Regulation of microRNA biogenesis in stem cells. This project aims to understand the mechanisms by which microRNA (miRNA) processing is regulated in embryonic stem cells (ESC). ESCs have the remarkable capacity to differentiate into all cell types and are of potential therapeutic value for numerous degenerative diseases. However, the molecular foundations of ESC biology remain poorly defined. Functioning as negative regulators of gene expression, miRNAs are critical for diverse cell fate decisions during normal development; miRNA dysregulation is linked with several diseases including cancer. It is increasingly well appreciated that posttranscriptional mechanisms play an important role controlling miRNA expression. Lin28a selectively represses let-7 miRNA biogenesis in ESCs. This pathway helps maintain an undifferentiated cell state and is often reactivated in cancer. Although much progress has been made elucidating the mechanism of the Lin28-mediated control of let-7 expression, several outstanding questions remain.
Aim 1 will explore the regulation of the Lin28/let-7 pathway. A combination of biochemical and cell-based assays will explore the molecular determinants for let-7 regulation and will use state-of-the-art genetic engineering to explore the impact of a Lin28/let-7 developmental switch mechanism in ESCs. A variety of approaches will be used to investigate the mechanism by which Lin28b represses let-7 expression and to explore the possible TUTase-independence of this pathway. In vitro binding and cell-based assays will be employed to investigate the regulation of the Lin28/let-7 pathway by an identified lncRNA.
Aim 2 is focused on understanding the posttranscriptional mechanisms controlling miR-17~92 biogenesis. The precise control of miR-17~92 is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. New data implicate a novel biogenesis step upstream of Microprocessor that controls miR-17~92 expression in ESCs. A variety of biochemical and cellular assays will be used to test this and will reveal the RNA features and regulatory factors that control miR-17~92 expression in ESCs. Illuminating the molecular details of the Lin28/let-7 pathway and miR-17~92 regulation promises to impact broad areas of biology and disease, reveal novel stem cell regulatory factors, and offer strategies for therapeutic targeting to treat cancer, obesity, and diabetes.
Stem cells hold great promise for the development of new approaches to combat disease. However, the molecular basis for stem cell self-renewal and differentiation remains incompletely understood. The proposed work will provide novel insight into embryonic stem cell gene regulation, and may lead to new therapies to manipulate microRNAs. These studies are relevant to the treatment of cancer, diabetes, developmental disorders and numerous degenerative diseases.
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