©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
RNA Ligase and Its Involvement in Guide RNA/mRNA Chimera Formation
EVIDENCE FOR A CLEAVAGE-LIGATION MECHANISM OF TRYPANOSOMA BRUCEI mRNA EDITING (*)

(Received for publication, December 7, 1994; and in revised form, January 23, 1995)

Robert Sabatini (1) Stephen L. Hajduk (1)(§)

From the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 cosediment 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.


INTRODUCTION

In kinetoplastid protozoa, RNA editing is a post-transcriptional RNA-processing event that occurs in the mitochondrion and results in the addition and deletion of uridine residues at specific sites in mRNA transcripts. Editing produces the sequence information necessary for functional RNAs by correcting frameshifts, creating start codons, or in some cases, forming complete reading frames(1, 2, 3, 4) . The genetic information necessary to direct the insertion or deletion of uridine residues in these mRNAs is present in small, 55-70-nucleotide primary transcripts called guide RNAs (gRNAs). (^1)The gRNAs are complementary to the edited mRNAs and contain a nonencoded poly(U) tail of 5-15 nucleotides(5, 6, 7, 8, 9) . The uridine tail may donate or accept uridines during the editing process through the formation of a chimeric gRNA/mRNA molecule consisting of the gRNA covalently linked via the poly(U) tail to the 3` segment of the mRNA(10, 11, 12, 13) . The gRNA also contains a short 7-10-nucleotide anchor region at its 5`-end which is complementary to the pre-edited sequences immediately 3` to the editing site. The anchor region recognizes and base pairs with unedited mRNA and directs the initiation of the editing process. Uridine addition and deletion continues until the mRNA is fully complementary to the gRNA.

Based on the identification of chimeric molecules in vivo(10) and in vitro(11, 12, 13) , two models have been proposed for the mechanism of kinetoplastid mRNA editing. The first, similar to the mechanism of mRNA splicing, involves successive rounds of trans-esterification(10, 14) . In this model the 3`-hydroxyl of the poly(U) tail of the guide makes a nucleophilic attack on the phosphodiester bond at the editing site, producing a free 5` mRNA segment and a chimeric RNA. This chimera is resolved in a second trans-esterification event involving the 3`-hydroxyl of the free 5` mRNA segment to produce an edited mRNA that has one or more uridines inserted or deleted and a gRNA that has the corresponding change in the length of its poly(U) tail.

The second model proposes multiple rounds of cleavage-ligation and draws analogies from the mechanism of tRNA splicing. In this scheme, the gRNA-mRNA chimera is formed by endonuclease and RNA ligase activities(15) . The pre-edited mRNA is cleaved by an endonuclease at the editing site and RNA ligase joins the gRNA to the 3`-fragment of the mRNA producing the chimeric molecule. The chimera is resolved by a second round of cleavage and ligation.

Support for the cleavage-ligation mechanism comes from the identification of mitochondrial RNPs which are thought to be involved in RNA editing(16) . These were shown by glycerol gradient sedimentation of mitochondrial extract to consist of two ribonucleoprotein particles which sedimented as 19 S and 35-40 S complexes containing RNA ligase, chimera formation, and terminal uridylyl-transferase activities. Uridylyl-transferase presumably is required for addition of the nonencoded poly(U) tail to the gRNA. These results led us to examine the role of the RNA ligase in chimera formation.

The mechanism of RNA ligation has been studied for ligases from wheat germ(17, 18) , yeast(19) , and T4-infected Escherichia coli cells (reviewed in (20) ). Even though the enzymes differ in the structures of their substrates and products, their overall mechanisms are quite similar. The T4 enzyme, for example, joins RNAs with a 5`-phosphate to 3`-hydroxyl termini in three distinct and reversible steps:1. E + ATP &lrhar2; E-AMP + PP(i)

2. E-AMP + pN- &lrhar2; E + AppN-

3. -N + AppN- &lrhar2; -NpN- + AMP

The first step involves the adenylylation of the enzyme (E), by the transfer of AMP from ATP, via phosphoamide linkage with a lysine residue on the protein. In the second step, AMP from the adenylylated enzyme is transferred to the 5`-phosphate of the RNA molecule forming an activated RNA molecule with a 5`,5`-phosphoanhydride bond. In the third and final step, the 3`-hydroxyl of the same or different RNA molecule attacts the activated RNA forming a phosphodiester bond and releasing AMP.

