Characterisation of the Rex1 exonuclease domain and RYS domain

Gene expression requires the accurate production of RNA from DNA. Raw transcription products are usually non-functional, requiring processing by nucleases to reach a functional, mature state. If a transcript is not processed correctly it will be targeted by nucleases for degradation; in some cases the same nuclease required for maturation can mediate degradation. By understanding the mechanisms nucleases use to bind their substrates, it is possible to understand nucleases features that direct a transcript towards maturation or degradation. This study aims to dissect the substrate recognition mechanisms of Rex1, a 3´ exonuclease belonging to the DEDD nuclease superfamily found in the model eukaryote Saccharomyces cerevisiae. Rex1 is unique in the yeast proteome for its ability to precisely trim short stem-loop adjacent 3´ overhangs found at the end of small RNA precursors including tRNA and 5S rRNA. The equivalent processing event is performed in Escherichia coli by a related DEDD exonuclease RNase T, however the sequence features responsible for substrate binding in RNase T are not found in Rex1. This study uses phylogenetic analysis, sequence conservation, and structure prediction to identify candidate substrate binding features within the Rex1 exonuclease domain, and defines a novel Alphafold2-predicted ‘RYS’ domain that is conserved throughout eukaryotes. An in vitro biochemical analysis of recombinant Rex1 is performed by observing the trimming of single-stranded oligo substrates. These trimming assays are used alongside in vivo complementation and RNA crosslinking to examine the contribution of Rex1 prospective substrate-binding features to function. This study demonstrates that the RYS domain is central to Rex1 substrate binding, which may be conserved in the structural homologues of Rex1 that are found in eukaryotes spanning plants to humans.