Purification and enzymatic characterization of the RNA ligase RTCB from Thermus thermophilus.

OBJECTIVE: To identify the key residues of Thermus thermophilus (T. thermophilus) RTCB in RNA ligation and DNA activation. RESULTS: The biochemical activities of RTCB from T. thermophilus were purified, characterized, and compared. Structure and sequence alignment between T. thermophilus RTCB and Pyrococcus horikoshii (P. horikoshii) RTCB identified six conserved residues (D64, D95, N203, H204, E207, H399) that were essential for RNA ligation. Mutation analysis showed that the expression levels of mutants D95A, N203A, H204A, E207A and H399A were relatively low. Compared to wide-type RTCB, variant D64A protein had no RNA ligation and DNA activation activity. In addition, T. thermophilus RTCB showed acceptable catalytic activity of 3'-phosphate DNA activation at 37 °C. CONCLUSION: D64 was proved to be essential for RTCB-catalyzed RNA ligation and DNA activation (from 37 to 70 °C) in T. thermophilus.

Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology.

With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

Analysis of orthologous groups reveals archease and DDX1 as tRNA splicing factors.

RNA ligases have essential roles in many cellular processes in eukaryotes, archaea and bacteria, including in RNA repair and stress-induced splicing of messenger RNA. In archaea and eukaryotes, RNA ligases also have a role in transfer RNA splicing to generate functional tRNAs required for protein synthesis. We recently identified the human tRNA splicing ligase, a multimeric protein complex with RTCB (also known as HSPC117, C22orf28, FAAP and D10Wsu52e) as the essential subunit. The functions of the additional complex components ASW (also known as C2orf49), CGI-99 (also known as C14orf166), FAM98B and the DEAD-box helicase DDX1 in the context of RNA ligation have remained unclear. Taking advantage of clusters of eukaryotic orthologous groups, here we find that archease (ARCH; also known as ZBTB8OS), a protein of unknown function, is required for full activity of the human tRNA ligase complex and, in cooperation with DDX1, facilitates the formation of an RTCB-guanylate intermediate central to mammalian RNA ligation. Our findings define a role for DDX1 in the context of the human tRNA ligase complex and suggest that the widespread co-occurrence of archease and RtcB proteins implies evolutionary conservation of their functional interplay.

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases.

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 required for sealing by a ligase domain. Here, we use short HORNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting HORNA>p to HORNA2'p, which is then phosphorylated to pRNA2'p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2',3'-cyclic-PO4 moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.

T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis.

BACKGROUND: T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA. RESULTS: With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction. CONCLUSIONS: Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.

Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro.

Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2', 3'-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.

HSPC117 is the essential subunit of a human tRNA splicing ligase complex.

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.

Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules.

Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5'-kinase, and 2',3'-cyclic phosphate 3'-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5'-tri/diphosphate RNAs is dependent on ATP, a 2'-phosphate-3'-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5' terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5'-phosphate and 2'-phosphate-3'-hydroxyl ends. Two RNA molecules having 5'-hydroxyl and 2',3'-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation.

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5'-PO(4) single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65 degrees C, AppRNA was predominantly ligated to a circular product. In contrast, at 35 degrees C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases.

Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Purification and sequence determination of an RNA ligase from wheat embryos.

Wheat embryos have an RNA ligase activity that catalyses ligation of RNA molecules between the 5'-hydroxyl and 2',3'-cyclic phosphate termini. In contrast to yeast tRNA ligase, which ligates tRNA half molecules specifically, the wheat enzyme acts on various RNA substrates. It has been suggested that RNA ligase plays some unknown roles in vivo other than the maturation of tRNA. Here, we report purification of the enzyme and determination of the protease-digested peptide sequences. The sequences showed homologies to three database cDNA clones from Oryza sativa and Arabidopsis thaliana.

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.

Purification of histidine-tagged T4 RNA ligase from E. coli.

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.

RNA ligase and its involvement in guide RNA/mRNA chimera formation. Evidence for a cleavage-ligation mechanism of Trypanosoma brucei mRNA editing.

RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is thought to be directed by guide RNAs and may proceed via a chimeric guide RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprotein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylylated proteins of 50 and 57 kDa co-sediment on glycerol gradients with RNA ligase activity as components of the ribonucleoprotein particles. The two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addition of ligatable RNA substrate with the concomitant release of AMP and by the addition of pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate results in an accumulation of the adenylylated RNA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable analogs indicates that both RNA ligase and chimera-forming activities require alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera formation. These results strongly suggest the involvement of RNA ligase in in vitro chimera formation and support the cleavage-ligation mechanism for kinetoplastid RNA editing.

Isolation of new ribozymes from a large pool of random sequences [see comment].

An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the attack of a 3'-hydroxyl on an adjacent 5'-triphosphate--a reaction similar to that employed by the familiar protein enzymes that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction rates 7 million times faster than the uncatalyzed reaction rate.

Conserved mechanism of tRNA splicing in eukaryotes.

The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms. Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide. This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity. In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate. We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells. Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA. These results argue that the mechanism of tRNA splicing is conserved among eukaryotes.