Molecular Mechanisms Governing Non-canonical miR-146a Biogenesis

About This Grant

Aberrant inflammation drives the development of autoimmune diseases and cancer. The incomplete understanding of molecular mechanisms controlling inflammatory activation and resolution remains a significant obstacle to developing effective treatments for inflammatory disorders. Therefore, the main objective of this proposal is to identify and characterize novel molecular pathways that regulate inflammatory responses. MicroRNAs (miRNAs) have emerged as essential regulators that fine-tune immune homeostasis through both activating and inhibitory functions. Among these, miR-146a serves as a paradigmatic example of an immunomodulatory miRNA that functions primarily as a negative feedback regulator of NFκB signaling. The indispensable role of miR-146a in immune homeostasis is demonstrated by miR-146a-deficient mice, which develop systemic autoimmunity, hematological malignancies, and bone marrow failure. Similarly, altered miR-146a expression in humans is associated with inflammatory diseases and cancer. Despite its clinical significance, the mechanisms controlling miR-146a biogenesis remain poorly understood. Our preliminary studies revealed an unexpected post-transcriptional mechanism regulating miR-146a production. We discovered that active protein synthesis is specifically required for miR-146a maturation, as translation inhibition arrests miR-146a processing at the precursor stage while other miRNAs are unaffected. Through an unbiased biochemical approach, we identified the tRNA synthetase EPRS1 as a specific pre-miR- 146a binding protein that recognizes its conserved apical loop. EPRS1 depletion significantly impairs miR-146a processing without affecting other miRNAs, establishing a novel regulatory axis linking protein synthesis machinery to inflammatory control. Our central hypothesis is that miR-146a biogenesis is controlled post- transcriptionally by EPRS1 and additional, yet-to-be-identified, translation-dependent factors. In Aim 1, we will define how EPRS1 regulates miR-146a biogenesis by characterizing their molecular interaction and identifying short-lived proteins required for EPRS1-dependent processing. In Aim 2, we will leverage CRISPR screen methodology to identify additional regulators of miR-146a biogenesis and determine their functional relationship to EPRS1. The proposed research is significant because it will reveal previously unappreciated mechanisms controlling miR-146a production and potentially explain its dysregulation in inflammatory and hematological diseases. Understanding these regulatory mechanisms may enable the development of innovative therapeutic strategies to restore miR-146a levels in patients, circumventing the limitations of direct miRNA therapeutics.