Structural and functional basis of the non-canonical human Dicer-tRNA complex

Human Dicer (hDicer) is a key enzyme in the RNA interference (RNAi) pathway generating 21-22 nt micro-RNA (miRNA) or small interfering RNA (siRNA). We have previously shown that hDicer also generates tRNA-derived small RNA (tsRNA) that mediate nuclear gene silencing and regulate hundreds of disease-associated genes. tsRNAs have emerged as powerful and conserved cellular regulators. Therefore, it is important to understand their biogenesis. However, the molecular and structural basis of tRNA cleavage by hDicer, and the role of chemical modifications, such as 5-methylcytosine (m5C), in this process, remain unknown. Here, we present the first structural insights into hDicer in complex with tRNA, obtained by cryo-electron microscopy (cryo-EM), selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) and molecular dynamics (MD) simulations. Our results reveal that tRNAs adopt alternative conformations that are recognized and processed by hDicer. Furthermore, we show that tRNA cleavage by hDicer is facilitated by m5C modification deposited by Nop2/SUN RNA methyltransferase 2 (NSUN2). Taken together, our findings redefine tRNAs as integral hDicer substrates and expose a modification-dependent biogenic pathway that reshapes current understanding of the origins and regulation of human small RNAs.