RNA substrate length as an indicator of exosome interactions in vivo

The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies have identified multiple pathways for RNA substrates into the exosome complex in vitro, but determining which pathways are preferentially followed by individual RNA species in vivo remains challenging. Here we attempted to address this important question by an RNase protection approach. We applied an in vivo RNA-protein crosslinking technique to the exosome component Rrp44/Dis2, which has both endonuclease and exonuclease activity. The resulting high-throughput sequence reads were stratified by length of the cDNA. This should reflect the length of the RNA fragments, and therefore the RNA region that was protected by exosome binding. Striking effects were seen for short fragments, which presumably arise from RNA species that follow the “direct access” route to the Rrp44 exonuclease active site. These were strongly elevated in strains lacking exonuclease activity and, surprisingly, suppressed by point mutations in the endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggest that they are generated by the endonuclease activity. Confident mapping of the short reads is challenging but it seems clear that they are derived from only a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads. Overall design: 1 sample was analysed from strains carrying HTP tagged protein.