Abstract
In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5′ UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5′ UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.
Highlights
TRNA molecules are key players within cells since they translate genetic information into protein
Haloferax volcanii encodes an RNA splicing endonuclease (HVO_2952, endA) in a bicistronic operon together with a tryptophanyl-tRNA synthetase (HVO_2951, trpS1) (Figure 3), the two genes overlap by four nucleotides
Proteome data clearly show that the splicing endonuclease is present in the proteome (Jevticet al., 2019; Schulze et al, 2020)
Summary
TRNA molecules are key players within cells since they translate genetic information into protein. Generation of functional tRNA molecules requires a plethora of processing steps starting with the removal of 5 leader and 3 trailer sequences from pre-tRNA [for a review see (Clouet-d’Orval et al, 2018; Figure 1)]. Some tRNA genes contain introns that must be removed from precursor RNA to yield mature functional tRNAs. While some bacterial tRNA genes contain self-splicing group I introns, archaeal and eukaryotic tRNA introns are removed by proteins. The initial step of intron removal in eukaryotes and archaea is catalyzed by an RNA splicing endonuclease. The resulting splice products are ligated by a tRNA ligase, thereby generating mature tRNA as well as a circular intron (Figure 1)
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