Role of newly discovered SLFN14 in megakaryopoiesis and platelet development

新的造血细胞特异的SLFN14内切核糖核酸酶是在寻找 负责一般翻译控制的核糖核酸酶。同时，错义突变在 一种名为SLFN14的新基因与遗传性血小板减少症相关 失血过多。考虑到SLFN14是巨核细胞生成的关键调节因子 对于血小板的发展，我们计划使用一种完整的方法来研究SLFN14在血小板中的作用 生物发生学。我们将使用新的SLFN14小鼠模型和诱导多能干细胞(IPSC)来源 表达SLFN14患者突变的巨核细胞(MK)与生化、分子 生物学、细胞生物学技术和低温电子显微镜(Cryo-EM)结构分析。更多 具体来说，我们将通过研究血小板和血小板来研究SLFN14如何控制血小板的形成和功能 巨核细胞特异性SLFN14条件性敲除小鼠和K218E和K219N敲入小鼠 点突变，与人类患者相似的初始表型，以及IPSC来源的 携带患者SLFN14突变的巨核细胞，使用CRISPR基因组编辑创建，我们有 已经生成。为SLFN14调控机制提供进一步的线索 巨核细胞的生成、信号传递、致密颗粒的形成、血小板的形成和激活，我们将使用RNA 用测序方法分析OUR来源的巨噬细胞的基因表达和基因调控的变化 SLFN14-KO和SLFN14-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 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，是SLFN14突变的结果。热力学和 突变小鼠和IPSCs来源的单核细胞和血小板中依赖SLFN14的RNA降解动力学 被研究。通过足迹分析鉴定rRNA中的SLFN14特异性切割位点 原代小鼠血小板和IPSC来源的MKs将揭示该蛋白的序列/结构切割特异性。 揭示SLFN14是否通过限制细胞质rRNA/tRNA/mRNA来扰乱翻译机制 IPSC来源的巨噬细胞和小鼠血小板、多聚体图谱和非规范氨基酸标记技术 将会被利用。对IPSCs中的主要降解系统使用选择性抑制剂将揭示 常染色体显性遗传性SLFN14相关的血小板减少症的降解途径。突变研究 SLFN14的S寡聚基序、内切核糖核酸酶核心、核糖体结合和解旋酶结构域耦合 通过这套体外和体内测试，将建立蛋白质的结构-功能关系。装订 将介绍SLFN14在MK中的合作伙伴的特征。SLFN14相关的80S核糖体的结构分析 还将进行冷冻-EM低聚形式的SLFN14。

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.