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Role of newly discovered SLFN14 in megakaryopoiesis and platelet development

新发现的SLFN14在巨核细胞生成和血小板发育中的作用

基本信息

批准号：
10158537
负责人：
Neil Morgan
金额：
$ 54.43万
依托单位：
SUNY DOWNSTATE MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-05 至 2022-08-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10158537
关键词：
Affect Alleles Amino Acid Substitution Amino Acids Binding Biochemical Biogenesis Bioinformatics Biological Assay Biology Blood Coagulation Disorders Blood Platelets C-terminal Candidate Disease Gene Cells Cellular biology Characteristics Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Coupled Cryoelectron Microscopy Cytoplasmic Granules Data Data Analyses Databases Defect Degradation Pathway Development Disease Dominant-Negative Mutation Employment Endoribonucleases Family member Functional disorder Gene Expression Gene Expression Regulation Gene Family Genes Hematopoietic Hemorrhage Hemostatic function Human Immature Platelet Impairment In Vitro Inherited Kinetics Knock-in Mouse Knockout Mice Label Libraries Light Longevity Mediating Megakaryocytes Megakaryocytopoieses Messenger RNA Missense Mutation Molecular Biology Mus Mutant Strains Mice Mutation Mutation Analysis N-terminal Patients Phenotype Physiological Platelet Count measurement Play Pluripotent Stem Cells Point Mutation Process Protein Biosynthesis Protein Family Proteins Proteomics RNA RNA Degradation RNA Helicase Regulation Reporting Ribonucleases Ribosomal RNA Ribosomes Role Sampling Signal Transduction Single Nucleotide Polymorphism Site Specificity Structure Structure-Activity Relationship System Techniques Technology Testing Thermodynamics Thrombocytopenia Thrombopoiesis Thrombosis Time Transfer RNA Translations Variant Visualization Work conditional knockout differential expression experience experimental study genome editing helicase improved in vitro Model in vivo inhibitor/antagonist insight mRNA Expression mouse model mutant new therapeutic target novel novel strategies platelet function polysome profiling reconstitution transcriptome transcriptome sequencing

项目摘要

The novel hematopoietic-specific SLFN14 endoribonuclease was initially discovered during the search for ribonucleases responsible for general translation control. Simultaneously, missense mutations were identified in a novel gene, SLFN14, in patients with a dominantly inherited form of thrombocytopenia, associated with excessive bleeding. Considering that SLFN14 is a key regulator of megakaryopoiesis and of structural development of platelets, we plan to use an integral approach to investigate the role of SLFN14 in platelet biogenesis. We will employ novel SLFN14 mouse models and inducible Pluripotent Stem Cell (iPSC) derived megakaryocytes (MKs) expressing SLFN14 patient mutations, in conjunction with biochemical, molecular biology, cellular biology techniques, and structural analysis via cryo-electron microscopy (cryo-EM). More specifically we will investigate how SLFN14 controls platelet formation and function by studying a platelet and megakaryocyte specific SLFN14 conditional knock-out mouse and knock-in mice with the K218E and K219N point mutations, with an initial phenotype analogous with human patients, alongside iPSC-derived megakaryocytes bearing patient SLFN14 mutations, created using CRISPR genome-editing, which we have already generated. To give further clues to the mechanism through which SLFN14 may regulate megakaryopoiesis, signaling, dense granule formation, platelet formation and activation, we will use RNA sequencing to analyze alterations in gene expression and the regulation of genes in MKs derived from our SLFN14-KO and SLFN14-KI mutant mice. Gene transcriptome and bioinformatic data analysis will define altered gene expression of upregulated/downregulated genes known or predicted to be associated with MK differentiation, maturation, platelet formation and function, as a result of SLFN14 mutation. Thermodynamics and kinetics of SLFN14-dependent RNA degradation in MKs and platelets derived from mutant mice and iPSCs will be studied. Identification of the SLFN14-specific cleavage sites within rRNA by footprinting analysis in the primary mouse platelets and iPSC-derived MKs will reveal sequence/structure cleavage specificity of the protein. To unveil whether SLFN14 disrupts the translational machinery by restricting cytoplasmic rRNA/tRNA/mRNA in iPSC-derived MKs and mouse platelets, polysome profiling and non-canonical amino acid labelling techniques will be utilized. Employment of selective inhibitors for the major degradation systems in iPSCs will reveal the degradation pathway underlying the autosomal dominant SLFN14-related thrombocytopenia. Mutational studies of SLFN14’s oligomerization motifs, endoribonuclease core, ribosomal binding and helicase domains coupled with the set of in vitro and in vivo assays will establish structure-function relationships of the protein. Binding partners of SLFN14 in MKs will be characterized. Structural analysis of SLFN14-associated 80S ribosomes and oligomeric forms of SLFN14 by cryo-EM will also be performed.

新的造血特异性SLFN 14核糖核酸内切酶最初是在寻找负责一般翻译控制的核糖核酸酶。与此同时，错义突变被确定在显性遗传性血小板减少症患者中的一种新基因SLFN 14与以下疾病相关出血过多。考虑到SLFN 14是巨核细胞生成和结构分化的关键调节因子，血小板的发育，我们计划使用积分方法来研究SLFN 14在血小板中的作用，生物起源。我们将采用新的SLFN 14小鼠模型和诱导型多能干细胞（iPSC）衍生的细胞。表达SLFN 14患者突变的巨核细胞（MK），结合生物化学、分子生物学和免疫组织化学，生物学、细胞生物学技术和通过冷冻电子显微镜（cryo-EM）进行的结构分析。更具体地说，我们将通过研究血小板来研究SLFN 14如何控制血小板的形成和功能，巨核细胞特异性SLFN 14条件性敲除小鼠和K218 E和K219 N敲入小鼠点突变，具有与人类患者类似的初始表型，以及iPSC衍生的携带患者SLFN 14突变的巨核细胞，使用CRISPR基因组编辑创建，我们已经已经生成。为了进一步了解SLFN 14可能通过何种机制调节巨核细胞生成，信号传导，致密颗粒形成，血小板形成和活化，我们将使用RNA 测序分析基因表达的改变和来自我们的MK中基因的调节， SLFN 14-KO和SLFN 14-KI突变小鼠。基因转录组和生物信息学数据分析将定义改变的已知或预测与MK相关的上调/下调基因的基因表达分化、成熟、血小板形成和功能，作为SLFN 14突变的结果。热力学和 SLFN 14依赖性RNA在衍生自突变小鼠和iPSC的MK和血小板中降解的动力学将被研究。通过足迹法分析鉴定rRNA内SLFN 14特异性切割位点，原代小鼠血小板和iPSC衍生的MK将揭示蛋白质的序列/结构切割特异性。为了揭示SLFN 14是否通过限制细胞质中的rRNA/tRNA/mRNA来破坏翻译机制， iPSC衍生的MK和小鼠血小板、多核糖体分析和非规范氨基酸标记技术将被利用。在iPSC中主要降解系统的选择性抑制剂的使用将揭示iPSC中的主要降解系统。这是常染色体显性SLFN 14相关血小板减少症的潜在降解途径。突变研究 SLFN 14的寡聚化基序、核糖核酸内切酶核心、核糖体结合和解旋酶结构域偶联通过一组体外和体内测定将建立蛋白质的结构-功能关系。结合将对MK中SLFN 14的合作伙伴进行表征。SLFN 14相关的80 S核糖体的结构分析和还将进行通过冷冻-EM检测SLFN 14的寡聚体形式。