这些RNA。我们将研究DDX41突变对使用DDX41缺陷型核提取物在体外系统中产生的DDX41缺陷核提取物的生化步骤的影响。我们将使用体内剪接测定法以及RNA-Seq分析哺乳动物细胞中DDX41突变赋予的剪接和细胞表型的改变。由于DDX41突变的MDS患者对Lenalidomide治疗的反应很好，因此我们将研究该药物对体内和体外DDX41的影响，以确定潜在的作用机理。该应用程序的成功完成将导致有关剪接体核心成分在基因表达和疾病中发挥作用的新信息。此外，该奖学金申请提供的机会将增强在协作转化研究过程中对年轻科学家的培训。