权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
9884402
负责人：
Neil Morgan
金额：
$ 59.83万
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 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9884402
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SLFN14核糖核酸内切酶最初是在寻找