©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Trypanosoma brucei RNA Editing
A FULL ROUND OF URIDYLATE INSERTIONAL EDITING IN VITRO MEDIATED BY ENDONUCLEASE AND RNA LIGASE (*)

(Received for publication, October 30, 1995; and in revised form, December 14, 1995)

Kenneth J. Piller (§) Laura N. Rusché Barbara Sollner-Webb (¶)

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.


INTRODUCTION

The term RNA editing describes a variety of processing events that generate RNAs that are different from those predicted by their genes (reviewed in (1) ). In trypanosomatids (Trypanosoma, Herpetomonas, Crithidia, and Leishmania) and other related kinetoplastids (Trypanoplasma), RNA editing refers to the specific insertion and deletion of uridylate (U) (^1)residues in mitochondrial mRNAs. Since its discovery in kinetoplastids(2) , RNA editing has been shown to create translational start and stop codons, correct frameshifts, and extend open reading frames (reviewed in (3, 4, 5, 6) ). The extent of editing can be small (e.g. the insertion of four U residues into Trypanosoma brucei cytochrome oxidase subunit 2 (COII) mRNA(2) ) or extensive (e.g. the creation of >50% of the total transcript length of T. brucei COIII mRNA (7) ). Because trypanosomatids diverged from one another 200-300 million years ago(8, 9) , this RNA editing is at least that old.

The genetic information directing RNA editing resides in short (50-70 nt) mitochondrial transcripts(10) . These guide RNAs (gRNAs) contain a 5` anchor sequence (4-18 nt) that is complementary to the pre-edited mRNA immediately 3` of the segment to be edited, a guiding region that is complementary (allowing for G:U base pairing) to the fully edited mRNA segment and a nonencoded (5-24 nt) 3` poly(U) tail (4) . Presumably the gRNA base pairs with the pre-edited mRNA and directs editing to progress 5` from the anchor region, inserting and deleting U residues to yield mRNA complementary to the guiding region(10) . The poly(U) tail may further stabilize the duplex of gRNA with the purine-rich mRNA (11) and/or serve as the source of U residues for insertion(12) .

Blum et al. (10) proposed that each cycle of U insertion or removal involves an endonuclease to cleave the RNA, a terminal uridylyl transferase to insert or an exonuclease to delete U residues, and an RNA ligase to rejoin the 5` and 3` half mRNAs. Indeed, these proposed enzymatic activities are present in trypanosome mitochondria (13, 14, 15, 16) . Editing was later proposed to involve sets of transesterification reactions(12, 17) . In the most popular of these schemes, the 3` hydroxyl of the U tail of the gRNA attacks the mRNA at the first gRNA:mRNA mismatch, creating a gRNA-mRNA chimeric molecule. The chimera is then attacked by the 3` hydroxyl of the 5` mRNA fragment again adjacent to the duplex region, within the poly(U) tract, generating a partially edited mRNA and liberating the gRNA. Although the transesterification model has been considered very appealing because of its common mechanism with RNA intron removal (reviewed in (18) ) and because of the in vivo existence of gRNA-mRNA chimeric molecules in trypanosomes(12) , identical chimeric intermediates and partially edited RNAs could alternatively be generated by endonuclease cleavages and RNA ligations ((19) ; see Fig. 1).


Figure 1: One complete cycle of CYb RNA editing. A model for U insertional editing mediated by endonuclease plus RNA ligase involving a gRNA-mRNA chimeric intermediate is illustrated. In the first half cycle of editing, the extract endonuclease (or MBN) cleaves CYb pre-edited mRNA within the 3` editing domain, and the extract ligase (or T4 RNA ligase) covalently joins the 3` oligo(U) end of a CYb gRNA to the 3` half mRNA, generating a chimera with U residues at the junction. In the second half cycle, the extract endonuclease (or MBN) cleaves the chimera within the oligo(U) tract, and extract ligase (or T4 RNA ligase) then covalently rejoins the mRNA halves, generating a partially edited mRNA with U residues transferred from the gRNA tail to an editing site of the mRNA. The white boxes represent the editing domain of the mRNA, and the black boxes represent the oligo(U) tail of the gRNA.