In this report we provide the first direct evidence for the involvement of mitochondrial RNA ligase in the production of RNA editing intermediates. The RNA ligase activity present in the mitochondria of Trypanosoma brucei has similar substrate requirements and the same overall mechanism as previously characterized RNA ligases. We identify adenylylated RNA ligase intermediates in the 19 S and 35-40 S complexes, along with chimera activity, and demonstrate their involvement in the mechanism of in vitro chimera formation. The involvement of RNA ligase in the in vitro reaction provides strong evidence for a cleavage-ligation type mechanism of RNA editing.


EXPERIMENTAL PROCEDURES

Materials

All radioisotopes were from DuPont NEN. Bacterial alkaline phosphatase and snake venom phosphodiesterase were from Worthington. T4 kinase and RNA ligase were from Life Technologies, Inc. Polyethyleneimine cellulose thin layer sheets and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate were obtained from Aldrich. Sodium periodate, tetrasodium pyrophosphate, sodium phosphate, yeast pyrophosphatase, and all nucleotides and ATP analogs were obtained from Sigma. Fetal bovine serum was obtained from BioWhittaker.

Preparation of T. brucei Mitochondrial Extract and Glycerol Gradient Sedimentation

Procyclic T. brucei TREU 667 were grown at 26 °C in semidefined medium (23) or in SDM-79 medium (24) supplemented with 10% heat-inactivated fetal bovine serum. Cells were collected when they reached a density of 1-1.5 times 10^7 cells/ml (or 2-2.5 times 10^7 cells/ml when grown in SDM-79).

Mitochondria were isolated as described by Braley et al.(25) and Rohrer et al.(26) . Mitochondrial extract was prepared and sedimented on a 10-30% glycerol gradient as described by Pollard et al.(16) except that 1 times 10 cell equivalents of mitochondria were loaded onto each gradient and centrifuged for 8 h.

Assay of EbulletAMP Complex

Reaction mixtures (30 µl) contained 20 µl of each glycerol gradient fraction, 6.5 µM [alpha-P]ATP (3000 Ci/mmol), and buffer (final concentrations, 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 0.5 mM DTT, and 50 mM potassium chloride). Reactions were incubated for 15 min at 26 °C and stopped with the addition of 3 volumes of acetone (-20 °C). Precipitated protein was recovered and washed with 70% acetone, and the pellet was dried. Pellets were resuspended in SDS sample buffer(21) , boiled for 3 min, and loaded onto a 12% SDS-polyacrylamide gel. Adenylylated proteins were visualized by autoradiography.

Isolation of EbulletAMP Complex

400 µl of a glycerol gradient fraction, corresponding to the peak of RNA ligase, was incubated with [alpha-P]ATP under the conditions described above for 15 min at 26 °C. The reactions were then loaded onto a Sephadex G-50 fine column (0.8 times 23 cm) previously equilibrated with 10% glycerol gradient buffer(16) . Fractions (200 µl) were collected, and radioactivity was determined by Cerenkov counting. The peak fractions eluting in the void volume (containing the adenylylated proteins as determined by SDS-PAGE and PEI cellulose TLC) were pooled and treated as described in the text. When investigating the release of AMP from the ligase, the incubation with [alpha-P]ATP was followed by an additional 15-min incubation with unlabeled ATP (0.5 mM final concentration). This was done to maximize the adenylylated ligase as well as to displace bound, labeled ADP. ADP generated by an ATPase activity present in the extract interferes with the TLC analysis.

Free nucleotides were also removed by dialysis. The extract, which was adenylylated with [alpha-P]ATP and chased with ATP as described above, was dialyzed against 1 liter (three changes) of 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 50 mM potassium chloride, 5% glycerol, 0.4 mg/ml bovine serum albumin, and 0.1% Triton X-100 at 4 °C for 16 h.

Formation of the Adenylylated RNA Intermediate

Reaction mixtures (30 µl) containing 20 µl of glycerol gradient extract (complex I), 6.5 µM [alpha-P]ATP, buffer (final concentrations: 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 0.5 mM DTT, and 50 mM potassium chloride), and 8 pM 5`-P nonligatable substrate (prepared as described below) were incubated for 90 min at 26 °C. Reactions were treated with proteinase K (100 µg/ml) for 10 min at 37 °C, and RNAs were run on an 8% polyacrylamide, 8 M urea gel.

