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.

直到最近，人们才发现环状RNA（circular RNAs, circRNAs）在所有生命界中广泛存在，包括嗜热古菌深海热球菌（Pyrococcus abyssi）。这促使我们探究此前观测到的Pab1020 RNA连接酶所具有的微弱分子内连接活性的生理学意义。本文证实，尽管该酶与DNA连接酶序列相似性较高，但其确为RNA特异性多核苷酸连接酶，可有效作用于具有生理学意义的底物。结合RNA免疫沉淀实验（RNA immunoprecipitation assays）与RNA测序（RNA-seq），我们的全基因组水平研究在深海热球菌中鉴定出133个独立的环状RNA基因位点。其中绝大多数位点可在细胞内与Pab1020发生相互作用，且我们通过生化实验验证了部分筛选得到的C/D盒（C/D Box）与5S核糖体RNA（5S rRNA）转录本的环化过程。综上，本研究证实Pab1020是RNA环化所必需的酶。我们的结果进一步表明，祖先核苷酸转移酶（nucleotidyltransferase, NTase）结构域和/或DNA连接酶可通过功能特化获得RNA连接酶活性，这也呼吁学界进一步开展原核生物中环状RNA广泛功能的相关研究与表征。深入解析Pab1020的细胞底物，或将助力开发全新的生物技术应用，例如将预腺苷化接头连接至RNA分子的相关技术。