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

<jats:p>Human Dicer (hDicer) is a key enzyme in the RNA interference (RNAi) pathway that generates ~21-22 nt micro-RNA (miRNAs) and small interfering RNAs (siRNAs). We have previously shown that hDicer also generates tRNA-derived small RNAs (tsRNAs), which mediate nuclear gene silencing and regulate hundreds of disease-associated genes. As powerful and evolutionarily conserved cellular regulators, tsRNAs emerged as an important class of small RNAs. Therefore, it is essential to understand their biogenesis. However, the molecular and structural basis of tRNA cleavage by hDicer, as well as 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 the m5C modification deposited by Nop2/SUN RNA methyltransferase 2 (NSUN2). Collectively, our findings redefine tRNAs as bona fide hDicer substrates and uncover a modification-dependent biogenetic pathway that reshapes the current understanding of the origins and regulation of human small RNAs.</jats:p>