Small RNA sequencing of in vitro Angiogenin-hydrolyzed tRNAs

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. After identifying RNA helicases that were able to unwind ‘nicked’ tRNAs in vitro, the specificity of one of those enzymes, DDX3X, was determined by RNA helicase assays on tRNAs, which had been subjected to recombinant Angiogenin. Both in vivo ‘nicked’ tRNAs, DDX3X-unwound ‘nicked’ tRNAs as well as heat-denatured ‘nicked’ tRNAs were subjected to small RNA sequencing. To understand DDX3X unwinding specificity towards specific cleaved tRNAs, we performed small RNA sequencing on input tracer tRNA duplexes, heat-denatured tsRNAs as well as tsRNAs displaced from tracer tRNA duplexes by DDX3X helicase.