In vitro studies on possible mechanisms of RNA editing have identified an editing domain-specific endonuclease in T. brucei that is single strand-specific (20) and cleaves cytochrome b (CYb) pre-edited mRNA predominantly at editing sites (ES) 2 and 3(14) . T. brucei mitochondrial extract was also shown to direct chimera formation, specifically joining a model CYb gRNA to pre-edited mRNA(21) , although the precise position of the junction (whether at ES1, ES2, and/or ES3) remained unclear. Investigations of the in vitro activity that forms chimeras have shown it to co-sediment with both RNA ligase (15) and the editing domain specific endonuclease(22) , demonstrating the feasibility of chimera formation through the enzymatic pathway(22) . Ruschéet al. (23) and Sabatini and Hajduk (24) then demonstrated that gRNA-mRNA chimeras are formed in trypanosome mitochondrial extract by a pathway that involves RNA ligase. Furthermore, data in Ruschéet al.(23) , in conjunction with results in this paper (see Fig. 2), demonstrate that the in vitro chimera formation pathway also utilizes the editing site-specific endonuclease of the extract. However, it remained unresolved whether the resultant chimeras could be intermediates in a complete cycle of editing.


Figure 2: The junction sequences of CYb gRNA-mRNA chimeras. A, CYb pre-edited mRNA (0.5 pmol) and P-labeled CYb gRNA (1 pmol) were incubated with mitochondrial extract (lane 2) or with MBN and T4 RNA ligase (lane 3). The arrow indicates the chimeric product. The P-labeled gRNAs are off the bottom of this photograph. The sizing markers used in this and subsequent figures are a HpaII digest of pBR322 (lane 1). B, the gRNA-mRNA junction sequences from 37 cDNA clones of chimeras generated using mitochondrial extract are shown, grouped according to their mRNA junction sites. N is the nonediting site immediately upstream of ES3, and the bold T represents 0, 1, or 2 T residues. Arrows show the locations of ES1, ES2, and ES3. C, the gRNA-mRNA junction sequences from 34 cDNA clones of chimeras generated using MBN and T4 RNA ligase are shown. Because the clones of C derived from PCR amplification of 10 times more chimeric RNA than was used in the amplifications of B, it is likely that each clone represents a separate original RNA molecule. The 3` ends of the gRNAs used in A had 1-3 U residues, whereas those in B and C had 3 U residues.



In this study we report a full cycle of T. brucei U insertional editing, using CYb substrates and mitochondrial extract, as predicted by the enzymatic model involving a gRNA-mRNA chimeric intermediate (Fig. 1). The editing pattern of the resultant CYb partially edited mRNAs is similar to that of CYb partially edited RNAs generated in vivo. Furthermore, the utilization of ATP analogs indicates that previously reported U deletion (25) and U insertion (26) activities, like the above described activities, all involve RNA ligase.


EXPERIMENTAL PROCEDURES

In Vitro RNA Production

The CYb pre-edited mRNA (208 nt), model CYb gRNA (30 or 42 nt; containing a 3` tail of 3 or 15 U residues), and plL RNA (82 nt; used for RNA ligase assays) were synthesized from DNA templates as described(22) . CYb-I gRNA[558] (70 nt) was synthesized by T7 RNA polymerase using a DraI-linearized plasmid kindly provided by the Stuart laboratory(27) . CYb chimeric RNA (CML15-2; 196 nt) and CYb partially edited mRNA (PE5-2; 212 nt) were synthesized by T3 RNA polymerase using HindIII-digested templates. All mRNAs were uniformly labeled with either P or ^3H for RNA quantification. RNA purification, 5` end-labeling, and 3` end-labeling were carried out as described(22) .

In Vitro Mitochondrial Endonuclease Reactions

Mitochondrial extracts (2 times 10 cell equivalents/ml) were prepared from procyclic T. brucei strain TREU 667 as described(22) . Cleavage reactions (20 µl) contained substrate RNA (50-200 fmol) and 2 µl of mitochondrial extract in MRB buffer (25 mM Tris-HCl pH 7.5, 60 mM KCl, 10 mM Mg(OAc)(2), 1 mM EDTA, 0.5 mM dithiothreitol, and 5% glycerol). Following incubation at 25 °C for 30 min, reactions were brought to 0.3 mM NaOAc, supplemented with 1 µg each of tRNA and glycogen, phenol-chloroform extracted, ethanol precipitated, electrophoresed, and visualized as described(22) . Where indicated, cleavage was augmented by the addition of heparin (0.5 mg/ml final). For sizing standards, substrate RNAs were treated with RNaseT1 (USB) or RNaseU2 (Pharmacia Biotech Inc.) for 5-10 min at 50 °C.

