Identification of RNA helicases that mediate processing of tRNAs into distinct tRNA fragments by LC-MS/MS.

Specific environmental insults cause the limited fragmentation of transfer RNAs (tRNAs) into tRNA-derived small RNAs (tsRNAs), which have been implicated in a wide range of biological processes. tRNA fragmentation results from endonucleolytic activities targeting single-stranded tRNA regions. However, how a tRNA with a single hydrolyzed phosphodiester bond in the anticodon loop (‘nicked’ tRNA) gives rise to distinct tsRNAs remains poorly understood. By utilizing biochemical fractionation of tsRNA-containing ribonucleoprotein complexes (RNPs) coupled with LC-MS/MS, we identified several RNA helicase enzymes that represent putative tRNA/tsRNA processing enzymes. Furthermore, a combination of biochemical and computational approaches revealed that specific RNA helicases indeed bind specific tRNAs with high affinity, as well as process nicked tRNAs into individual tsRNAs. In summary, these findings reveal that tRNA-derived duplexes can be substrates of well-known RNA helicases, thereby expanding their potential for cellular function to the creation of individual tsRNAs during the cellular stress response.