High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

Abstract

It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 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 P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 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 Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules.

Keywords: Archaea; RNA ligase; RNA-Seq; circular RNA; computational biology.

Figure 1.

Pab1020 RNA ligase binds single-stranded DNA and RNA, but only circularizes single-stranded RNA oligonucleotides. (A) EMSA assays were performed with internally labeled (Cy5) single stranded DNA or RNA oligonucleotides using increasing amounts of wild-type (WT) Pab1020 RNA ligase. The relative amount of bound DNA or RNA was plotted against the protein concentration. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab1020-nucleic acid complexes, increased as a function of the protein concentration. (B) RNA and DNA ligation assays with WT and mutant K95G of Pab1020 RNA ligase. Standard ligation reactions containing 10 pmol Cy5-RNA or -DNA molecules and 200 pmol RNA ligase Pab1020 were incubated 90 min at 50°C. Reaction products were resolved on denaturing PAGE and a 700 nm scan of the gel was performed on Licor Odyssey Infrared Imager. While no activity was observed with DNA substrate, Pab1020 RNA ligase circularized an RNA oligoribonucleotide as shown on the gel with the apparition of a lower band corresponding to circular RNA molecules. Expectedly, a control reaction with an inactive enzyme (mutant K95G) presented no lower band. (C) Identical to panel (A), except that the enzymes used in the EMSA assays corresponded to the mutant G296A (dimerization domain) and the amino-terminal domain of 250 residues carrying a nucleotide transferase (NTase) domain. Both mutants were able to form RNA-Protein complexes with 18-mers single-stranded RNA. (D) Identical to panel (B), except that circularization was performed only with RNA substrate and with G296A mutant and NTase domain. No circRNAs were observed (positive control is indicated in panel B).

Figure 2.

Identification of P. abyssi circRNAs using high throughput sequencing. (A) The workflow for identification of circularization junctions using RNA samples isolated from P. abyssi cells using IonTorrent semiconductor-based sequencing technology is shown. “ ± RIP” refers to the fact that identical computational approach was used for total and RNA immunoprecipitation (RIP) samples. Obtained linear and circular RNA molecules were fragmented at least once (indicated by a double arrow in panel A) using RNase III treatment. Following reverse transcription, samples were sequenced and obtained reads were aligned to the P. abyssi reference genome using Blastn. Reads were considered circular if 2 permuted matches covering the whole read was detected. (B) Number and percentage of the different functional classes (loci) considered circular in our sequencing experiments. (C) Number and percentage of the sequencing reads (total 28 279) supporting circularization of the different functional groups. (D) Percentage of the reads supporting RNA circularization (supportive circular reads) of the different RNA categories. Only intron containing tRNAs as identified as circular were included in the analysis. “Other circular reads” refers to a minority of putative circular reads that fulfill all the computational criteria indicated in panel A without supporting the junctions identified in panel B. Samples used were: A, circular reads after RIP assays using Pab1020 antibodies; B, circular reads after RIP assay and ribonuclease R treatment; C, circular reads in total RNA samples treated with ribonuclease R. New (NA) refers to previously non-annotated loci.

Figure 3.

Pab1020 RNA ligase circularizes physiologically relevant RNA molecules. (A) RNA binding between Pab1020 RNA ligase (0.2 to 4.5 μM) and the in vitro transcripts (0.4 μM) corresponding to BoxC/D RNAs SR4 (▪) and SR29 (▴) and 5S rRNA (▾) was analyzed by EMSA. A fraction of protein-RNA complex formed was plotted as a function of input protein. Insert: On the EMSA gel, the amount of the higher molecular weight bands, corresponding to Pab1020-nucleic acid complexes, increased as a function of the protein concentration. (B) In vitro transcript of 5S rRNA was incubated (right panel) or not (left panel) with Pab1020 RNA ligase (WT) for 120 min at 55°C. After incubation, recovered RNAs were treated or not with exoribonuclease RNase R for 120 min at 37°C before analysis on a 7% acrylamide 8M urea gel. (C) Schematic illustration of RT-PCR experiments on linear and circular RNAs with divergent primers to distinguish linear RNAs from circular RNAs products after incubation with Pab1020 RNA ligase. Only reverse transcription and PCR reactions on a circular RNA template will lead to the total amplification of the substrate sequence. (D) cDNA generated using outward facing primers on RNAs previously incubated (+) or not (−) with Pab1020 RNA ligase and in the presence (+) or absence (−) of RNase R were separated by gel electrophoresis. A full-length product attesting to amplification of circular RNA molecules, indicated by the asterisk, was observed for 5S rRNA (128 bp), Box C/D SR4 RNA (68 bp) and Box C/D SR29 RNA (66 bp). Circularization was observed only in the presence of Pab1020 RNA ligase.

Figure 4.

Venn diagram summarizing the results of our RNA-seq experiments. Samples used were: A, circular reads after RIP assays using Pab1020 antibodies; B, circular reads after RIP assay and ribonuclease R treatment; C, circular reads in total RNA samples treated with ribonuclease R. Black numbers refer to the categories of 133 junctions (Fig. 2B) and white numbers to 42 junctions with increased enrichment in RNase R experiments (Table 3).