Generation of Chimeras and Partially Edited mRNAs

The 20-µl chimera-forming reactions contained ^3H-labeled CYb pre-edited mRNA (0.5-1 pmol), P-labeled CYb gRNA (0.5-2 pmol), and 2 µl of mitochondrial extract in MRB buffer supplemented with 1 mM ATP and were incubated for 30 min at room temperature. CYb chimeras were also formed using MBN and T4 RNA ligase as described(22) .

CYb partially edited mRNAs were formed using the 5` half of CYb pre-edited mRNA (isolated from MBN-digested 5` end-labeled CYb pre-edited mRNA), the 3` half of CML15-2 RNA (prepared by digestion of pCp-labeled CML15-2 RNA with either mitochondrial extract or MBN), and 2 µl of mitochondrial extract or 15 U T4 RNA ligase as described for chimera formation. The samples were phenol-chloroform extracted, ethanol precipitated, electrophoresed, and visualized by autoradiography.

PCR Amplification, Subcloning, and Sequencing of Junctions

The chimeric and partially edited RNA products were gel isolated from reactions scaled up 6-20-fold and reverse transcribed and PCR amplified using the thermostable rTth reverse transcriptase RNA PCR kit (Perkin-Elmer) and a Hybaid Omnigene Thermocycler (Marsh Biomedical). Both of the reverse transcription reactions (20 µl) contained primer I (CCACGCGTCAAGCTAAACACACTCCAC) and were incubated for 20 min at 70 °C and then placed on ice. They were then brought to 0.75 mM EGTA (to chelate the Mn) and 1.5 mM Mg and either primer II (GTCCGGGACTGATCATTAAAAGAC) or primer III (GAGAATTCGAGCTCGGTACCCG) was added for amplification of chimeric and partially edited cDNAs, respectively. Chimeric cDNA was amplified by incubation at 92 (2 min) and then 95 °C (2 min), followed by 40 cycles alternating between 95 (30 s) and 68 °C (30 s). Partially edited cDNA was amplified by incubation at 90 (1 min) and then 94 °C (1 min) followed by 42 cycles alternating between 94 (30 s), 68 (30 s), and 72 °C (30 s). The PCR products were digested with MluI (primer I) and either BclI (primer II) or EcoRI (primer III), gel-purified, mixed with MluI/BamHI- or MluI/EcoRI-digested pIBI31 vector (International Biotechnologies Inc.), and incubated with T4 DNA ligase for 1 h at 37 °C. Ligation mixtures were transformed into E. coli JM109 competent cells using the transformation and storage solution procedure(28) . Plasmid DNA from 3-ml cell cultures was isolated using the alkaline lysis procedure (29) and suspended in 20 µl of water. Sequencing was carried out with the Sequenase kit (U. S. Biochemical Corp.) using 2 µl of the miniprep plasmid DNA and 1 pmol of universal (-20) sequencing primer.

Secondary Structure Modeling of RNAs

Secondary structure calculations were performed with the University of Wisconsin Genetics Computer Group program FOLD (30) using updated energy values on a VAX model 8530.

Enzyme Assays Using ATP Analogs

Mitochondrial extracts were depleted of endogenous ATP by incubation with 5 mM glucose and 1 unit of hexokinase (Sigma)/20 µl for 30 min at room temperature (23) . Chimera assays (described above) and ligase assays (described in (22) and (23) ) were carried out without added nucleotides or in the presence of 1 mM ATP, AMP-CPP (Boehringer Mannheim), AMP-PNP (Boehringer Mannheim), or AMP-PCP (Sigma). Analogous ligase assays were also performed using 3 units of T4 RNA ligase.


