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. 2011 Mar 22;108(12):4782-7.
doi: 10.1073/pnas.1100862108. Epub 2011 Mar 7.

Evolution of Introns in the Archaeal World

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Free PMC article

Evolution of Introns in the Archaeal World

Giuseppe D Tocchini-Valentini et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The self-splicing group I introns are removed by an autocatalytic mechanism that involves a series of transesterification reactions. They require RNA binding proteins to act as chaperones to correctly fold the RNA into an active intermediate structure in vivo. Pre-tRNA introns in Bacteria and in higher eukaryote plastids are typical examples of self-splicing group I introns. By contrast, two striking features characterize RNA splicing in the archaeal world. First, self-splicing group I introns cannot be found, to this date, in that kingdom. Second, the RNA splicing scenario in Archaea is uniform: All introns, whether in pre-tRNA or elsewhere, are removed by tRNA splicing endonucleases. We suggest that in Archaea, the protein recruited for splicing is the preexisting tRNA splicing endonuclease and that this enzyme, together with the ligase, takes over the task of intron removal in a more efficient fashion than the ribozyme. The extinction of group I introns in Archaea would then be a consequence of recruitment of the tRNA splicing endonuclease. We deal here with comparative genome analysis, focusing specifically on the integration of introns into genes coding for 23S rRNA molecules, and how this newly acquired intron has to be removed to regenerate a functional RNA molecule. We show that all known oligomeric structures of the endonuclease can recognize and cleave a ribosomal intron, even when the endonuclease derives from a strain lacking rRNA introns. The persistence of group I introns in mitochondria and chloroplasts would be explained by the inaccessibility of these introns to the endonuclease.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Neighbor-joining tree of 23S rRNA from Archaea calculated as described in the materials and methods section. The number of each node is the result of the bootstrap analysis including 1,000 replicates. Presence and location of the intron are indicated in gray according to the text. ARCFU, Archaeoglobus fulgidus; CANKO, Candidatus Korarchaeum cryptofilum OPF8; HYPBU, Hyperthermus butilicus; IGNHO, Ignicoccus hospitalis; METKA, Methanopyrus kandleri; METSE, Metallosphaere sedula; METSM, Methanobrevibacter smithii ATCC 35061; NANEQ, Nanonarcheum equitans; PYRAE, Pyrobaculum aerophilum; PYRAR, Pyrobaculum arsenaticum DSM 13514; PYRIS, Pyrobaculum islandicum DSM 4184; STAMA, Staphilothermus marinus F1; SULAC, Sulfolobus acidocaldarius; SULSO, Sulfolobus solfataricus; SULTO, Sulfolobus tokodaii; THENE, Thermoproteus neutrophilus V24Sta; THEPE, Thermofilum pendens Hrk 5; THEKO, Thermococcus kodakarensis KOD1; PYRFU, Pyrococcus furiosus; PYRHO, Pyrococcus horikoshii; PYRAB, Pyrococcus abyssi.
Fig. 2.
Fig. 2.
Neighbor-joining tree of homing endonuclease homologs calculated using the method of observed divergence. The number on each node is the result of the bootstrap analysis including 1,000 replicates. Only the first 18 hits resulting from the blast search described in the text were included. Archaea, bacterial, chloroplast, and mitochondrial genes are indicated with a letter “A,” “B,”“C,” and “M” respectively. ACACA, Acanthamoeba castellanii; AERPE, Aeropyrum pernix; CANKO, Candidatus Korarchaeum cryptofilum OPF8; CARLU, Carteria lunzensis; CHAGL, Chaetosphaeridium globosum; CHLBR, Chlorosarcina brevispinosa; CHLEC, Chlorococcum echinozygotum; CHLMU, Chlamydomonas mutabilis; LOBSE, Lobochlamys segnis; MONOM, Monomastix sp. M722; SCEOB, Scenedesmus obliquus; SYNE, Synechococcus sp. C9; THELT, Thermotoga lettingae; THEM, Thermotoga sp. RQ7; TREJA, Trebouxia jamesii; VULDI, Vulcanisaeta distributa.
Fig. 3.
Fig. 3.
View of the 23S rRNA crystallographic structure in the Thermus thermophilus 70S ribosome (shown in gray) with H69 and H71 (red and yellow) forming the bridge (B2a) with helix H34 of the 16S rRNA (not shown) that interacts with tRNA (cyan) bound in the P site of the ribosome.
Fig. 4.
Fig. 4.
(A) Predicted secondary structure of the intron-exon motif present at position 23P3 in STAMA. The sequence of H69 is red, the sequence of H71 is yellow, the sequence between the two helices is green and blue, and the intron sequence is black. Gray dots indicate the phosphates that are cleaved by the S1 nuclease, and the black dots the phosphates that are also cleaved by RnaseT1. (B) Secondary structure of domain IV of the 23S rRNA following intron removal. (C) Crystallographic structure of the same domain where a black arrow shows where the intron is inserted.
Fig. 5.
Fig. 5.
Enzymatic RNA protection assay after a short (A) and long (B) electrophoresis run. The substrate 95 nucleotides long is described in Fig. 4A, and the fragments are numbered accordingly. The nucleases used are labeled S1 (nuclease S1) and T1(Rnase T1), in native and denaturing conditions. The controls (C) were performed in the reaction buffer specific for each nuclease (T1B, S1B) in the absence of enzyme. A partial alkaline hydrolysis (OH-) was performed to determine the size of each fragment.
Fig. 6.
Fig. 6.
tRNA splicing endonuclease assay. Lane 1, control (no enzyme) in archaeal enzyme buffer (AB); lane 2, homotetrameric METJA endonuclease; lane3, homodimeric ARCFU endonuclease; lane 4, heterotetrameric SULSO endonuclease; lane5, heterotetrameric SCHPO enzyme; lane 6, control (no enzyme) in yeast buffer (YB). The bands corresponding to each reaction product are indicated. ARCFU, Archaeoglobus fulgidus; METJA, Methanocaldococcus jannaschii; SCHPO, Schizosaccharomyces pombe; SULSO, Sulfolobus solfataricus.
Fig. 7.
Fig. 7.
Model proposed by Goddard and Burt (39). The model describes the life cycle of the ORF of the homing endonuclease associated with the group I introns. There are three states: functional ORF, non functional ORF, and empty. The figure shows (A) the life cycle in bacteria and organelles, and (B) the life cycle of the ORF associated with a group I intron in Archaea.

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