Small non-coding RNAs (sRNAs) constitute a significant portion of the transcriptome in eukaryotes. Many of these sRNAs regulate gene expression. Next-generation sequencing (NGS) has revealed a plethora of previously uncharacterised sRNAs with potential biological function, a number of which originate from longer RNAs. Here, the biogenesis of sRNAs derived from the non-coding Y RNAs (YsRNAs) was characterised as a model for understanding this emerging class of sRNA fragments. Y RNAs are highly conserved, 100 nt long molecules involved in DNA replication which bind to the autoimmune proteins Ro60 and La. YsRNAs are produced in cells undergoing apoptosis. Here, it was demonstrated that YsRNAs are generated from the 5’ and 3’ ends of all four Y RNAs in stressed and unstressed cells. Furthermore, production of these fragments was observed in both cancerous and non-cancerous cells. Although YsRNAs have been proposed to have gene silencing activity, experiments done here found that YsRNAs do not enter the microRNA pathway and are not generated by the gene silencing-related protein Dicer. Furthermore, experiments established that the enzyme which produces fragments from tRNAs, angiogenin, was also not responsible for YsRNA generation. Using mammalian cultured cells along with gene knockout and RNA interference (RNAi) technology, it was determined that RNase L contributed to YsRNA generation. Furthermore, the Y RNA binding protein Ro60 was shown to be essential for YsRNA production through a model of RNase protection. Analysis of deep sequencing data in Ro60 knockout cells revealed that many other sRNAs are also dependent on Ro60. Finally, a ‘high definition’ (HD) protocol to improve NGS detection of sRNAs was tested. The HD protocol was found to be better at detecting sRNAs than current methods. This will facilitate more efficient detection of novel sRNAs in the future.
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