Post-transcriptional regulation of hematopoietic stem cell function and cell fate determination during hematopoiesis

细胞命运的决定是干细胞生物学的一个基础领域，对理解功能器官的发育和维持至关重要。造血，血液形成的过程，是由造血干细胞（hsc）驱动的。造血干细胞是一小群细胞，它们具有独特的自我更新能力，可以在一生中重建系统中所有终末分化的细胞。在造血过程中，细胞命运决定的每一步都是由特定基因表达程序的激活精心策划和调节的，导致mrna的转录和随后功能蛋白的产生。然而，关于蛋白质合成过程，即转录后控制如何影响干细胞自我更新和分化，我们知之甚少。天然mRNA合成后，会加入聚腺嘌呤（poly-A）尾部，这有助于保护mRNA不被降解，并支持mRNA转化为蛋白质。聚A去死化介导聚A尾部的缩短并启动mrna的RNA衰变过程。CCR4-NOT （CNOT）复合物是两个多亚基聚a死基化复合物之一。最近，我发现RNA甲基化m6A“writer”METTL3是造血干细胞（hematopoietic stem cells, hsc）和翻译的重要调控因子。CNOT复合体被招募到m6A转录本中，介导mRNA降解。在CNOT复合物的所有亚基中，CNOT3在造血干细胞中表达最高，在祖细胞和分化细胞中表达下调。鉴于CNOT复合物的核心作用以及翻译和mRNA衰变之间的调节联系，我们的目标是研究CNOT复合物，特别是CNOT3在调节造血干细胞和造血中的作用，以揭示细胞命运决定的新机制。我的研究计划的总体目标是确定在正常造血过程中控制HSC功能和细胞命运的基因表达调控的转录后和翻译机制。在接下来的5年里，我们将揭示CNOT3和CCR4-NOT介导的死蛋白化在造血干细胞和造血中的作用。我们假设CNOT复合物介导的mRNA衰变和翻译的控制对造血系统中细胞命运的决定至关重要。具体来说，我们提出了3个目标：定义CNOT3在正常造血中的作用，AIM 2。确定CNOT3在调节HSC细胞命运（即HSC静止、细胞周期及非对称和对称分裂的控制）中的功能明确CNOT3在造血干细胞中功能的分子机制。这些研究将确定血细胞维持和再生的新介质，以及mRNA降解和翻译串扰的新分子机制。这些结果将推进我们在干细胞生物学方面的知识，同时为难以捉摸的转录后研究领域提供见解。

Cell fate determination is a fundamental area in stem cell biology and critical to understanding the development and maintenance of functional organs. Hematopoiesis, the process of blood formation, is driven by hematopoietic stem cells (HSCs). HSCs are a small population of cells, which possess unique self-renewal ability to reconstitute all terminally differentiated cells in the system throughout a lifetime. During hematopoiesis, each step of cell fate determination is orchestrated and regulated by activation of a specific gene expression program, resulting in transcription of mRNAs and subsequently generation of functional proteins. However, little is known about how processes of protein synthesis i.e. post-transcriptional control impacts stem cell self-renewal and differentiation. After a native mRNA is made, it undergoes addition of a polyadenine (poly-A) tail, which helps protect mRNA from degradation and supports translation of mRNA to protein. PolyA deadenylation mediates the shortening of the poly(A) tail and starts the RNA decay process of mRNAs. The CCR4-NOT (CNOT) complex is one of two multi-subunit polyA deadenylation complexes. Recently, I discovered that RNA methylation m6A "writer" METTL3 is an important regulator of hematopoietic stem cells (HSCs) and translation. CNOT complex was recruited to m6A transcripts to mediate the mRNA degradation. Among all subunits of the CNOT complex, CNOT3 exhibits the highest expression in HSCs and is downregulated in progenitors and differentiated cells. Given central roles of the CNOT complex and the regulatory link between translation and mRNA decay, we aim to investigate a role for the CNOT complex, in particular CNOT3 in regulation of HSCs and hematopoiesis to uncover novel mechanisms underlying cell fate decision. The overarching goal of my research program is to determine post-transcriptional and translational mechanisms of gene expression regulation in control of HSC function and cell fate determination during normal hematopoiesis. Over the next 5 years, we will uncover the role of CNOT3 and the CCR4-NOT mediated deadenylation in HSCs and hematopoiesis. We hypothesize that control of mRNA decay and translation mediated by the CNOT complex is critical for cell fate determination in hematopoietic systems. Specifically, we propose 3 aims: AIM 1. Define the role of CNOT3 in normal hematopoiesis, AIM 2. Determine CNOT3 function in the regulation of HSC cell fate (i.e. HSC quiescence, cell cycle and control of asymmetric and symmetric division) AIM 3. Define the molecular mechanism for CNOT3 function in hematopoietic stem cells. These studies will identify novel mediators of blood cell maintenance and regeneration, as well as novel molecular mechanisms of mRNA degradation and translation cross-talk. The results will advance our knowledge in stem cell biology while providing insights to the elusive post-transcriptional research field.