Incubation of the isolated adenylylated proteins (as described in Fig. 2, B and C) also resulted in the formation of an adenylylated RNA intermediate. However, with the use of extract directly off the glycerol gradient and excess [alpha-P]ATP, we were able to increase the yield for further analysis.


Figure 2: Identification of the adenylylated proteins as RNA ligase intermediates. Preadenylylated extract (with [alpha-P]ATP and ATP) was isolated by gel filtration and utilized in the RNA ligase assay as described under ``Experimental Procedures.'' A, reactions contained 15,000 cpm of 5`-P ligatable (Lig.) RNA substrate (lanes 1 and 3) or 5`-P nonligatable (non-Lig.) RNA substrate (lanes 2 and 4). Control reactions consisted of the RNAs incubated in the absence of extract (lanes 3 and 4). Ligation products corresponding to linear (L) and circular (C) molecules are indicated. B and C, ligase reactions were performed as above except the extract was incubated in the absence (lanes 1 and 2) or presence of unlabeled 5`-P ligatable (lanes 3 and 4) or nonligatable (lanes 5 and 6) RNA substrates. Aliquots (10 µl) were taken at the start (lanes 1, 3, and 5) and end (lanes 2, 4, and 6) of the 90-min incubation at 26 °C and stopped with the addition of 5 µl of stop buffer (EDTA, 8 mM final concentration) followed by heating to 80 °C. 2 µl of each reaction were spotted onto a PEI-cellulose plate (B) while 13 µl were run on a 10% SDS-PAGE (C). An autoradiograph of each is shown. Regions of the TLC analysis corresponding to authentic marker compounds AMP, ADP, and ATP, located by ultraviolet illumination, are indicated. Background release of AMP is presumably due to the action of the ligase on endogenous RNAs.



The adenylylated substrate was also chemically synthesized essentially as described by Sninsky et al.(22) . [5`-P]RNA substrate (1.5 times 10^5 cpm) was dried down and dissolved in 100 µl of 0.1 M AMP, 0.1 M MgCl(2) (pH 5.0) and mixed with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (40 mg). The reaction was incubated for 24 h at 26 °C and diluted with 100 µl of H(2)O, and the RNA was recovered by ethanol precipitation. The resulting RNA was then treated with 0.1 unit of bacterial alkaline phosphatase for 1 h at 37 °C. After proteinase K treatment, extraction with phenol-chloroform, followed by chloroform, the RNA was recovered by ethanol precipitation.

Preparation of RNA Ligase Substrates

Substrates for the RNA ligase assay (containing 5`-P and 3`-OH) consisted of an in vitro generated T3 transcript of BSSK plasmid cleaved by AccI. The 5`- and 3`-terminal nucleotides were identified as G and A, respectively, by nuclease P1 and T2 analysis (data not shown). The 100-nucleotide RNA was bacterial alkaline phosphatase-treated and kinased with T4 polynucleotide kinase and in the presence of either ATP or [-P]ATP. The substrate was made nonligatable by oxidation of the free 3`-terminal hydroxyl group with a 100-fold excess of sodium periodate (NaIO(4)) in 0.15 M sodium acetate pH 5.3(27, 28) . After 2 h at 4 °C, sucrose (500-fold molar excess over NaIO(4)) was added to inactivate excess NaIO(4), and the RNAs were precipitated two times with ethanol.

