In vitro dissection revealed that the kinase domain of wheat RNA ligase is physically isolatable from the flanking domains as a non-overlapping domain enzyme

Wheat RNA ligase contains 5′-hydroxyl kinase, 2′,3′-cyclic phosphate 3′-phosphodiesterase, and 5′-phosphate 2′-phosphate-3′-hydroxyl RNA ligase activities in a 110-kDa polypeptide. Taking advantage of a wheat cell-free protein production system, we prepared various fragments containing a part of the enzyme. The method allowed us to check the activities of the fragments rapidly, eliminating the time-consuming cloning and sequencing steps for the expression of the fragment proteins. The results showed that each of the three activities can be assigned to a non-overlapping domain that does not require the presence of the other part(s) of the enzyme for its activity. This contrasts to the case of yeast tRNA ligase, in which the central kinase domain has been suggested to require to be tethered to one of the flanking domains for its activity.     

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg²⁺ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.     

Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme

We previously reported the in vitro selection of several Mg²⁺-dependent deoxyribozymes (DNA enzymes) that synthesize a 2′–5′ RNA linkage from a 2′,3′-cyclic phosphate and a 5′-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure–function relationships of catalytic nucleic acids.     

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA^3′pp^5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA ^3′pp^5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates.     

Coevolution of RtcB and Archease created a multiple-turnover RNA ligase

RtcB is a noncanonical RNA ligase that joins either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.     

Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg²⁺-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5′-hydroxyl group with a 2′,3′-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2′–5′ phosphodiester bonds rather than native 3′–5′ linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2′–5′ linkages. This includes deoxyribozymes with an arrangement that favors 3′–5′ linkages for a different chemical reaction, that of a 2′,3′-diol plus 5′-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2′–5′ RNA linkages upon opening of a 2′,3′-cyclic phosphate with a 5′-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2′,3′-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.[Reviewing Editor: Niles Lehman]     

Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.     

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

RNA editing in trypanosomes

Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4–14 nucleotide anchor sequence embedded in the 5 region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5–24 nucleotide 3 terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3 U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.Abbreviations gRNA guide RNA     

A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.     

Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme

Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2′,3′ cyclic phosphate and 5′-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2′,3′ cyclic phosphate to form a 3′-OH and phosphorylation of the 5′-OH to form a 5′-PO₄. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3′ end-healing domain (H189D) that yields a 2′-PO₄ product instead of a 2′-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.     

2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3′-phosphate RNA end to a 2′,3′-cyclic phosphate via covalent enzyme-(histidinyl-Nϵ)-AMP and RNA(3′)pp(5′)A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2′-phosphate RNA end in high yield. The rate of 2′-phosphate cyclization by RtcA is five orders of magnitude slower than 3′-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA_3′p and RNA_2′p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5′-OH ends to either 3′-phosphate or 2′,3′-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2′-phosphates, which cannot be spliced by RtcB, to 2′,3′-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ⁵⁴ coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.     

Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.     

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO₄ duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO₄, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization.     

A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine

tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2′,3′-cyclic phosphate and 5′-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2′-PO₄ specificity by showing that AtRNL is deficient in transferring AMP to pRNA_OH to form AppRNA_OH but proficient at sealing pre-adenylylated AppRNA_OH. This strategy for discriminating 2′-PO₄ versus 2′-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific “capping” of 5′-PO₄ ends.     

Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon–exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.     

Cyclases of the 3'-terminal phosphate in RNA: a new family of RNA processing enzymes conserved in eucarya, bacteria and archaea.

The 2',3'-cyclic phosphate termini are produced, as either intermediates or final products, during RNA cleavage by many different endoribonucleases. Likewise, ribozymes such as hammerheads, hairpins, or the hepatitis delta ribozyme, generate 2',3'-cyclic phosphate ends. Discovery of the RNA 3'-terminal phosphate cyclase has indicated that cyclic phosphate termini in RNA can also be produced by an entirely different mechanism. The RNA 3'-phosphate cyclase converts the 3'-terminal phosphate in RNA into the 2',3'-cyclic phosphodiester in the ATP-dependent reaction which involves formation of the covalent cyclase-AMP and the RNA-N3' pp5' A intermediates. The findings that several eukaryotic and prokaryotic RNA ligases require the 2',3'-cyclic phosphate for the ligation of RNA molecules raised a possibility that the RNA 3'-phosphate cyclase may have an anabolic function in RNA metabolism by generating terminal cyclic groups required for ligation. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity. This article reviews properties of the human and bacterial cyclases, their mechanism of action and substrate specificity. Possible biological functions of the enzymes are also discussed.     

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I

Many high-throughput small RNA next-generation sequencing protocols use 5′ preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter''s 5′ end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5′ adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3′ blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3′ ends to be 5′ adenylylated at >90% efficiency.