Genetic and Functional Analyses of Archaeal ATP-Dependent RNA Ligase in C/D Box sRNA Circularization and Ribosomal RNA Processing

Abstract

RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon Thermococcus kodakarensis to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates. Recombinant archaeal Rnl could circularize C/D box sRNAs with a mutation in the conserved C/D box sequence element but not when the terminal stem structures were disrupted, suggesting that proximity of the two ends could be critical for intramolecular ligation. Furthermore, T. kodakarensis accumulates aberrant RNA fragments derived from ribosomal RNA in the absence of Rnl. These results suggest that Rnl is responsible for C/D box sRNA circularization and may also play a role in ribosomal RNA processing.

Keywords: C/D box sRNAs; RNA ligase; circular RNA; rRNA processing; thermococcus kodakarensis KOD1.

FIGURE 1

Differentially expressed genes by deletion of TkoRnl gene. (A) Generation of RNA ligase knockout in T. kodakarensis. The TK1545 gene was replaced with the pyrF selectable marker (763 bp). The positions of four pairs of PCR primers and their expected product sizes are depicted in (1–4). The lower panel shows an agarose gel electrophoresis of PCR products derived from WT and tk1545 KO genomic DNAs. (B) Total RNA extracted from WT and tk1545 KO strains were separated using 10% denaturing polyacrylamide gel electrophoresis. Ethidium bromide staining of the gel is shown. (C) Transcriptosome analysis of WT and tk1545 KO. Total RNA isolated from WT and tk1545 KO cells were subjected to RNA-Seq analysis. Each dot represents an individual gene and are depicted according to read per million mapped reads (RPKM) values. The RPKM value with less than one RPKM were set to 0. Genes with RPKM of <2.0 in both WT and tk1545 KO were omitted. Differentially expressed genes are depicted as colored dots. Green dots represent the 10 genes that are up-regulated, and red dots represent 13 genes that are down-regulated by more than 2-fold in tk1545 KO (excluding the tk1545 gene). The RPKM for tk1545 from the WT and tk1545 KO were 9.05 and 0.04, respectively. Genes that show greater than two-fold change are listed in (D) and in Supplementary Table S2.

FIGURE 2

Effect on RNA circularization by TK1545 knockout. (A) Sequence alignment of circRNAs which were reduced or absent in tk1545 KO (see Table 1). Putative circular C/D box RNAs are listed on top and other circRNAs are listed on the bottom. The positions of conserved motifs (C box, C′ box, D box, and D′ box) are indicated. Identical nucleotides are highlighted in black with white text and conserved nucleotides are highlighted in gray. (B) Expression of C/D box RNA. Expression analysis of sixty-one T. kodakarensis C/D box RNA listed on Supplementary Table S3. Red dots represent the six C/D box RNAs reduced by more than 2-fold in tk1545 KO. (C) RT-PCR analysis was used to detect circRNAs from total RNA isolated from WT and tk1545 KO. The primer set used to detect circular RNA species is indicated on the right of the gel. A circular tRNA^Trp intron primer was used as a positive control (tRNA-Trp). As a negative control, assay was performed without reverse transcriptase treatment using tRNA^Trp intron primers [tRNA^Trp (no RT)], or with the TK0705 primers (Gene ID 3234407: NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, which is not circularized in both WT and tk1545 KO). The size of the molecular weight markers is indicated on the left. PCR product that migrates ∼40 nts most likely represented a product derived from a primer dimer.

FIGURE 3

Characterization of RNA circularization activity of MthRnl on C/D box sRNA. (A) MthRnl (360 ng) was incubated with 2 pmol of indicated pRNA at 70°C in a reaction mixture (40 μL) that contained 50 mM Tris-HCl (pH 6.5), 1 mM MgCl₂, in the presence or absence of 1 mM ATP. Aliquots (3 μL) were withdrawn at the times indicated and the products were separated by denaturing polyacrylamide gel. Positions of pRNA, AppRNA, and circRNA are indicated on the left. The sequences of pRNA substrates are illustrated on the right. The conserved C/D box sequence elements in sR42 and sR31 are highlighted, and the variant nucleotides in a mutant form of sR42 and sR31 RNAs are colored in red. (B) The yield of circular RNA products by MthRnl in the presence (+ATP) and absence (−ATP) of ATP is plotted as a function of time. The data shown represent the average of three separate experiments with SE bars.

FIGURE 4

Effect of TK1545 knockout on rRNA circularization and processing. (A) Circular RNAs derived from T. kodakarensis 16S-tRNA^Ala-23S rRNA operon. A diagram on the bottom represents a 5 kb segment of T. kodakarensis 16S-tRNA^Ala-23S rRNA operon, corresponding to 2022701–2027700 of NC_006624.1 reference sequence. The 16S rRNA, tRNA^Ala, and 23S rRNA genes are colored in orange. Positions of SRL and H98 are annotated. Arrowheads indicate predicted positions of bulge-helix-bulge motifs (BHB) on the primary transcript. The black lines above the diagram represent the expected size and position of highly represented circRNAs (Supplementary Data S2). Predicted length and position were determined from total RNA-Seq reads containing the junction sequence. Small RNAs fragments derived from WT and T. kodakarensis were cloned into TOPO vector via adaptor-mediate RNA ligation. The cloned fragments derived from WT (WT: green) or tk1545 KO (rnl: red) were aligned to the reference genome. The number on the right represents number of identical RNA fragments recovered in the RNA cloning experiment (i.e., 2 x indicates twice). See Supplementary Figure S5 for circular junction and alignment of small RNA fragments with the 16S-tRNA^Ala-23S rRNA operon. (B) Read-coverage in 23S rRNA at the 3′-end from total RNA-Seq dataset from WT (top) and tk1545 KO (bottom). (C) Read-coverage in 23S rRNA at the 3′-end from small RNA-Seq dataset from WT (top) and tk1545 KO (bottom). The read counts (y-axis) for the tk1545 KO were normalized to the WT read counts. The position of H98 is marked below the WT read-coverage.