Enzyme Assays

Two RNA ligase assays were used. The first, measured by the ability to label an RNA substrate with [5`-P]pCp, was performed essentially as described by Pollard et al.(16) . Synthetic pre-edited cytochrome b mRNA (1-5 µg) was incubated with 20 µl of extract, 10 µCi of pCp and buffer (final concentrations: 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 0.5 mM DTT, 50 mM KCl, 15% glycerol, and 0.5 mM ATP, except were noted) in a final volume of 30 µl at 26 °C for 1.5 h. Reactions were terminated by the addition of 170 µl of stop buffer (0.5% SDS, 100 µg/ml proteinase K, and 10 mM HEPES, pH 7.9) and incubated at 37 °C for 10 min. After extraction with phenol-chloroform, and chloroform, the RNAs were precipitated and then run on an 8% polyacrylamide, 8 M urea gel. Products were visualized by autoradiography. In the second RNA ligase assay, reaction mixtures (40 µl) contained 35 µl of the G-50 fraction incubated with 15,000 cpm of [5`-P]RNA substrate (BSSK transcript described above) plus buffer (final concentration, 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 0.25 mM DTT, 50 mM KCl, and 10% glycerol) at 26 °C for 90 min. Reactions were terminated, and RNAs were recovered and electrophoresed as above. This second RNA ligase assay allowed the visualization of AMP release without interference from [5`-P]pCp in the TLC analysis. Chimera formation assays were performed as described by Harris and Hajduk(12) . Synthetic cytochrome b gRNA (100,000 cpm), uniformly labeled with [alpha-P]UTP (3,000 Ci/mM), was incubated with unlabeled synthetic pre-edited cytochrome b mRNA (10 pM) in the presence of 20 µl of extract in buffer (final concentrations: 25 mM HEPES, pH 7.9, 10 mM magnesium acetate, 50 mM KCl, 0.5 mM DTT, 10% glycerol, and 0.5 mM ATP, except where noted) in a final volume of 30 µl at 26° for 1.5 h. Reactions were terminated with stop buffer, the RNAs extracted and precipitated as above and electrophoresed on an 8% polyacrylamide, 8 M urea gel. The gel was visualized by autoradiography.

Nuclease Incubations

Nuclease P1 incubations used 1 unit of enzyme in 5 µl of 20 mM NH(4)OAc, pH 5, for 2 h at 37 °C. The reactions were then heated at 80 °C for 4 min, dried down, and resuspended in 20 mM Tris-HCl, pH 9.0, 10 mM MgCl(2). Subsequent digestion with snake venom phosphodiesterase was with the addition of 0.02 unit of enzyme and incubation for 1 h at 37 °C. The samples were applied to thin layer plates for analysis upon completion of the incubations.

Thin Layer Chromatography

Nucleotide analyses were carried out by thin layer chromatography on polyethyleneimine cellulose plates developed with 1.5 M LiCl. After application of the samples, the plates were washed in methanol prior to development.


RESULTS

Presence of Two Distinct Adenylylated Proteins in the 19 S and 35-40 S Complexes

Previous studies have shown that RNA ligase and chimera forming activities cosediment on glycerol gradients (16) (Fig. 1, A and B). Thus, it was important to further characterize the ligase and investigate its possible involvement in the generation of the chimeric molecule. To determine whether an adenylylated ligase intermediate could be detected, mitochondrial extracts were separated on glycerol gradients and proteins were adenylylated by incubation of the fractions with [alpha-P]ATP. Adenylylated proteins were analyzed by SDS-PAGE followed by autoradiography (Fig. 1C). Two labeled proteins of approximately 57 and 50 kDa were visualized. Endoprotease mapping and ATP affinity experiments indicated that the two proteins have different amino acid sequences and binding affinities for ATP (data not shown). The stability of the label to heating in the presence of SDS, during sample preparation for SDS-PAGE, suggests a covalent linkage. The labeled molecules are proteinase K-sensitive and do not become labeled following incubation with [-P]ATP (data not shown). The distribution of these adenylylated proteins in the glycerol gradients coincides with RNA ligase and chimera activities (Fig. 1, A-C). These results show that the adenylylated proteins are components of mitochondrial RNPs, possibly representing ligase intermediates.


Figure 1: Cosedimentation of adenylylated proteins, RNA ligase and chimera-forming activities. T. brucei mitochondrial extract was partially purified by glycerol gradient sedimentation and aliquots of each fraction were assayed for: A, in vitro chimera formation; B, RNA ligase activity; C, protein adenylylation as described under ``Experimental Procedures.'' Samples from the chimera and ligase assays were subjected to PAGE and autoradiography, while samples from protein adenylylation were subjected to SDS-PAGE and autoradiography. A, the chimeric molecule (155 nt) is indicated by an arrow. Also visible are aberrant chimeras consisting of the gRNA attached at cryptic sites in the cytochrome b mRNA substrate and ligation products (230 nt) consisting of the gRNA ligated to the 5`- or 3`-end of the cytochrome b mRNA substrate. B, RNA ligase is assayed by the ability to [5`-P]pCp label the synthetic cytochrome b pre-mRNA. C, two proteins which incorporate [alpha-P]ATP are approximately 50 and 57 kDa in size. Fractions corresponding to complex I and II, as described previously by Pollard et al.(16) , are indicated.



