X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α′₂, α₄ and α₂β₂) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε₂ homodimer and has broad substrate specificity like the α₂β₂ type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε₂ homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (α^N, α and β^C) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε₂ and α₂β₂ EndAs were separately acquired through different evolutionary processes.     

Structural characterization of the catalytic subunit of a novel RNA splicing endonuclease

The RNA splicing endonuclease is responsible for recognition and excision of nuclear tRNA and all archaeal introns. Despite the conserved RNA cleavage chemistry and a similar enzyme assembly, currently known splicing endonuclease families have limited RNA specificity. Different from previously characterized splicing endonucleases in Archaea, the splicing endonuclease from archaeum Sulfolobus solfataricus was found to contain two different subunits and accept a broader range of substrates. Here, we report a crystal structure of the catalytic subunit of the S.solfataricus endonuclease at 3.1 angstroms resolution. The structure, together with analytical ultracentrifugation analysis, identifies the catalytic subunit as an inactive but stable homodimer, thus suggesting the possibility of two modes of functional assembly for the active enzyme.     

RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

tRNA核酸内切酶的研究进展

tRNA在蛋白质合成过程中起着极其重要的作用。在所有的生物体内,tRNA首先以前体形式转录,然后必需经过一系列的加工后才能成为有功能的tRNA分子。tRNaseZ、RNaseP和tRNA剪接内切酶是参与tRNA前体加工的三种主要的核酸内切酶,分别参与tRNA前体3′末端、tRNA前体5′末端和内含子剪接的加工。这三种酶具有不同的结构特征,并且利用完全不同的催化机制水解磷酸二酯键。tRNaseZ和RNaseP都是金属酶,活性中心分别需要Zn^2＋和Mg^2＋的参与;而tRNA剪接内切酶活性中心不需要金属离子,是一个由不同催化亚基上的关键氨基酸残基构成的组合式活性中心。     

Molecular cloning and characterization of a nuclear gene encoding a putative subunit of tRNA splicing endonuclease from Arabidopsis thaliana.

tRNA splicing endonuclease is required to produce mature tRNAs from intron-containing tRNA precursors. To characterize the structural features of plant endonuclease, we have isolated a cDNA and a corresponding genomic DNA clone from libraries of Arabidopsis thaliana which encode a putative subunit of the endonuclease. The gene product has an apparent mass of 27 kDa and contains a homologous domain of approximately 130 amino acids at the C-terminal region commonly found in other eucaryal and archaeal counterparts. Southern hybridization analysis of Arabidopsis genomic DNA utilizing the cDNA clone as probe indicates the presence of at least two related genes.     

tRNA 3' end maturation in archaea has eukaryotic features: the RNase Z from Haloferax volcanii

Here, we report the first characterization and partial purification of an archaeal tRNA 3' processing activity, the RNase Z from Haloferax volcanii. The activity identified here is an endonuclease, which cleaves tRNA precursors 3' to the discriminator. Thus tRNA 3' processing in archaea resembles the eukaryotic 3' processing pathway. The archaeal RNase Z has a KCl optimum at 5mM, which is in contrast to the intracellular KCl concentration being as high as 4M KCl.The archaeal RNase Z does process 5' extended and intron-containing pretRNAs but with a much lower efficiency than 5' matured, intronless pretRNAs. At least in vitro there is thus no defined order for 5' and 3' processing and splicing. A heterologous precursor tRNA is cleaved efficiently by the archaeal RNase Z. Experiments with precursors containing mutated tRNAs revealed that removal of the anticodon arm reduces cleavage efficiency only slightly, while removal of D and T arm reduces processing effciency drastically, even down to complete inhibition. Comparison with its nuclear and mitochondrial homologs revealed that the substrate specificity of the archaeal RNase Z is narrower than that of the nuclear RNase Z but broader than that of the mitochondrial RNase Z.     

Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum

Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.     

Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates

The 2′-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2′-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level.     

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.     

Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: evolutionary implications

Most introns of archaeal tRNA genes (tDNAs) are located in the anticodon loop, between nucleotides 37 and 38, the unique location of their eukaryotic counterparts. However, in several Archaea, mostly in Crenarchaeota, introns have been found at many other positions of the tDNAs. In the present work, we revisit and extend all previous findings concerning the identification, exact location, size, and possible fit to the proposed bulge-helix-bulge structural motif (BHB, now renamed hBHBh') of the sequences spanning intron-exon junctions in intron-containing tRNAs of 18 archaea. A total of 103 introns were found located at the usual position 37/38 and 33 introns at 14 other different positions, that is, in the anticodon stem and loop, in the D-and T-loops, in the V-arm, or in the amino acid arm. For introns located at 37/38 and elsewhere in the pre-tRNA, canonical hBHBh' motifs were not always found. Instead, a relaxed hBH or HBh' motif including the constant central 4-bp helix H flanked by one helix (h or h') on either side generating only one bulge could be disclosed. Also, for introns located elsewhere than at position 37/38, the hBHBh' (or HBh') structure competes with the three-dimensional structure of the mature tRNA, attesting to important structural rearrangements during the complex multistep maturation-splicing processes. A homotetramer-type of splicing endonuclease (like in all Crenarchaeota) instead of a homodimeric-type of enzyme (as in most Euryarchaeota) appears to best fit the requirement for splicing introns at relaxed hBH or HBh' motifs, and may represent the most primitive form of this enzyme.     

Identification of two catalytic subunits of tRNA splicing endonuclease from Arabidopsis thaliana

tRNA splicing endonuclease is essential for the correct removal of introns from precursor tRNA molecules of Archaea and Eucarya. The only well-characterized eucaryotic enzyme until now is the endonuclease from yeast (Saccharomyces cerevisiae). This protein has a heterotetrameric structure. Two of the four subunits, i.e. Sen34 and Sen44, contain the active sites for cleavage at the 3'- and 5'-splice sites, respectively. We have identified three novel genes from Arabidopsis thaliana, encoding putative subunits of tRNA splicing endonuclease. They are designated as AtSen1, AtSen2, and AtpsSen1. Both genes AtSen1 and AtSen2 seem to be functionally active, as deduced from corresponding cDNA sequences. Comparison of the amino acid sequences of the these two Arabidopsis proteins revealed 72% identity. However, AtpsSen1 is more similar to AtSen1, but is very likely a pseudogene, as concluded from extended stretches of deletions and the presence of in-frame stop codons. All putative proteins contain a conserved domain at their C-terminus common to counterparts from other organisms. Interestingly, they are more similar to the yeast catalytic subunit Sen44 than to Sen34. Southern analysis with various probes revealed that each gene is present as single copies in the nuclear genome. The evolutionary implications of these findings are discussed.     

Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.     

Functional reconstitution of a crenarchaeal splicing endonuclease in vitro

Sulfolobus tokodaii strain 7 is one of Crenarchaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to EndA, a protein responsible for splicing endonuclease activity in Archaea. Interestingly, one of the two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORF products were individually expressed in Escherichia coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursor as a substrate, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease for the organism is a multi-subunit complex composed of the two endA gene products.     

Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.     

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.     

Two archaeal tRNase Z enzymes: similar but different

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3' trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2-4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2-3 M (NH(4))(2)SO(4) do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn(2+) ions per dimer. In addition tRNase Z requires Mn(2+) ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5' extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-D: -lactoylglutathion (SLG).     

Evolution of the Archaea

Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status. The archaeal ancestor was probably a hyperthermophilic anaerobe. Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota. Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups. Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota. Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples. The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution.     

Archaea in the Gulf of Aqaba

Using a polyphasic approach, we examined the presence of Archaea in the Gulf of Aqaba, a warm marine ecosystem, isolated from major ocean currents and subject to pronounced seasonal changes in hydrography. Catalyzed reported deposition FISH analyses showed that Archaea make up to >20% of the prokaryotic community in the Gulf. A spatial separation between the two major phyla of Archaea was observed during summer stratification. Euryarchaeota were found exclusively in the upper 200 m, whereas Crenarchaeota were present in greater numbers in layers below the summer thermocline. 16S rRNA gene-based denaturing gradient gel electrophoresis confirmed this depth partitioning and revealed further diversity of Crenarchaeota and Euryarchaeota populations along depth profiles. Phylogenetic analysis showed pelagic Crenarchaeota and Euryarchaeota to differ from coral-associated Archaea from the Gulf, forming distinct clusters within the Marine Archaea Groups I and II. Endsequencing of fosmid libraries of environmental DNA provided a tentative identification of some members of the archaeal community and their role in the microbial community of the Gulf. Incorporation studies of radiolabeled leucine and bicarbonate in the presence of different inhibitors suggest that the archaeal community participates in autotrophic CO₂ uptake and contributes little to the heterotrophic activity.