长链非编码RNA-Lincred1参与"GATA因子转换"调节及红系发育的功能和机制研究

A major fraction of the transcriptome of higher organisms comprised an extensive repertoire of long non-coding RNA (lncRNA) which are recognized as essential regulators in various cellular processes. Up to date, although numerous lncRNA has been identified, an existing challenge is to understand how the molecular functions of lncRNAs affect the phenotypes of the organisms. Limited by screening strategies and technological advances, the main problem in lncRNA study remains the identification of biologically functional lncRNAs. In this work, we started from an erythroid landmark event termed "GATA switch", which involves GATA-1-mediated displacement of GATA-2 from chromatin. Given that "GATA switch" often occurs at loci with critical functions, we aim to identify important lncRNAs that are under the control by this developmental control tools. As a result, we focused on the candidate Lincred1 to further analyze its regulatory roles during erythropoiesis in cell lines, primary cultured hematopoietic stem/progenitor cells and conditional transgenic mouse models. Since the SUMO E3 ligase Pc2 (polycomb protein 2), harboring an RNA binding domain, was shown to be associated with Lincred1 in our previous study, we will further confirm the interaction between Lincred1 and Pc2, assess their cooperativity in regulating GATA-1 sumoylation, thus evaluating their influence on GATA factors switching. By clarifying the function and molecular mechanism of Lincred1 in erythropoiesis, this strategy will be successfully used to identify lncRNAs that are relevant to the specific biological question. These results will also provide new insights into lncRNA study and gene regulatory networks in hematopoiesis.

长链非编码RNA分子（lncRNA）是哺乳动物转录组的重要组分，也是高等生物重要的遗传性调控因子。目前，虽有大量lncRNA被不断发现，但对其功能和机制的阐释仍相对滞后，尤其是缺乏功能性lncRNA筛选和研究的独特视角。本课题以造血红系发育过程中标志性的基因表达驱动事件"GATA switch"为切入点，系统性鉴定受红系关键转录因子GATA-1和GATA-2"转换"调节的lncRNA分子；在确定Lincred1为研究对象后，重点在造血干/祖细胞和基因敲除小鼠中考察Lincred1调节红系发育的生物学功能；同时通过研究Lincred1与多梳蛋白Pc2的相互作用、Pc2对GATA-1的SUMO化修饰作用、以及SUMO化修饰对"GATA switch"的影响，阐明Lincred1的作用机制。这不仅为功能特异性lncRNA的鉴定提供新的筛选策略，更为揭示和丰富lncRNA的作用机制提供新的依据。

RNA结合蛋白（RBP）和长链非编码RNA（lncRNA）是真核生物基因表达调控的重要参与者。由RBP和lncRNA所介导的转录后调控，不仅作为转录调控的重要补充，更在很大程度上决定基因的最终表达。但相对于转录层面调控而言，转录后调控研究较少，尤其在造血分化领域。本课题初始阶段，以两条线索出发：i. 在发现和鉴定受“GATA-1/-2 switch”调控的lncRNA：Lincred1基础上，进一步研究其在造血发育中的具体功能和详细机制；ii. RNA结合蛋白通过调控primary-miRNA（本质上也是一种lncRNA）加工参与造血发育。在项目实施过程中，Lincred1在小鼠红系分化中的功能先于我们的研究报道。因此，我们将研究重心转向RNA结合蛋白参与primary-miRNA（pri-miRNA）加工调节的机制及其生物学意义。我们发现：i. RNA结合蛋白QKI5可以与primary-miR-124-1（pri-miR-124-1）结合，增强了Drosha/DGCR8复合物的募集，该调控作用为体内pri-miR-124-1正常加工所必需，会最终影响成熟miR-124-1的产生。而该调控平衡被打破会直接影响正常红系发育的进行。QKI5的调控作用是通过距离miR-124-1 stem-loop上游~300nt的一个功能性RNA元件所介导，这也是首次发现的miRNA stem-loop之外的远距离作用元件可以调节pri-miRNA的加工，乃至成熟miRNA的产生。RNA分子的这种远距离作用机制与DNA十分相似，均是通过RNA loop使功能性元件在空间上相互接近，并因此命名为：“RNA processing enhancer”（Cell Research, 2017）。ii. 造血粒系和单核系发育过程中，RNA结合的蛋白KSRP，通过与pri-miR-129-1上三个功能性RNA元件的识别和结合，介导了其加工成熟。并且KSRP和miR-129-1在粒系和单核系分化过程中的执行相反功能，即促进粒系发育，抑制单核系发育，说明该调控作用直接影响了造血谱系分化命运的选择（Nature Communications, 2017）。以上研究不仅丰富了造血分化调控的转录后层面内容，更揭示出RBP通过与lncRNA互作参与基因表达调控的新机理。