Identification of Adenylylated Proteins as RNA Ligase

The stability of the labeled proteins to heating in SDS suggested a covalent linkage of the AMP to the protein as predicted for ligase intermediates (Fig. 1C). Aliquots of the isolated adenylylated proteins were subjected to various treatments to determine the nature of the E-AMP linkage (Table 1). The AMP-protein linkage was stable to boiling at neutral or alkaline pH, while boiling in 0.15 N HCl rendered the labeled material acid soluble. The protein-nucleotide linkage was hydrolyzed by hydroxylamine but was stable in sodium acetate at the same temperature and pH. The linkage was also hydrolyzed by pyrophosphate (releasing ATP, as visualized by TLC; data not shown). These results suggest that the AMP is linked to the protein via a phosphoamide bond as characterized for other ligases(20, 29) .



To confirm the identification of the adenylylated proteins as RNA ligases, we examined the release of label upon ligation. Adenylylated proteins from the 19 S fraction (complex I) were incubated with either 5`-P-labeled ligatable RNA substrate or the same [5`-P]RNA substrate that was periodate-treated to remove the 3`-hydroxyl groups (Fig. 2A). Ligation of the RNA substrate results in linear and circular molecules (Fig. 2A), identified by nearest neighbor and gel mobility shift experiments (data not shown). However, no ligation products are seen with the periodate-treated RNA (Fig. 2A). To determine whether ligation resulted in deadenylylation of the 50- and 57-kDa proteins, samples were incubated without RNA, with unlabeled ligatable RNA, or with unlabeled nonligatable RNA (Fig. 2, B and C). Aliquots of each reaction were analyzed by thin layer chromatography to visualize the nucleotides (Fig. 2B) and by SDS-PAGE to visualize the adenylylated proteins (Fig. 2C). The release of AMP from both proteins coincided with their incubation in the presence of ligatable versus nonligatable RNA substrate. Based on quantitation by phosphorimagery, there was a 5-fold difference in the AMP release between the ligatable and nonligatable reactions. Consistent with these results we found that the level of deadenylylation of both proteins is significantly greater in the reaction with ligatable RNA. We attribute this difference to an increase in turnover of the ligase in the presence of the ligatable substrate. There is no deadenylylation of either the 50- or 57-kDa protein or release of AMP following their incubation with DNA (data not shown), indicating that both proteins are RNA ligases.

Adenylylated RNA Intermediates

The mechanism of RNA ligation outlined above predicts transfer of AMP from the ligase to the 5`-P of the RNA to form an adenylylated RNA intermediate. The final step of ligation results in the release of this AMP. Removal of the 3`-OH of the substrate RNA, by periodate oxidation, should block the final step of ligation and result in the accumulation of the adenylylated RNA intermediate. To address this question, complex I from the glycerol gradient was incubated with [alpha-P]ATP and either ligatable or nonligatable unlabeled RNA substrate. Under these conditions only the nonligatable substrate RNA is labeled (Fig. 3A). A similar product was obtained with the same RNA incubated with T4 RNA ligase and [alpha-P]ATP (data not shown). The labeled RNAs were gel purified and analyzed by P1 digestion for the presence of the AppN linkage (Fig. 3B). Nuclease P1 digestion of RNA adenylylated by T4 RNA ligase yielded a product that does not comigrate with pA or pG and represents the P1 resistant 5`-5`-phosphoanhydride-linked AppG dinucleotide(30) . P1 digestion of the chemically synthesized adenylylated RNA also resulted in the AppG dinucleotide (data not shown). The presence of a 5`-5`-phosphoanhydride linkage was confirmed by sequential digestion of the labeled RNA with P1 followed by snake venom phosphodiesterase (Fig. 3B) or tobacco acid pyrophosphatase (data not shown), yielding pA. RNA labeled by the trypanosome mitochondrial complex I showed an identical activated intermediate. Both P1 and snake venom phosphodiesterase digestion products of complex I-labeled RNA are identical to those from T4-labeled RNA (Fig. 3B). These results indicate that the RNA ligase activity in complex I forms activated RNA intermediates identical to those previously characterized for other RNA ligases(31, 32) .


