Saturday, September 7, 2024
9/7/2024
PROJECT SUMMARY/ABSTRACT MicroRNAs (miRNAs) are essential regulators of the human transcriptome and play a central role in tissue development. As the hematopoietic system is composed of a multitude of mature cell types constantly produced from stem and progenitor cells, it is not surprising that correct miRNA biogenesis, function, and decay are essential for hematopoietic differentiation. We have recently identified U6 Biogenesis 1 (USB1) as being a novel 3·_ end miRNA deadenylase that regulates degradation of different miRNAs. As mutations in USB1 cause the bone marrow failure syndrome poikiloderma with neutropenia (PN), it becomes clear that the correct 3· - end processing of miRNAs by USB1 is critical for hematopoiesis. Due to a lack of adequate models and intrinsic difficulties in studying mi RNA processing, the pathways that control miRNA degradation remain largely unknown. A better understanding of the posttranscriptional regulation of miRNA processing, and how it relates to activation of different miRNA degradation pathways is essential to decipher the role of these non-coding RNAs during hematopoiesis. The focus of this proposal is to use the targeted hematopoietic differentiation of human pluripotent stem cells to decipher molecular pathways controlling posttranscriptional regulation and degradation routes of miRNAs. We have generated a large panel of human pluripotent stem cell lines harboring pathogenic mutations in USB1 and other miRNA 3'-end deadenylases, as well as mutations in different components of the exosome RNA decay complex. From these, we derive hematopoietic cells in vitro, following established protocols that recapitulate the in vivo formation of these cell types. Two specific aims are proposed that utilize this platform to identify novel regulators of miRNA processing and decay, and to determine their function in the hematopoietic system. Aim 1 will determine the mechanisms regulating target specificity and overlap of different deadenylases necessary for development, during several stages of hematopoiesis. We will complement these assays by determining which miRNA decay routes are activated in these cellular populations, focusing on exosome activation and target-directed miRNA degradation (TDMD). We will determine to which extent TDMD is modulated by incorrect miRNA 3'- end adenylation and deadenylation in WT and USB1 mutants. Aim 2 will investigate the specific mechanisms leading to impaired neutrophil development in USB 1 mutant cells, as PN causes severe non-cyclic neutropenia. We have identified potential targets of USB 1 that modulate neutrophil yield and will test their functional role in different processes, such as cellular replication and apoptosis, that could lead to a failure of myeloid progenitors to efficiently generate neutrophils. These studies will decipher novel effectors of miRNA degradation in the hematopoietic system, a tissue where miRNA-regulated gene expression plays a central role. Our unique molecular and cellular tools, combined with our expertise in DNA and RNA biology, as well as in hematopoiesis puts us in an ideal position to make a significant impact in this field.
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