Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.

Abstract

tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.