Moores Cancer Center.
Department of Biological Sciences.
Blood Adv. 2021 Mar 9;5(5):1310-1323. doi: 10.1182/bloodadvances.2020002090.
The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and is disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined central role, and mutations promote malignant transformation. More recently, studies have illuminated the importance of posttranscriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and the breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between posttranscriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the posttranscriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a vs RUNX1b/c) that mediate HSC self-renewal vs differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detect changes in RNA processing. We used this reporter in a clustered regularly interspaced short palindromic repeats (CRISPR) screen consisting of single guide RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the posttranscriptional regulation of a key hematopoietic transcription factor and identifies RBPs that may have widespread and important functions in hematopoiesis.
造血干细胞 (HSC) 自我更新和分化的适当平衡对于正常造血至关重要，并且在血液恶性肿瘤中被破坏。在 HSC 命运的调节因子中，转录因子起着明确的核心作用，并且突变会促进恶性转化。最近的研究阐明了 RNA 结合蛋白 (RBP) 在造血和白血病发展中的转录后调控的重要性。然而，涉及的 RBP 及其调控的广度才刚刚开始阐明。此外，转录后调控与造血转录因子功能之间的交集还知之甚少。在这里，我们研究了 RUNX1 的转录后调控，RUNX1 是一种关键的造血转录因子。RUNX1 的可变多聚腺苷酸化 (APA) 产生功能拮抗的蛋白异构体 (RUNX1a 与 RUNX1b/c)，介导 HSC 的自我更新与分化，这是一种在恶性肿瘤中失调的 RNA 加工事件。因此，调节此事件的 RBP 直接有助于健康和异常造血。我们使用分裂 GFP 迷你基因报告基因来模拟 RUNX1 APA，并证实我们的模型对检测 RNA 加工变化的敏感性。我们在由仅靶向 RBP 的单指导 RNA 组成的簇状规则间隔短回文重复 (CRISPR) 筛选中使用该报告基因，并发现 HNRNPA1 和 KHDRBS1 是 RUNX1a 异构体产生的拮抗调节剂。总的来说，我们的研究提供了对关键造血转录因子转录后调控的机制见解，并确定了 RBP 在造血中可能具有广泛而重要的功能。