RESULTS AND DISCUSSION

Junctions of Extract-generated CYb Chimeras Map to Endonuclease Cleavage Sites

Although recent data are consistent with the CYb chimera formation in T. brucei mitochondrial extract being catalyzed by the editing site-specific endonuclease of the extract (23) , the possibility that these chimeras may have their junctions at ES1 (21) would be inconsistent with this hypothesis, because the site-specific endonuclease cleaves CYb RNA predominantly at ES2 and ES3 (14) . We therefore determined the mRNA junction site in these CYb chimeras (Fig. 2A). Chimeras formed in extract using CYb pre-edited mRNA and a model CYb gRNA were reverse transcribed using thermostable polymerase (to enable extension through the stable anchor region duplex), PCR amplified, subcloned, and sequenced. In almost all chimera clones, the gRNA was joined to the mRNA either at ES2 or ES3 (Fig. 2B); only in one was it joined at ES1. Using a different, natural input CYb gRNA (g[558]; (27) ), we obtained a similar profile of ES junctions, with 19 of the 22 sequence junctions at either ES2 or ES3, two at ES1 and one that is one nucleotide upstream of ES3 (data not shown). Notably, in both cases the location and frequency of the mRNA junctions agree with the previously reported sites of endonuclease cleavage on CYb pre-edited mRNA - predominantly at ES2 and ES3, with minor cleavages at ES1 and one nucleotide upstream of ES3(14, 20) . Thus, extract-generated CYb chimeras contain junctions at precisely the positions predicted if these molecules resulted from cleavage of the pre-edited mRNA by the extract endonuclease, followed by ligation to the gRNA. This result, in conjunction with previous data(20, 22, 23, 24) , provides very strong evidence that extract-generated chimeras are formed via the endonuclease/ligase pathway.

Chimeras can also be generated using MBN and T4 RNA ligase(20) . Their junctions should be at the same sites as those generated using mitochondrial extract because MBN also cleaves CYb pre-edited mRNA at ES2 and ES3(14) . As expected, in all 34 sequenced MBN/T4 RNA ligase-generated CYb chimera clones examined, the gRNA is joined to the CYb mRNA at either ES2 or ES3 (Fig. 2C). Obtaining the same profile of mRNA junction sites in CYb chimeras generated using either mitochondrial extract or a heterologous single strand-specific endonuclease plus RNA ligase provides additional support that the chimeras generated in the mitochondrial extract are formed by the concerted action of the extract endonuclease and RNA ligase.

Unlike the heterologous enzyme-generated CYb chimeras (Fig. 2C), most of the extract-generated chimeras did not retain all three U residues from the 3` tail of the gRNA (Fig. 2B). This observation explains the small size difference of the extract-generated and MBN/T4 RNA ligase-generated chimeras (Fig. 2A). Furthermore, chimeras generated in mitochondrial extract using a CYb gRNA with a 3` tail of 15 U residues retained 0-6 U residues at the gRNA-mRNA junction, and the 3` U tail of the free gRNAs was also observed to decrease in length (data not shown). These results suggest that a 3` exonuclease is active in T. brucei mitochondrial extracts. The observation that this 3`-terminal nucleotide removal appears to stop at the end of the oligo(U) tail (Fig. 2B) suggests that it may be a U-specific exonuclease. An analogous loss of U residues observed in the Leishmania tarentolae NADH dehydrogenase subunit 7 chimera-forming system (31) suggests that a similar exonuclease activity may be active in other kinetoplastids. In vivo, such a U-specific exonuclease activity could function in deleting genomically encoded U residues (10) or in removing excess U residues that were transferred to an editing site.

Extract Cleaves within the Oligo(U) Region of Chimeras

The nuclease-ligase model of editing via a chimeric intermediate (Fig. 1) predicts that endonuclease would cleave the chimera within its poly(U) region at the gRNA-mRNA junction, transferring one or several U residues to the 5` end of the cleaved 3` mRNA fragment. To continue examining the feasibility of this model, we assessed whether the endonuclease of mitochondrial extract cleaves a CYb chimera with this specificity (Fig. 3A). The chimeric RNA CML15-2, which contains 15 U residues (an average size oligo(U) tract for in vivo CYb chimeras(27) ) at the ES2 junction was 3` pCp-labeled to follow the mRNA region. Incubation of CML15-2 RNA with mitochondrial extract indeed resulted in specific cleavage (Fig. 3A, lane 8). Relative to sizing standards (Fig. 3A, lanes 4 and 5), the liberated mRNA portion of the chimera (Fig. 3A, lane 8) migrated 8-11 nt slower than the analogous 3` cleavage product from CYb pre-edited mRNA (Fig. 3A, lane 2). These 8-11 extra residues represent a portion of the U tract that derived from the gRNA tail (see also Fig. 4B). Thus, T. brucei mitochondrial extract specifically cleaves the CYb chimera within the poly(U) region at the gRNA-mRNA junction.


