High-Throughput Sequencing Reveals Circular Substrates for an Archaeal RNA ligase

It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon <i>Pyrococcus abyssi</i>, has emerged. This led us to investigate the physiological significance of a previously observed weak intramolecular ligation activity of <i>Pab</i>1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in <i>P. abyssi</i>. The large majority of these loci interacted with <i>Pab</i>1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that <i>Pab</i>1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase towards RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of <i>Pab</i>1020 may facilitate the development of new biotechnological applications <i>e.g.</i> in ligation of preadenylated adaptors to RNA molecules.