Small non-coding RNAs (sRNAs) are important regulators of gene expression and play fundamental roles in many different organisms. During the last decade, next generation sequencing (NGS) has become a powerful tool for sRNA profiling and several classes of novel regulatory sRNAs with potential biological functions have been discovered. Among them, sRNAs derived from longer non-coding Y RNAs were identified.
Y RNAs are non-coding RNAs (~ 100 nt) that are evolutionarily conserved in vertebrates. Human Y RNAs bind to the autoimmune proteins Ro60 and La forming ribonucleoprotein complexes. Y RNAs are involved in the initiation of chromosomal DNA replication, regulation of RNA stability and cellular stress response. During apoptosis, Y RNAs produce smaller RNA fragments from the 3’ and 5’ ends. However, the biogenesis, processing and the biochemical functions of Y RNA derived sRNAs remain elusive.
In previous work a high throughput mutagenesis analysis on the 3’ end of hY5 RNA was performed. In this work, I confirmed that Y RNA cleavage on the 3’ end of hY5 RNA correlated with the secondary structure of the mutated hY5 RNA. Furthermore, I clearly showed that the nucleotide sequence in the loop was not important and a bulge of one nucleotide was sufficient for Y RNA cleavage to occur.
In this thesis I designed and performed a high throughput mutagenesis approach on the 5’ end of hY5 RNA. This revealed a nucleotide motif UUAU located in an internal loop at the position 22-25 which contributed to Y RNA cleavage at both the 3’ and 5’ end of hY5 RNA.
In order to identify ribonucleases involved in Y RNA cleavage, I confirmed that Ro60 was essential for YsRNA production in mouse embryonic stem cells. Interestingly, I could show that RNase L contributed to Y RNA cleavage in mouse embryonic fibroblast cells and human lung cancer cells