Figure 3: Endonuclease specifically cleaves CYb gRNA-mRNA chimeras. A, endonuclease cleavage reactions contained 0.2 pmol of pCp-labeled CYb pre-edited mRNA (lanes 1-5) or 0.2 pmol of pCp-labeled CML15-2 RNA (lanes 7-9). (CML15-2 is chimeric RNA transcribed in vitro from a reverse transcriptase-PCR amplified and cloned CYb chimera that was generated by MBN and RNA ligase using a CYb gRNA with a 3` tail of 15 U residues.) The reactions were incubated without additions (lanes 1 and 7) or with 2 µl of mitochondrial extract (lanes 2 and 8), 25 units of MBN (lanes 3 and 9), RNase T1 (lane 4), or RNase U2 (lane 5). The reactions of lanes 2 and 8 also contained 50 mg/ml of heparin. The bracket denotes the 15 U residues of CML15-2 RNA that originally derived from the gRNA tail. B, the predicted secondary structure for CML15-2 mRNA (DeltaG = -40.5 kcal/mol) was determined by the FOLD algorithm(30) . The major cleavage sites for mitochondrial endonuclease (solid arrows) and for MBN (open arrows) are within the gRNA-derived U tract (bold line). The first 29 nt and last 18 nt of this RNA are vector sequences.




Figure 4: Generation of CYb partially edited mRNA in vitro. A, pCp-labeled CML15-2 RNA was cleaved using MBN (lane 3) or extract (lane 4) and the isolated 3` half mRNAs (25 fmol) were mixed with P-labeled 5` half of CYb pre-edited mRNA (25 fmol, MBN-derived) and treated with either 15 units of T4 RNA ligase (lane 3) or 2 µl of mitochondrial extract (lane 4). The arrow denotes partially edited mRNAs. 3` CML indicates the 3` cleavage product of CML15-2 RNA; 5` pre indicates the 5` cleavage product of CYb pre-edited RNA. Markers are CYb pre-edited mRNA (lane 2) and HpaII cleaved pBR322 (lane 1). Lane 4 was exposed 4 times longer than lane 3. B, the sequences of cDNAs derived from extract-generated (solid bars) and MBN/T4 RNA ligase-generated (stipled bars) CYb partially edited mRNAs, as in A, are represented. The downstream sequence corresponds to CYb mRNA up to ES2, at which point the number of inserted U residues and frequency of representation are shown in the histogram. The upstream sequence also corresponds to CYb mRNA, beginning at ES2 in most clones but at ES3 in a small fraction of clones, including that used in Fig. 5.




Figure 5: Endonuclease specifically cleaves CYb partially edited mRNA. A, 0.2 pmol of pCp-labeled CYb pre-edited mRNA (lanes 2-4) or 0.2 pmol of pCp-labeled PE5-2 mRNA (lanes 5-8) were incubated without additions (lanes 2 and 5) or with 2 µl of mitochondrial extract in the presence of 0.5 mg/ml heparin (lanes 3 and 6), 25 units of MBN (lanes 4 and 7), RNase T1 (lane 8), or RNase U2 (lane 9). The 5 U residues transferred to ES2 in PE5-2 are indicated by the bracket. B, analysis of extract cleavage sites using 3` labeled PE5-2 (left) or 5` labeled PE5-2 (right) is shown on a gel that was run approximately twice as far as that of A. C, the secondary structure for PE5-2 mRNA (DeltaG = -41.1 kcal/mol) predicted by the FOLD algorithm (30) is illustrated. The 5 transferred U residues are indicated by the bold line, and the arrows indicate the endocnuclease cleavage sites.



To address whether the structure of the CYb chimeric RNA determines its cleavage pattern, pCp-labeled CML15-2 RNA was incubated with MBN, a probe for single-stranded structure. This RNA was also specifically cleaved by MBN (Fig. 3A, lane 9), generating a family of 3` mRNA products containing 10-13 U residues at their 5` ends. This MBN cleavage within the poly(U) tract of CML15-2 RNA indicates that this region of the chimera is single-stranded, a result also predicted by analysis of this RNA using the FOLD algorithm (30) (Fig. 3B).

Such a stem-loop structure may well be a general feature of gRNA-mRNA chimeras, because the duplex involving the anchor region of the gRNA and its complement from the mRNA region presumably remains base paired, whereas the segment between those intervening regions shows only limited self-complementarity. Thus, preferential cleavage by single strand-specific nuclease in the oligo(U) region may be a general feature of chimeric molecules. The predicted structures for 20 trypansome pre-edited mRNA sequences also suggested a single strand-specific nuclease sensitivity in the region where editing begins (20) . Therefore, a common mitochondrial single strand-specific activity could recognize both the pre-edited mRNA and the gRNA-mRNA chimera, based on their secondary structures.

