Figure 2. Diagram of models for U insertion/deletion RNA editing. The vertical lines indicate base pairs. The arrowheads indicate sites of cleavage. (A) Double transesterification models (27,28). Only U insertions are shown. (B) Modified enzyme cascade model (22). The 3'-oligo(U) tail of the gRNA is shown as a single-stranded overhang, but it is possible that the tail can interact with the purine-rich pre-edited sequence (16,61) and the gRNA may have secondary structure (81). In the U deletion model three unpaired Us (in gray) to be deleted are shown as an example. It is possible that the U addition activity adds Us to the 3'-end of the cleavage fragment at the deletion site, which are then trimmed back, but this scenario is not indicated. In the U addition model 13 Us are shown added to the 5'-fragment, but the evidence indicates that the number of added Us is actually heterogeneous (60). In the `guided' diagrams the exonuclease nucleotide trimming is complete, yielding the correct -3 or +3 guided products. If trimming is incomplete or excessive prior to ligation, gRNA-dependent misedited products are produced, as shown in the `misedited' diagram.

In recent years, however, evidence has been accumulating in support of the cleavage-ligation model for RNA editing (42 ), in which a series of protein-mediated cleavages, uridine insertions and ligations leads to the production of edited mRNA (Fig. 2 B).

Frech et al. (63 ) described an in vitro editing-like activity in which a synthetic pre-edited mRNA substrate could be labeled internally with [[alpha]-³²P]UTP by incubation with a L.tarentolae mitochondrial extract. Although the insertion of Us occurs predominantly within the pre-edited region, addition of exogenous gRNA was not required and, in fact, inhibited the reaction (63 ), possibly by titrating away [[alpha]-³²P]UTP for the more favored gRNA 3'-uridylylation reaction (G.Connell and L.Simpson, unpublished results). Evidence against a transfer of Us from the gRNA 3'-oligo(U) tail to the insertion site in this reaction was provided by showing that internal incorporation of (Sp)-[alpha]-S-UTP proceeded by an inversion of the stereoconfiguration, as predicted by a ligase-mediated reaction, and not by the net retention of the stereoconfiguration predicted by a transfer model (64 ). Connell et al. (65 ) also showed, by primer extension assay, that the in vitro U insertion reaction occurring in sites 1 and 2 of a Cyb pre-edited mRNA substrate was unaffected by mutating the anchor sequence of the mRNA substrate and therefore was also independent of endogenous gRNA. Nevertheless, the fact that U is the predominant nucleotide incorporated and that its incorporation is limited to the pre-edited region suggests a connection between this type of U insertion and bona fide RNA editing activity. This U insertion requires free UTP and ATP. ATP analogs non-hydrolyzable at the [alpha]-[beta] phosphate could not satisfy the ATP requirement.

Connell et al. (65 ) also showed that the in vitro gRNA-independent U insertion activity in L.tarentolae is dependent on the presence of an intramolecular RNA duplex formed by complementary sequences upstream and downstream of the mRNA pre-edited region. They proposed that this duplex may structurally and functionally mimic the gRNA-mRNA anchor duplex in a gRNA-mediated system in vitro, but left the question open as to whether this also occurs in vivo.

An intriguing observation made by Frech et al. (63 ) was that U insertion activity was selectively inhibited by digestion of the extract with micrococcal nuclease. Since the insertion activity is independent of endogenous gRNA, at least for guiding the insertions, the suggestion is that either the endogenous gRNA is required for a non-coding function or that there is another RNA component in the editing complex required for U insertions. This remains an open question and requires confirmation by identification and characterization of the putative RNA component(s).

