RNA Ligase Activity Research Articles

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

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.

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

The Leishmania kinetoplast-mitochondrion contains terminal uridylyltransferase and RNA ligase activities

Purified mitochondria of Leishmania tarentolae contain 3'-terminal uridylyltransferase and RNA ligase activities which can be solubilized by detergent lysis of the organelle. Run-off transcription of maxicircle and minicircle DNA also occurs in intact and Triton-lysed mitochondria, using [32P]GTP as the labeled precursor. Heparin inhibits the solubilized terminal uridylyltransferase activity but does not affect the labeling of endogenous RNAs in intact mitochondria with [32P]UTP. Clarification of the mitochondrial Triton lysate causes an increase in terminal uridylyltransferase activity with exogenous substrates. These two activities are candidates for involvement in a post-transcriptional RNA editing process of mitochondrial transcripts.

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo.

Purification of wheat germ RNA ligase. I. Characterization of a ligase-associated 5'-hydroxyl polynucleotide kinase activity.

An RNA ligase that catalyzes the formation of a 2'-phosphomonoester-3',5'-phosphodiester bond in the presence of ATP and Mg2+ was purified approximately 6000-fold from raw wheat germ. A 5'-hydroxyl polynucleotide kinase activity copurified with RNA ligase through all chromatographic steps. Both activities cosedimented upon glycerol gradient centrifugation even in the presence of high salt and urea. RNA ligase and kinase activities sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. The purified polynucleotide kinase activity required dithiothreitol and a divalent cation for activity and was inhibited by pyrophosphate and by ADP. The kinase phosphorylated a variety of 5'-hydroxyl-terminated polynucleotide chains including some that were substrates for the RNA ligase (e.g. 2',3'-cyclic phosphate-terminated poly(A)) and others that were not ligase substrates (e.g. DNA or RNA containing 3'-hydroxyl termini). RNA molecules containing either 5'-hydroxyl or 5'-phosphate and 2',3'-cyclic or 2'-phosphate termini were substrates for the purified RNA ligase activity. The rate of ligation of 5'-hydroxyl-terminated RNA chains was greater than that of 5'-phosphate-terminated molecules, suggesting that an interaction between the wheat germ kinase and ligase activities occurs during the course of ligation.

Studies on RNA ligase activity in the brain and liver cells of mouse.

RNA ligase in eukaryotic mammalian cells was studied by using mouse brain and liver cell extracts as enzyme sources and Oligo A as substrates. RNA ligase activity was determined by measuring the formation of alkaline phosphate-resistant product from 5'-32P-terminated Oligoribonucleotides. Under appropriate conditions, the activity of this enzyme in brain and liver cells may vary between 16-49 mU/ml. The joining way between donor and acceptor is 5'-P----3'-OH. Further studies were carried out by using synthetic UpCpU and 32pNp as substrates and crude enzyme preparations from extracts of cell nuclei of brain and liver as enzyme sources. RNA ligase activity was examined by homochromatography and autoradiography. A clear joining product was demonstrated and then isolated from the reaction mixture by DEAE-Sephadex A25 column chromatography. The eluted fractions were identified by DEAE-cellulose thin layer chromatography. The joining product was hydrolyzed either with KOH or with alkaline phosphatase, the autoradiographic spot of the product disappeared. In this case the joining way between donor and acceptor is 3'-P----5'-OH instead of 5'P----3'-OH. All this indicated that in extracts of mouse brain and liver cells most probably exists some other kind of RNA ligase, which differs from the T4 RNA ligase in the joining way.

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities.

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.

An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro.

Transfer RNA half-molecules are intermediates in the splicing of tRNA precursors containing intervening sequences. We have utilized yeast tRNA half-molecules to identify and partially purify an ATP-dependent RNA ligase activity from extracts of wheat germ. This activity can complement a yeast tRNA endonuclease in vitro to efficiently splice 10 different yeast tRNA precursors. The products of in vitro splicing are a covalently joined tRNA and a circular intervening sequence RNA. The internucleotide bond formed at the splice junction is a 2'-phosphomonoester, 3',5'-phosphodiester structure. The 2'-phosphate originates from the 2',3'-cyclic phosphate at the 3' terminus of the 5' half-tRNA. The phosphodiester phosphate is derived from the gamma-phosphate of ATP.