Formation of CYb Partially Edited Molecules

The nuclease-ligase model for editing next predicts that the 5` half mRNA becomes covalently joined to the 3` half mRNA liberated from the chimeric RNA, thereby transferring U residues to an editing site and resulting in a partially edited mRNA (Fig. 1). To determine whether partially edited mRNA could be generated via this pathway in vitro, the 3` extract cleavage product (P-pCp-labeled) from the chimera and the 5` cleavage product (P-labeled) from the original CYb pre-edited mRNA cleavage reaction were incubated in mitochondrial extract (Fig. 4A, lane 4). A large product was generated that migrated several nucleotides slower than CYb pre-edited mRNA (Fig. 4A, lane 2), as would be expected from a partially edited mRNA. A similarly sized RNA product was generated by incubation of the 3` MBN cleavage product (P-pCp-labeled) of the chimera plus the 5` cleavage product (P-labeled) from the original CYb pre-edited mRNA cleavage reaction in the presence of T4 RNA ligase (Fig. 4A, lane 3).

To verify the identity of these putative partially edited mRNAs, both the extract-generated and the T4 RNA ligase-generated products were reverse transcriptase-PCR amplified, subcloned, and sequenced. Partially edited mRNAs were indeed obtained (Fig. 4B). The majority of extract-generated partially edited mRNAs contained 8-9 U residues at ES2, whereas the majority of MBN plus T4 RNA ligase-generated mRNAs contained 10-11 U residues at ES2. These partially edited mRNAs represent products of a complete cycle of in vitro editing, demonstrating that it is indeed possible to transfer U residues from a gRNA tail, through a chimeric intermediate, and to an editing site within pre-edited mRNA. The fact that a full round of U insertional editing can also be mimicked by the structure-specific heterologous enzymes suggests that RNA secondary structural features direct the reaction. These results (Fig. 4B) also confirm the cleavage site data of Fig. 3A.

Partially Edited Molecules Are Susceptible to Endonuclease Cleavage

If the partially edited RNAs generated in vitro are intermediates in an RNA editing pathway (Fig. 1), they should be substrates for re-editing, beginning with a specific endonuclease cleavage within the editing domain. This was tested using a CYb partially edited mRNA (PE5-2) containing 5 U residues at ES2 and otherwise identical to CYb pre-edited mRNA. Incubation of PE5-2 mRNA (pCp-labeled) in mitochondrial extract indeed resulted in specific cleavage (Fig. 5A, lane 6), and this cleavage is adjacent to ES2, mapped relative to sizing markers (Fig. 5A, lanes 3, 8, and 9). Higher resolution mapping of the 3` and 5` cleavage products (Fig. 5B, left and right) demonstrates a major cleavage at the 3` end of the oligo(U) tract and minor cleavages after each U residue. The complementary cleavage profiles observed using 3` and 5` end-labeled mRNA (Fig. 5B) indicate that the minor bands represent initial cleavages in the extract and not secondary degradation products. Thus, extract endonuclease specifically cleaves partially edited CYb mRNA at the incompletely edited site, demonstrating that CYb partially edited mRNA is a potential substrate for re-editing via an enzymatic pathway. MBN also cleaves PE5-2 mRNA at each site within the oligo(U) region but not preferentially at the oligo(U)/ES2 junction (Fig. 5A, lane 7; data not shown). This suggests that the U residues are single-stranded, an outcome also predicted by application of the FOLD algorithm (30) on PE5-2 mRNA (Fig. 5C).

It is notable that this PE5-2 RNA as well as CML15-2 RNA and CYb pre-edited mRNA is cleaved by the extract endonuclease predominantly 2 nt from the 3` end of the loop, whereas they are cleaved by MBN predominantly at the central positions of the single-stranded loop. A cleavage preference of mitochondrial endonuclease for a site a fixed distance from the 3` end of a single-stranded loop might be predicted for an endonuclease involved in editing.

