Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment

In this study, we use small RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. Complemented and extended by the use of northern blots, our results demonstrate that tRNAs and Y RNAs in the vesicle-depleted extracellular compartment are released from cells as full-length precursors. Following their release, tRNAs and Y RNAs are processed by RNase 1 into distinct fragments. In addition, our findings show that standard sequencing methods employed to detect tRNA fragments leave many of such fragments undetected and that a combination of end healing, 3’ deacylation and RNA modification removal before library preparation can substantially improve detection of tRNA halves and reduce biases. An RNASE1 null mutation was generated in K562 human chronic myelogenous leukemia cells. RNA from extracellular vesicle-depleted conditioned medium was sequenced using a small RNA sequencing workflow. Four replicates each of RNA from RNASE1 mutant and wild type cells grown in RPMI 1640 were sequenced with pretreatment of the RNA with T4 polynucleotide kinase (PNK). Two replicates of RNA from RNASE1 wild type cells grown in RPMI 1640 were sequenced without RNA pretreatment with PNK. One replicate each of mutant and wild type cells grown in QBSF-55 medium were sequenced with RNA pretreatment with PNK. Two replicates of RNA from RNASE1 wild type cells grown in RPMI 1640 were sequenced with RNA pretreatment including deacylation followed by PNK and demethylation with AlkB.