Ligation of endogenous tRNA 3′ half molecules to their corresponding 5′ halves via 2′-phosphomonoester,3′,5′-phosphodiester bonds in extracts of Chlamydomonas 1

tRNA preparations from Chlamydomonas and wheat germ contain small amounts of tRNA 5' halves and corresponding 3' halves. Incubation of cell-free extracts from the two sources with [gamma-P]ATP yielded 5'-P-labeled tRNA 3' halves which were joined to their corresponding 5' counterparts to form mature tRNA containing 2'-phosphomonoester,3', 5'-phosphodiester bonds. tRNA 3' halves labelled with T4 kinase were purified, sequenced and also joined to their 5' counterparts. It is proposed that these tRNA halves may be intermediates of the tRNA splicing process, and that the RNA kinase and ligase activities observed here are part of the tRNA splicing complex.

RNA ligation via 2'-phosphomonoester, 3'5'-phosphodiester linkage: requirement of 2',3'-cyclic phosphate termini and involvement of a 5'-hydroxyl polynucleotide kinase.

Extracts of wheat germ contain a RNA ligase activity that catalyzes the conversion of linear polyribonucleotides into covalently closed circles. As reported previously, this enzyme joins two ends of a RNA substrate via a 2'-phosphomonoester, 3',5'-phosphodiester linkage. In the present work we provide evidence that a 2',3'-cyclic phosphate group at the 3' terminus is required for RNA ligation and that the 5'-hydroxyl end is phosphorylated before the two RNA ends are joined. We report on the presence of 5'-hydroxyl polynucleotide kinase and polynucleotide 2',3'-cyclic phosphate 3'-phosphodiesterase activities in wheat germ extracts. A possible involvement of these enzymes in the ligation process and a potential role of the newly described ligation pathway in RNA processing are discussed.

Genetic and physiological studies of the role of the RNA ligase of bacteriophage T4

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro. We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.

Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.

Formation of a 2'-phosphomonoester, 3',5'-phosphodiester linkage by a novel RNA ligase in wheat germ.

Extracts of wheat germ contain an RNA ligase activity that catalyses the conversation of linear polyribonucleotides into covalently closed circles. The newly formed 3',5'-phosphodiester bridge is resistant to alkali and a number of ribonucleases. This unusual stability is due to the presence of a phosphoryl group esterified to the 2'-position of the 3'-nucleotide joined. This is the first demonstration of an RNA ligase in higher plants and of a natural 2'-phosphomonoester, 3',5'-phosphodiester linkage.

Suppressors of mutations in the bacteriophage T4 gene coding for both RNA ligase and tail fiber attachment activities.

The protein product of T4 gene 63 catalyzes both the attachment of tail fibers to fiberless phage particles and the ligation of single-stranded RNA (Snopek at al., Proc. Natl. Acad. Sci. U.S.A. 74:3355-3359, 1977). To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of amM69 in gene 63. We screened for revertants that could grow at 30 degrees C but not at 43 degrees C on Escherichia coli OK305 when nucleotides were limiting. These false revertants contained the original mutation in gene 63 and new suppressor mutations. Some of these suppressor mutations caused temperature sensitivity by themselves, allowing single mutants carrying the suppressor to be recognized and isolated. The results of mapping and complementation studies indicated that most of these ts suppressors were in the t gene (lysis), one was in gene 5 (baseplate), and one was in gene 18 (sheath). The mutation in gene 18, tsDH638, suppressed three different amber mutations in gene 63 but did not suppress amber mutations in several other genes. None of the suppressors that were characterized were in genes with known functions in nucleotide metabolism. However, an intriguing property of these false revertants was that they were very sensitive to hydroxyurea, an inhibitor of nucleotide metabolism.

Bacteriophage T4 RNA ligase is gene 63 product, the protein that promotes tail fiber attachment to the baseplate.