Comparison of in Vitro and in Vivo Partially Edited mRNAs

Is the CYb partial editing that we observe in vitro reflective of in vivo editing-like activities? Sequence analysis of in vivo generated partially edited RNAs from L. tarentolae and of in vivo generated chimeric RNAs have implicated a model in which editing begins at the 3` most editing site (ES1), so the relevance of editing-like activities that start predominantly at ES2 or ES3 (Fig. 2B) leaving ES1 unaltered must be questioned. Interestingly, sequence analysis of in vivo generated partially edited CYb mRNAs from T. brucei(32) revealed that almost all the molecules were aberrantly edited and many of these molecules have patterns consistent with the in vitro editing reported above. Specifically, over one-third of the steady state T. brucei CYb mRNAs that are partially edited in vivo are unedited at ES1 and instead have partial editing starting at ES2 or ES3 (Fig. 6). This would be expected to occur if their editing modifications were initiated by the editing site-specific endonuclease that initiates in vitro editing at ES2 and ES3. Also like the in vitro generated partially edited molecules (Fig. 4B), the in vivo generated partially edited molecules contain more U residues at the initiating editing site than are present in the correctly edited mRNA (Fig. 6). Thus, the CYb in vivo generated partially edited RNAs may have initiated their editing by events much like those we observe in vitro. Whether these partially edited molecules can be subsequently re-edited to generate the canonical mature mRNA sequence remains to be determined, although their in vitro endonuclease sensitivity suggests that this could be the case(20) .


Figure 6: Partially edited CYb mRNAs generated in vivo. Partially edited CYb mRNAs from in vivo T. brucei cellular RNA were cloned and sequenced(32) . Their sequences are grouped according to whether editing begins at ES2, at ES3, immediately 5` of ES3 (ES3`), or at ES1. The unedited sites of the editing domain 3` of the partially edited region are boxed. The unedited and fully edited CYb mRNA sequences are shown at the top with the editing sites numbered and the edited region shaded.



There are other examples in the literature of in vivo edited RNAs in which the editing also begins at ES2 rather than ES1. These include half of the reported Crithidia fasciculata MURF2 partially edited RNAs (33) and an additional T. brucei CYb RNA(27) . The literature also contains numerous examples of cDNAs containing an unedited site or sites within a larger otherwise correctly edited region (e.g. CYb of Trypanoplasma borrelli(34) , NADH dehydrogenase subunit 7 of T. brucei(35) , and G6(36) , CYb, and COIII of L. tarentolae(37) ), suggesting that editing in vivo may also permit a more relaxed versus a strict 3`-5` propagation.

Chimera Formation, U Insertion, and U Deletion Activities May All Involve RNA Ligase

There are two previously published examples of complete cycles of editing in vitro: a specific gRNA-directed deletion of U residues from ES1 of A6 pre-edited mRNA in T. brucei extracts (25) and an insertion of multiple U residues at sites within the editing domain of CYb pre-edited mRNA in L. tarentolae extracts(26) . Might endonuclease and RNA ligase also mediate these reactions, as they do the full round of editing reported here? The potential role of extract endonuclease can be addressed by examining whether the sites of U insertion or deletion also correlate with the sites of endonuclease cleavage. Indeed, the T. brucei extract and gRNAs that direct specific U deletion from ES1 of A6 mRNA also direct cleavage of this RNA at ES1. (^2)(^3)Furthermore, for the L. tarentolae extract that inserts U residues into CYb pre-edited mRNA, the insertions are at sites within the editing domain and upstream of ES1(26) , and although they are not yet precisely mapped they could well be at the sites of preferential cleavage by the extract endonuclease(16) . Thus, endonuclease may be important for all the reported editing-like activities.

Available data also imply that RNA ligase is critical for the T. brucei A6 U deletion (25) and the L. tarentolae CYb U insertion(26) , as it is for the T. brucei CYb chimera formation (23, 24) and the U insertion examined here. All of these activities are ATP-dependent, and all are unable to utilize AMP-CPP (an ATP analog with a nonhydrolyzable alpha-beta linkage) (23, 24, 25, 26) , like RNA ligases in general(38) . Although the report that the U deletion activity does not utilize AMP-PCP (an ATP analog with a nonhydrolyzable beta- linkage) suggested that U deletion may also require beta- bond hydrolysis(25) , AMP-PCP also does not efficiently support L. tarentolae CYb U insertion(26) , T. brucei chimera-forming action (Fig. 7B), T. brucei RNA ligase activity (Fig. 7B), or even T4 RNA ligase activity (Fig. 7B). However, the L. tarentolae U insertion, T. brucei chimera formation, and T. brucei RNA ligase activities all function with AMP-PNP (another ATP analog with a nonhydrolyzable beta- linkage) ( (23) and (26) and Fig. 7), and therefore none of these reactions require beta- bond hydrolysis. (This difference in analog utilization presumably reflects the known structural differences between ATP or AMP-PNP and AMP-PCP(39) .) S. Seiwert has kindly assayed the T. brucei U deletion activity in the presence of AMP-PNP and also found it to be active, (^4)demonstrating that this reaction also does not require beta- bond hydrolysis. Thus, the T. brucei A6 U deletion activity and the L. tarentolae CYb U insertion activity, like the T. brucei CYb chimera formation that can lead to U insertion, all show the same specificity for various ATP analogs as do T. brucei and T4 RNA ligases (Fig. 7A). These activities all require alpha-beta bond hydrolysis but not beta- bond hydrolysis and use AMP-PCP inefficiently. The above data suggest that all these in vitro editing-like activities utilize some form of endonuclease- and RNA ligase-based mechanisms.


