Identification and Functional Characterization of Divergent 3'-Phosphate tRNA Ligase From Entamoeba histolytica.

HSPC117/RtcB, 3'-phosphate tRNA ligase, is a critical enzyme involved in tRNA splicing and maturation. HSPC117/RtcB is also involved in mRNA splicing of some protein-coding genes including XBP-1. Entamoeba histolytica, a protozoan parasite responsible for human amebiasis, possesses two RtcB proteins (EhRtcB1 and 2), but their biological functions remain unknown. Both RtcBs show kinship with mammalian/archaeal type, and all amino acid residues present in the active sites are highly conserved, as suggested by protein alignment and phylogenetic analyses. EhRtcB1 was demonstrated to be localized to the nucleus, while EhRtcB2 was in the cytosol. EhRtcB1, but not EhRtcB2, was required for optimal growth of E. histolytica trophozoites. Both EhRtcB1 (in cooperation with EhArchease) and EhRtcB2 showed RNA ligation activity in vitro. The predominant role of EhRtcB1 in tRNAIle(UAU) processing in vivo was demonstrated in EhRtcB1- and 2-gene silenced strains. Taken together, we have demonstrated the conservation of tRNA splicing and functional diversification of RtcBs in this amoebozoan lineage.

Cleavage of 3'-terminal adenosine by archaeal ATP-dependent RNA ligase.

Methanothermobacter thermoautotrophicus RNA ligase (MthRnl) catalyzes formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl termini of single-stranded RNAs. It can also react with RNA with a 3'-phosphate end to generate a 2',3'-cyclic phosphate. Here, we show that MthRnl can additionally remove adenosine from the 3'-terminus of the RNA to produce 3'-deadenylated RNA, RNA(3'-rA). This 3'-deadenylation activity is metal-dependent and requires a 2'-hydroxyl at both the terminal adenosine and the penultimate nucleoside. Residues that contact the ATP/AMP in the MthRnl crystal structures are essential for the 3'-deadenylation activity, suggesting that 3'-adenosine may occupy the ATP-binding pocket. The 3'-end of cleaved RNA(3'-rA) consists of 2',3'-cyclic phosphate which protects RNA(3'-rA) from ligation and further deadenylation. These findings suggest that ATP-dependent RNA ligase may act on a specific set of 3'-adenylated RNAs to regulate their processing and downstream biological events.

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis.

An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl-AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5'-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl-AMP complex.

Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7.

The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism. However, the actual cleavage site in the splicing process was not determined for 13 interrupted tRNAs because of the presence of the same nucleotides at both 5' and 3' border regions of each intron. The cleavage sites for all the introns, which were determined by an in vitro cleavage experiment with recombinant splicing endonuclease as well as cDNA sequencing of the spliced tRNAs, indicated that non-canonical BHB structure motifs were also recognized and processed by the splicing machinery in this organism. This is the first report to empirically determine the actual cleavage and splice sites of introns in the whole set of archaeal tRNA genes, and reassigns the exon-intron borders with a novel and more plausible non-canonical BHB structure.

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity.

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi.

Translation elongation factor G (EF-G) in bacteria plays two distinct roles in different phases of the translation system. EF-G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post-termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF-G are distributed over two different EF-G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF-G is also found in bacteria. Two EF-G paralogues are found in the spirochaete Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-G1 is a translocase, while EF-G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF-G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF-3) is present in the reaction. These results indicate that two B. burgdorferi EF-G paralogues are close relatives to mitochondrial EF-G paralogues rather than the conventional bacterial EF-G, in both their phylogenetic and biochemical features.

Detection of enzymatic activities related to the tRNA splicing pathway in Methanosarcina acetivorans cell extract.

At least two separate enzymes, an endonuclease and a ligase, are thought to be involved in the tRNA splicing pathway. The yeast and archaeal endonucleases acting in the first step of tRNA splicing commonly produce 2', 3'-cyclic phosphate and 5' hydroxy group at the exon-intron borders. Despite this similarity in the first step of tRNA splicing, the subsequent mechanism of archaeal splicing pathway has not been elucidated yet. We have been searching for the archaeal ligase activity from Methanosarcina acetivorans. Here, we report the distinct activity of a splicing endonuclease detected in its cell extract.

Gain and loss of an intron in a protein-coding gene in Archaea: the case of an archaeal RNA pseudouridine synthase gene.

BACKGROUND: We previously found the first examples of splicing of archaeal pre-mRNAs for homologs of the eukaryotic CBF5 protein (also known as dyskerin in humans) in Aeropyrum pernix, Sulfolobus solfataricus, S. tokodaii, and S. acidocaldarirus, and also showed that crenarchaeal species in orders Desulfurococcales and Sulfolobales, except for Hyperthermus butylicus, Pyrodictium occultum, Pyrolobus fumarii, and Ignicoccus islandicus, contain the (putative) cbf5 intron. However, the exact timing of the intron insertion was not determined and verification of the putative secondary loss of the intron in some lineages was not performed. RESULTS: In the present study, we determined approximately two-thirds of the entire coding region of crenarchaeal Cbf5 sequences from 43 species. A phylogenetic analysis of our data and information from the available genome sequences suggested that the (putative) cbf5 intron existed in the common ancestor of the orders Desulfurococcales and Sulfolobales and that probably at least two independent lineages in the order Desulfurococcales lost the (putative) intron. CONCLUSION: This finding is the first observation of a lineage-specific loss of a pre-mRNA intron in Archaea. As the insertion or deletion of introns in protein-coding genes in Archaea has not yet been seriously considered, our finding suggests the possible difficulty of accurately and completely predicting protein-coding genes in Archaea.

Functional importance of crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease.

Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-A resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.

Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease.

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.

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.

Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes.

In the present study, mitochondrial ribosomes of the nematode Ascaris suum were isolated and their physiochemical properties were compared to ribosomes of Escherichia coli. The sedimentation coefficient and buoyant density of A. suum mitochondrial ribosomes were determined. The sedimentation coefficient of the intact monosome was about 55 S. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.40 g/cm(3), which suggests that the nematode mitoribosomes have a much higher protein composition than other mitoribosomes. The diffusion coefficients obtained from dynamic light scattering measurements were (1.48 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for 55 S mitoribosomes and (1.74 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for the 70 S E. coli monosome. The diameter of mitoribosomes was measured by dynamic light-scattering analysis and electron microscopy. Though the nematode mitoribosome has a larger size than the bacterial ribosome, it does not differ significantly in size from mammalian mitoribosomes.

Functional reconstitution of an archaeal splicing endonuclease in vitro.

Sulfolobus tokodaii strain 7 is one of archaea 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 these two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORFs were individually expressed in E. coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursors as substrates, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease of the organism is a multi-subunit complex composed of the two ORF products.

eEF1A binding to aminoacylated viral RNA represses minus strand synthesis by TYMV RNA-dependent RNA polymerase.

The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3'-end that can be valylated and then form a stable complex with translation elongation factor eEF1A.GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3'-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A.GTP to the valylated viral RNA. eEF1A.GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A.GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A.GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A.GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNAHis, tRNAAla, and tRNAPhe transcripts. It is proposed that minus strand repression by eEF1A.GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA.

Potato virus X: a model system for virus replication, movement and gene expression.

SUMMARY Considerable research has focused on the cis- and trans-acting components required for various aspects of the potato virus X (PVX) infection process. In addition, the development of PVX-based vectors has facilitated analyses of the PVX infection process and provided diverse technological applications. As a result, the PVX system will continue to serve as a model for analyses of processes such as virus movement, RNA replication, and gene silencing, and as a tool for protein expression.