Figure 3: Formation of an adenylylated RNA intermediate. Reaction mixtures (30 µl) containing 20 µl of glycerol gradient fraction (complex I) and components described under ``Experimental Procedures'' were incubated with [alpha-P]ATP and the indicated RNA substrate for 90 min at 26 °C. A, samples were proteinase K-treated and subjected to PAGE and autoradiography. Lanes: 1 and 8, pM of 5`-P-ligatable RNA substrate; 2 and 8 pM 5`-P nonligatable RNA substrate. B, the nucleic acids from the gel of Panel A were eluted, digested with nuclease P1, and examined by thin layer chromatography on PEI plates and 1.5 M LiCl, lane 3. Identical RNA that was labeled upon incubation with T4 RNA ligase and [alpha-P]ATP was also digested with P1, lane 1. Sequential digestion by P1 and snake venom phosphodiesterase (SVPDE) of each of these RNAs is shown in lanes 2 and 4. The identity of digestion products are indicated on the left. The AppG dinucleotide was identified by P1 digestion of the synthetic adenylylated substrate.



Involvement of RNA Ligase in Chimera Formation

The release of AMP upon ligation is indicative of the adenylylated proteins representing ligase intermediates (Fig. 2). If RNA editing proceeds according to a cleavage-ligation mechanism, we would expect a similar release of AMP during chimera formation. Thus, we investigated whether AMP is released from the ligase upon the in vitro formation of the chimeric molecule. The RNA ligases in complex I were preadenylylated with [alpha-P]ATP, free nucleotides were removed by dialysis and the labeled proteins were then utilized in a chimera assay containing unlabeled cytochrome b pre-edited mRNA and [alpha-P]UTP uniformly labeled gRNA. Aliquots of the reaction were spotted on a PEI plate to visualize the AMP release by TLC and RNAs electrophoresed to visualize the generation of the chimeric molecule (Fig. 4). During the production of the chimeric molecule (Fig. 4A) AMP is released (Fig. 4B). This release corresponds to a loss of AMP from both ligases, as indicated by SDS-PAGE (data not shown). However, because of other low level ligation events that occur during the in vitro reaction, such as gRNA self ligation (60 nucleotides), we cannot conclude that this AMP release is due solely to chimera formation.


Figure 4: Release of AMP from RNA ligase concomitant with chimera formation. A and B, complex I from the glycerol gradient was pre-adenylylated, dialyzed, and utilized in a chimera assay as described under ``Experimental Procedures.'' Reactions containing 30 µl of extract, 0.5 mM ATP, and components described under ``Experimental Procedures'' (40 µl final volume) were incubated for 90 min at 26 °C. A, RNAs were recovered from the reaction and separated by PAGE; an autoradiograph is shown. The arrow denotes the chimeric molecule. B, aliquots (5 µl) were taken at the start (lane 1) and completion (lane 2) of the reaction, were stopped as indicated in Fig. 2, and spotted onto a PEI-cellulose plate that was developed with 1.5 M LiCl. An autoradiograph is shown. The positions of AMP, ADP, and ATP, located by ultraviolet illumination, are indicated.



The predicted involvement of an RNA ligase in the mechanism of chimera formation requires an adenylylated ligase intermediate. Formation of this intermediate requires the hydrolysis of ATP at the alpha-beta bond. To investigate the involvement of this reaction intermediate in chimera formation, we examined the nucleotide requirements of RNA ligase and chimera activities. We found that both activities share a requirement for ATP which could not be substituted with CTP, GTP, UTP, or dideoxy-ATP (data not shown). To determine the requirement for ATP hydrolysis, either at the alpha-beta or beta- position, each of these activities were assayed in the presence of the alpha,beta-methylene (AMP-CPP) and beta--imino (AMP-PNP) analogs of ATP (Fig. 5). AMP-CPP, which is nonhydrolyzable at the alpha-beta bond was not utilized in either assay. AMP-PNP, which is hydrolyzable at the alpha-beta but not the beta- bond, supported both ligase and chimera formation. AMP-CPP was not utilized but was able to compete with ATP in both the RNA ligase and chimera assays. Thus, the nonutilization of this analog is due to the importance of alpha-beta bond hydrolysis and not the inability of the enzyme to bind the analog. These results indicate that both RNA ligase and chimera activities require the hydrolysis of ATP at the alpha-beta bond but not the beta- bond. The differential ability of the AMP-CPP analog to compete with ATP in the RNA ligase versus chimera assay indicates additional ATP requirements, other than the adenylylation of the ligase, in the mechanism of chimera formation.