Figure 7: Utilization of ATP analogs by editing-like activities. A, mitochondrial extracts were depleted of ATP using hexokinase (23) and tested for their ability to support chimera formation (top panel) or RNA ligation (middle panel) in the absence of exogenously added ATP (lane 1) or in the presence of ATP (lane 2) or the indicated ATP analogs (lanes 3-5). The added ATP, but not the AMP-PNP, is a substrate for the residual hexokinase(23) . T4 RNA ligase (bottom panel) was also tested for ligation under the above conditions. B, the ability of ATP and ATP analogs to support the in vitro T. brucei A6 U deletion(25) , L. tarentolae CYb U insertion(26) , and T. brucei CYb chimera formation and RNA ligase activity (23, 24) are summarized in the white boxes. The data in the shaded boxes were determined in this study, and the box with the asterisk indicates unpublished data kindly determined for us by S. Seiwert^4 and reproduced in our laboratory.^3



Conclusions

We have demonstrated the feasibility of a model for U insertional RNA editing in which an endonuclease and RNA ligase function to transfer U residues from the 3` end of a gRNA to an editing site in the cognate mRNA via a chimeric intermediate. The endonuclease and RNA ligase activities of the T. brucei mitochondrial extract (i) specifically cleave CYb pre-edited mRNA predominantly at ES2 and ES3(14) , (ii) specifically join a cognate gRNA to the 3` cleavage product generating chimeras (23, 24) with junctions predominantly at ES2 and ES3 (Fig. 2), (iii) cleave the chimeras specifically within the oligo(U) region of the gRNA tail (Fig. 3), (iv) rejoin the mRNA halves to generate a partially edited mRNA with U residues transferred to an editing site (Fig. 4), and (v) continue the cycle with another round of specific cleavage (Fig. 5). These reactions are all mimicked by the single strand-specific MBN and T4 RNA ligase, indicating that RNA secondary structure is an important determinant. The observation that many T. brucei in vivo generated partially edited mRNAs (32) show editing patterns that are similar to the in vitro generated partially edited CYb mRNAs with editing beginning at ES2 or ES3 and not at ES1 (compare Fig. 2and Fig. 4with Fig. 6) suggests that these in vivo RNAs may also arise from a similar endonuclease-dependent route. Furthermore, it appears that extract endonuclease and RNA ligase are also the catalytic basis of previously reported in vitro systems that support T. brucei A6 U deletion (25) and L. tarentolae CYb U insertion(26) .


FOOTNOTES

*
This work was funded by National Institutes of Health Grant GM34231 (to B. S.-W.) and American Cancer Society postdoctoral fellowship PF3674 (to K. J. P.). 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.

§
Howard Hughes Medical Institute predoctoral fellow.

To whom correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-7419; Fax: 410-955-0192.

(^1)
The abbreviations used are: U, uridylate; nt, nucleotide; gRNA, guide RNA; CYb, cytochrome b; ES, editing site; PCR, polymerase chain reaction; AMP-CPP, adenosine 5`-(alpha,beta-methylene)triphosphate; AMP-PNP, adenosine 5`-(beta,-imino)triphosphate; AMP-PCP, adenosine 5`-(beta,-methylenetriphosphate); MBN, mung bean nuclease; pCp, [alpha-P]5`,3`-cytidine bisphosphate.

(^2)
S. Seiwert and K. Stuart, personal communication.

(^3)
J. Cruz-Reyes and B. Sollner-Webb, unpublished results.

(^4)
S. Seiwert, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Drs. Ken Stuart and Scott Seiwert for sharing unpublished results, Jorge Cruz-Reyes for comments on the manuscript, and members of the Sollner-Webb laboratory for helpful discussions.


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