The first direct evidence for the mechanism of the editing process came from the work of Seiwert et al. (66 ), who initially visualized gRNA-directed U deletions at site 1 of the A6 mRNA by a primer extension assay using unfractionated T.brucei mitochondrial extract. This was the first confirmation of the hypothesis that gRNA mediated U deletion editing by base pairing. Seiwert et al. (61 ) then directly visualized putative intermediates in U deletion editing using a 73 nt end-labeled synthetic pre-edited A6 mRNA substrate incubated in a gradient-fractionated mitochondrial extract in the presence of synthetic cognate gRNA. The number of residues deleted from the mRNA could be manipulated in a predictable manner by affecting the extent of base pairing with the guiding nucleotides in the added gRNA. When 3'-end-labeled substrate RNA was incubated with the 20S glycerol gradient fraction four minor products were observed, which were shown to represent a molecule with the expected U insertions at site 1, a fragment representing the 3'-half of the substrate RNA cleaved adjacent to the Us to be deleted and two types of gRNA-mRNA chimeric molecules with different lengths of connecting U residues. In a time course experiment the 3'-cleavage fragment and edited product appeared a few minutes prior to the chimeric molecules and this was taken as evidence for the chimeric molecules not representing intermediates but by-products of the editing reaction. However, kinetics are difficult to interpret in a system in which all the components are not characterized and identified. The 5'-cleavage products were detected by 5'-end-labeling the substrate RNA (61 ). However, the major initial cleavage fragment visualized after 10 min incubation already had the -4 U deletion, but with further time of incubation molecules with four, three, two and one U were also visible, suggesting that these may not represent intermediates in successive U deletions but rather aberrant products of the reaction.

Mutations in the added gRNA were used to define important sequence elements required for the in vitro reaction. The anchor sequence was required, as was the 3'-oligo(U) tail, which had previously been proposed to hybridize with the purine-rich pre-edited sequence and stabilize the initial gRNA-mRNA interaction (42 ). However, artificially increasing the stability of the interaction between the 3'-tail of the gRNA and the mRNA prevented chimera formation but had no effect on generation of the edited mRNA, suggesting that chimeras represent aberrant by-products of the editing reaction (61 ). However, definitive evidence for this could not be obtained since blockage of the 3'-end of the gRNA by periodation, which would be expected to have no effect, inhibited the production of edited mRNA, as well as chimeras.

Cruz-Reyes et al. (67 ) analyzed the gRNA-directed U deletion mechanism in the T.brucei system in more detail. They showed that the cleavage occurs precisely at the first mismatched nucleotide upstream of the gRNA anchor duplex, as predicted by the enzyme cascade model; this cleavage site is incompatible with a chimera-based model mechanism. Evidence was presented that the 3'-exonuclease activity is not just a reversal of the TUTase activity and appears to be U-specific, although this must be confirmed with purified enzyme and model substrates.

Soon after the report of U deletion editing, gRNA-dependent U insertion editing was demonstrated to occur in vitro in T.brucei and L.tarentolae mitochondrial extracts(60 ,62 ). U insertion requires UTP, ATP, pre-edited mRNA, gRNA, Mg²⁺ and mitochondrial extract (60 ,62 ). Hydrolysis of the [alpha]-[beta] bond of ATP is required for the gRNA-dependent editing reaction (60 ,62 ), as is the case for the in vitro gRNA-independent U insertion activity in L.tarentolae extracts (63 ). In spite of an earlier report to the contrary (66 ), hydrolysis at the [beta]-[gamma] bond of ATP does not seem to be required for U deletion editing in vitro. The [alpha]-[beta] bond hydrolysis requirement is entirely consistent with an RNA ligation reaction, in which the charged intermediate has a covalently linked AMP residue, and is inconsistent with a transesterification model, in which the energy derived from breakage of the phosphodiester bond at the editing site is used to form the new bond with the incoming uridine. It should be cautioned, however, that the use of ATP analogs is only suggestive of a requirement for hydrolysis. A direct measurement of AMP production in the same stoichiometry as product formation would be required as definitive evidence.

