RNase R in Haloferax volcanii. RNaseR specializes in rRNA processing in Haloferax volcanii

Ribonucleases (RNases) are central to RNA metabolism, driving transcript processing, maturation, and degradation. In archaea, RNA degradation integrates bacterial and eukaryotic-like pathways, with most archaea relying on exosome complexes for RNA turnover. However, halophilic archaea, including Haloferax volcanii, lack exosome subunits and encode homologs of the bacterial RNase R instead. Previously reported to be essential and the primary 3’-5’ exoribonuclease in this biological system, the precise function and substrate specificity of RNase R in H. volcanii remain unclear. To unravel its function and RNA substrates, we successfully generated an H. volcanii RNase R deletion strain (Δrnr) and found that its absence had only mild effects on cellular adaptability. Using long-read RNA sequencing, we performed transcriptome-wide mapping of RNA degradation, end-processing, and rRNA maturation. While RNase R deletion had minimal effects on mRNA stability, it led to a substantial accumulation of late 16S rRNA precursors, suggesting a defined role in rRNA processing rather than general RNA turnover. These findings contrast with those observed for bacterial RNase R, which is more broadly involved in mRNA degradation. Using full-length RNA sequencing, our study highlights the evolutionary divergence of ribonuclease function and uncovers unexpected nuances in archaeal RNA metabolism.