Figure 5: Importance of alpha-beta bond versus beta- bond hydrolysis of ATP. ATP analogs that are nonhydrolyzable either at the alpha-beta bond or at the beta- bond were investigated for their utilization in the RNA ligase and chimera assays. Ligase (A) and chimera (B) assays were performed as described under ``Experimental Procedures'' in the presence of: lane 1, no ATP; lane 2, AMP-CPP (0.25 mM); lane 3, ATP (0.25 mM); lanes 4-11, ATP (0.25 mM) and increasing amounts of AMP-CPP, as indicated; lane 12, AMP-CPP (125 mM); lane 13, no ATP; lane 14, AMP-PNP (0.25 mM); lane 15, ATP (0.25 mM). Reaction products were separated by PAGE and autoradiographs are shown. Arrow denotes the chimeric molecule.



The formation of the adenylylated ligase intermediates resulted in the release of pyrophosphate. The finding that chimera formation also specifically requires the alpha-beta bond hydrolysis of ATP provides a strong argument for the involvement of a similar reaction intermediate. Further evidence was provided by the inhibition of chimera formation with pyrophosphate (Fig. 6) which deadenylylates the ligase (Table 1). Pyrophosphate inhibits both RNA ligase and chimera activities in a dose dependent manner (Fig. 6). On the other hand, inorganic phosphate at the highest concentration tested has no effect on either assay (Fig. 6). The inhibition by pyrophosphate can be partially reversed with the addition of pyrophosphatase (Fig. 6). The ability of pyrophosphatase to reverse the level of inhibition indicates that this inhibition is in fact due to the pyrophosphate, most likely by deadenylylating the ligase-AMP. The ability of pyrophosphate to deadenylylate the ligase and inhibit the formation of the chimeric molecule is indicative of ligase involvement in chimera formation.


Figure 6: Dose dependent inhibition of chimera formation and RNA ligase activity by pyrophosphate. Chimera assays were performed as described under ``Experimental Procedures'' in the presence of: lane 1, no sodium pyrophosphate (PP(i)); lanes 2-5, increasing amounts of PP(i) as indicated; lane 6, 2.0 mM PP(i) and 2 units of yeast pyrophosphatase; lane 7, 3.0 mM sodium phosphate. Ligase assays, lanes 8-14, were performed as described under ``Experimental Procedures'' with treatments as described above and as indicated. Reaction products were separated by PAGE; an autoradiograph is shown. Arrow denotes the chimeric molecule.




DISCUSSION

We have previously shown that the gRNA/mRNA chimeric molecules produced in vitro are analogous to those observed in steady-state kinetoplastid mitochondrial RNA(12) . The chimeras formed in vitro consist of the poly(U) tail of the gRNA linked to the mRNA specifically at editing sites and are dependent upon the correct anchor sequence(11, 12, 13) . It is believed that the chimeric molecule represents an intermediate in the editing process. Thus, determination of the mechanism of its formation would provide an indication of the mechanism of kinetoplastid RNA editing.

Previous work by Pollard et al.(16) characterized mitochondrial RNPs that contained potential editing activities including RNA ligase and chimera forming activities. The cosedimentation of RNA ligase and chimera activities suggested that ligase was involved in the production of chimeric molecules. This paper reports on the direct involvement of this RNA ligase activity in the mechanism of chimera formation. We find two distinct proteins which become adenylylated with ATP, cosediment with the ligase activity in glycerol gradients, and have been identified as ligase intermediates by the release of AMP concomitant with ligation. Deadenylylation of these intermediates results in the inhibition of chimera formation. In addition, AMP release from the ligase is concomitant with production of the chimeric molecule. These results argue for a cleavage ligation-type mechanism of chimera formation in vitro and suggests a similar mechanism for RNA editing in kinetoplastids.

The identification of the adenylylated proteins as RNA ligase intermediates is consistent with the mechanism of other ligases. Thus, it is not surprising that the RNA ligase activity present in the chimera-forming complexes specifically requires the alpha-beta bond hydrolysis of ATP. The requirement for alpha-beta bond hydrolysis of ATP in chimera formation could be explained by the involvement of the RNA ligase intermediate. The finding that pyrophosphate, which deadenylylates the intermediate, inhibits chimera formation suggests its direct involvement in the chimera-forming mechanism. Release of AMP from the ligases upon chimera formation is also consistent with this argument.