RNA ligase and tail fiber attachment activities, normally induced following bacteriophage T4 infection of Escherichia coli, are not induced when gene 63 amber mutants of T4 infect nonpermissive host cells. Both activities are induced when these mutants infect permissive hosts, or when revertants of these mutants infect nonpermissive hosts. When one of these mutants infects a host that carries supF, both activities are more than normally heat labile. RNA ligase, purified to homogeneity, promotes the tail fiber attachment reaction in vitro with a specific activity similar to that of the most highly purified preparations of gene 63 product isolated on the basis of tail fiber attachment activity. We conclude that T4 RNA ligase is gene 63 product. The RNA ligase and tail fiber attachment reactions differ in requirements and in response to some inhibitors, suggesting that the two activities of the gene 63 product may be mechanistically unrelated.

Absence of detectable RNA ligase activity in eukaryotic cells

Reports of the existence of eukaryotic RNA ligases may be incorrect. Evidence for this activity has been based upon the conversion of [5′-32p]-terminated oligoribonucleotides to an alkaline phosphatase resistant form and upon the detection of radioactive ribonucleoside monophosphates after alkaline hydrolysis of the reaction products. Although we have in part confirmed these observations, we find the labeled ribonucleoside monophosphate to be the 5′-isomer, and not the expected 2′ (3′)-isomer. In addition, roughly equivalent amounts of ribonucleoside monophosphate were observed whether or not alkaline hydrolysis was performed. We conclude that the existence of RNA ligase activity in eukaryotic cells is suspect.

Studies on Ribonucleic Acid Ligase: CHARACTERIZATION OF AN ADENOSINE TRIPHOSPHATE-INORGANIC PYROPHOSPHATE EXCHANGE REACTION AND DEMONSTRATION OF AN ENZYME-ADENYLATE COMPLEX WITH T4 BACTERIOPHAGE-INDUCED ENZYME

Abstract RNA ligase, isolated from bacteriophage T4-infected Escherichia coli, catalyzes the conversion of synthetic [5'-32P]polyribonucleotides to a form resistant to alkaline phosphatase. The reaction requires ATP and leads to the formation of AMP and PPi. The purification procedure for T4-induced RNA ligase has been modified to yield stable enzyme preparations. The influence of secondary structure on the RNA ligase reaction has been investigated with these fractions. The conversion of synthetic polynucleotides to complex secondary structures was either without effect or inhibitory in the RNA ligase system. Purified RNA ligase was shown to catalyze an exchange reaction between ATP and PPi, but not between ATP and AMP. The ATP-PPi exchange reaction occurred in the absence of a polyribonucleotide substrate. The formation of an RNA ligase-adenylate complex was demonstrated by gel filtration, polyacrylamide gel electrophoresis, and ultracentrifugation in a glycerol gradient. The complex was dissociated by 5'-P-poly(A) to yield AMP while ATP was generated in the presence of PPi. These findings suggest that an enzyme-AMP intermediate occurs during the course of the RNA ligase reaction. Evidence is presented for the occurrence of an RNA ligase activity in eukaryotic cells.

RNA Ligase Activity in Phage-Infected Bacteria and Animal Cells

An RNA ligase which is dependent on ATP and Mg2+ for maximal activity with 5′-32P-labeled poly(I) · poly(C) as substrate has been purified from Escherichia coli infected with phage T4. Ligase activity was demonstrated by the transfer of alkaline phosphatase sensitive 5′-32P termini into an alkaline phosphatase resistant form. The formation of phosphodiester bond was verified by hydrolysis of the reaction product and determining the transfer of 32P from the 5′ position to the 2′(3′) position by electrophoresis. An increase in sedimentation rate of poly(I) · poly(C) was also observed after reaction with RNA ligase. The enzyme also showed an ATP-PPi exchange activity. The activity on the synthetic polyribonucleotides poly(A), poly(U), poly(A) · poly(U) and poly(I) · poly(C) was examined and an increased efficiency of the substrate was obtained when [5′-32P]poly(U) formed a complex with poly(A). [5′-32P]poly(A) was a good substrate both with and without the complementary strand. With 5′-32P-labeled poly(I) · poly(C) as substrate RNA-ligase activity was detected in mammalian cells such as KB, HeLa and L-cells. The activity seems to increase after infection with encephalomyelocarditis (EMC) virus or poliovirus, but not after infection with adenovirus type 2. The RNA ligase activity was confined to the cytoplasmic fraction of the cells.