Mechanistic insights into non-coding Y RNA processing

Y RNAs (84-112 nt in length) are transcribed by RNA polymerase III and are characterised by a distinctive secondary structure. Human Y RNAs interact with the autoimmune proteins SSB/RO60. This ribonucleoprotein (RNP) complex is termed RoRNP. Y RNAs/RoRNP perform regulatory roles in DNA replication and RNA stability and have important implications for diseases such as cancer. During stress/apoptosis Y RNAs are cleaved into 3' and 5' end fragments termed Y RNA derived small RNAs (ysRNAs). Although ysRNAs were discovered a decade ago their mechanisms/biogenesis remains unclear. Here we report that 3' end RNY5 cleavage correlates with secondary structure. In mutagenesis experiments, cleavage occurred between the 2nd and 3rd nt above a double stranded stem comprising high guanosine/cytosine content. We show that an internal loop above stem S3 is critical for producing 3' end derived ysRNAs (31 nt) with mutants resulting in longer or no ysRNAs. We show that a UGGGU sequence motif at position 22 of RNY5 is critical for producing 5' end derived ysRNAs (22-25 nt). We demonstrate that RO60 is critical for ysRNA biogenesis. Finally, we conclude that ribonuclease L (RNASEL) contributes to Y RNA cleavage in mouse embryonic fibroblasts but is not essential in human cells.