In the initial characterization of the editing complexes the editing site-specific endonuclease did not appear to comigrate with either complex(16) . This enzymatic activity, which cleaves pre-edited mRNA at sites where editing occurs, would be an essential component of the editing complex for editing to occur by a cleavage ligation-type mechanism. Preliminary evidence indicates that our previous inability to detect the endonuclease within the complexes was due to the masking of endonuclease activity by RNA ligase. (^2)Work is currently being done to investigate the involvement of the endonuclease in chimera formation.

The involvement of RNA ligase in the production of the chimeric molecule is consistent with the type of mechanism proposed in Fig. 7. The first step in chimera formation would involve cleavage of the pre-edited mRNA at the editing site by the editing site-specific endonuclease. The adenylylated RNA ligase would then transfer the AMP to the 5`-phosphate of the 3`-cleavage fragment, forming a 5`,5`-phosphoanhydride bond. This activated phosphate is then attacked by the 3`-hydroxyl of the uridine at the end of the gRNA forming the chimeric molecule. It is conceivable that a second round of cleavage and ligation similar to that outlined above would produce an edited product.


Figure 7: Proposed mechanism of in vitro chimera formation. A model of the possible mechanism involving RNA ligase and endonuclease is presented.



The proposed cleavage-ligation mechanism outlined above is similar to the two stage mechanism of tRNA splicing in yeast(33) . In yeast, the mechanism involves polynucleotide kinase and cyclic phosphodiesterase activities as well as the endonuclease and ligase(19) . In the first stage the endonuclease cleaves the RNA generating the 5`-half, terminating with a 2`,3`-cyclic phosphate, the 3`-half, terminating with a 5`-hydroxyl, and the intervening sequence. In the second stage the 2`,3`-cyclic phosphate is opened by a cyclic phosphodiesterase and the 5`-hydroxyl is the substrate for a kinase activity. The resulting species are then finally substrates for the ligase forming a 2`-phosphomonoester 3`,5`-phosphodiester linkage where the phosphate in the newly formed bond comes from the position of the nucleotide cofactor.

The lack of requirement for beta- bond hydrolysis of ATP (Fig. 5), the inability to incorporate label from [-P]ATP into the chimera product, as well as the lack of detectable polynucleotide kinase activity in mitochondrial extract, (^3)describe an overall reaction scheme similar to tRNA splicing but with several distinct differences. The ability to digest the ligation products to mononucleotides with P1 nuclease^3 is consistent with a typical 3`-5`-phosphodiester linkage produced by the trypanosomal RNA ligase where the 2`-position is not blocked by a phosphoryl group. These results, along with the ability to pCp label the 5`-fragment with T4 RNA ligase,^3 suggest the endonuclease cleaves RNAs leaving 3`-hydroxyls and 5`-phosphates. The 3`-hydroxyl on the gRNA has previously been shown to be required for chimera formation(12, 13) . This may reflect the substrate requirement of the ligase for RNAs with 3`-hydroxyls and 5`-phosphates. Thus, the endonuclease may directly provide the substrates for the ligase in chimera formation as opposed to the mechanism of yeast tRNA splicing.

The involvement of RNA ligase in the in vitro reaction provides the first direct evidence for a cleavage-ligation mechanism of chimera formation. Further purification of the components of these complexes will allow the determination of other proteins involved in the in vitro reaction. The ability to resolve the chimeric molecule to the edited product would characterize them as true editing intermediates and verify the cleavage-ligation model as the mechanism of kinetoplastid RNA editing.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI21401 (to S. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Burroughs Wellcome Fund Scholar in Molecular Parasitology. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294. Tel.: 205-934-6033; Fax: 205-975-2547.

(^1)
The abbreviations used are: gRNA, guide RNA; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PEI, polyethyleneimine; RNP, ribonucleoprotein particle; AMP-CPP, adenosine 5`-(alpha,beta-methylene)triphosphate; AMP-PNP, adenosine 5`-(beta,-imino)triphosphate; AppG, adenylyl (5`,5`) guanosine; AppN, adenylyl (5`,5`) nucleoside.

(^2)
B. Adler, R. Sabatini, and S. Lottajduk, unpublished data.

(^3)
R. Sabatini and S. L. Hajduk, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Brian Adler and Dr. Victoria Pollard for many helpful discussions, and the rest of the Hajduk laboratory for criticisms of the manuscript.


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