High-throughput sequencing reveals circular substrates for an archaeal RNA ligase BeckerHubert F. HéliouAlice DjaoutKamel LestiniRoxane RegnierMireille MyllykallioHannu 2017 <p>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 physiologic 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 toward 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.</p>