The tRNA pseudouridine synthase TruB1 regulates the maturation of let‐7 miRNA

Let‐7 is an evolutionary conserved microRNA that mediates post‐transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let‐7 biogenesis is tightly regulated by several RNA‐binding proteins, including Lin28A/B, which represses let‐7 maturation. To identify new regulators of let‐7, we devised a cell‐based functional screen of RNA‐binding proteins using a let‐7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let‐7 family members. Rather than inducing pseudouridylation of the miRNAs, high‐throughput sequencing crosslinking immunoprecipitation (HITS‐CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem‐loop structure of pri‐let‐7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri‐let‐7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let‐7. Altogether, we reveal an unexpected function for TruB1 in promoting let‐7 maturation. Synopsis The tRNA pseudouridine synthase TruB1 positively and directly regulates the maturation step of miRNA let‐7 in an enzyme activity‐independent manner. This uncovered TruB1—let‐7 axis suppresses cell proliferation. A devised systematic cell‐based gain of function screen revealed that the tRNA pseudouridine synthase TruB1 promotes let‐7 expression. TruB1 selectively enhanced let‐7 family member maturation independent of its enzymatic activity. Endogenous TruB1 directly binds to the stem‐loop structure of pri‐let-7 and promotes the maturation steps of let‐7. Cell proliferation can be regulated by the TruB1 and let‐7 molecular cascade. Graphical Abstract TruB1 promotes let‐7 maturation independently of its enzymatic activity by enhancing the interaction of pri‐let‐7 and the microprocessor DGCR8.