The requirement for exogenously added UTP for in vitro gRNA-dependent U insertion activity, in both the T.brucei and the L.tarentolae systems, also appears to rule out models proposing that the non-templated U tails of the gRNAs are the reservoir for the Us to be inserted into the pre-edited mRNA, but does not exclude UTP acting as the nucleophile in RNA catalyzed editing, as proposed by Cech (27 ). However, the requirement for exogenous UTP could also reflect the normal maturation of the gRNA 3'-ends, leaving open the possibility that the inserted uridines are derived from the gRNA U tail. Definitive evidence against this has been provided by data from the L.tarentolae in vitro system, in which chemically blocking the 3'-end of the added gRNA by periodation did not interfere with U insertion into the pre-edited mRNA (60 ). In these experiments controls showed that ~70% of the 3'-block was retained after the in vitro reaction. This indicated that the lack of inhibition of in vitro U insertion was not due to removal of the 3'-block by nuclease activity during the reaction.

In the direct U deletion assay the addition of UTP was reported to inhibit accumulation of the mRNA 5'-cleavage fragment that completely lacks the 3'-terminal Us (67 ). However, fragments with one, two and three 3'-Us were present, suggesting that there was an equilibrium between addition of Us to the 3'-end of the 5'-fragment and exonuclease trimming of Us from this fragment or that UTP competitively inhibits the exonuclease activity. Of course, the in vivo concentrations of mRNA substrate and UTP are unknown and the in vitro situation may not reflect the in vivo conditions.

Kable et al. (62 ) examined U insertion editing at A6 editing site 2 in a substrate in which the Us at site 1 were already deleted. In the absence of UTP they saw accumulation of the 5'-fragment of the mRNA cleaved at the editing site dictated by the gRNA, but no uridines were incorporated into the mRNA. Upon addition of [[alpha]-³²P]UTP the 5'-fragment of the mRNA became labeled at its 3'-end. This observation is inconsistent with a double transesterification, which predicts uridine incorporation at the site of nucleophilic attack (i.e. the 5'-end of the 3'-fragment).

A gRNA-dependent U insertion activity was also demonstrated using L.tarentolae mitochondrial extracts (60 ). Due to the fact that the level of this activity was even lower than that reported using the T.brucei extracts, an indirect primer extension PCR assay had to be used. The substrate RNA contained the 5'-domain of the ND7 pre-edited mRNA, which normally has seven editing sites, the editing of which is mediated by a single gRNA. The mRNA substrate was mutated in the anchor region and a synthetic gRNA with compensatory mutations was used, to avoid interference with the endogenous wild-type edited RNA. U insertions at site 1 were assayed by PCR, amplifying the modified substrate using a 3'-primer specific to the mutated anchor sequence and a 5'-primer specific to the upstream mRNA sequence and then performing a primer extension through site 1 with [[alpha]-³²P]ATP as the only nucleotide provided.

Both gRNA-independent U insertions of 1-13 Us at site 1 and gRNA-dependent U insertions were detected by this assay (60 ). As was the case with the Cyb mRNA substrate (65 ), the extent of the gRNA-independent activity was dependent on 5' and 3' sequences in the mRNA. Addition of cognate gRNA directed the insertion of up to 13 Us in site 1, with the predominant number being determined by the number of guiding nucleotides. Use of a gRNA with no guiding nucleotides produced a decrease in the background ladder without any enhancement of specific bands.

Addition of low concentrations of heparin selectively inhibited the gRNA-independent background ladder but had no effect on either the gRNA-guided predominant band or on the gRNA-dependent background ladder (60 ). This suggests that the mechanism for creation of the background ladder in the presence of added gRNA differs from the mechanism for creation of the apparently similar ladder occurring in the absence of gRNA, possibly in the affinity of protein-RNA interactions. The authors speculated that the heparin-resistant gRNA-dependent non-specific U insertion activity may represent an integral part of the mechanism of editing. They suggested that multiple Us are first added to the 3'-end of the mRNA 5'-cleavage fragment at a U insertion site in a template-independent fashion. It is possible that this also occurs at U deletion sites, but as yet there is no evidence for this. The next event would be the formation of base pairs with the guiding nucleotides in the gRNA, followed by 3' -> 5' exonucleolytic trimming of the 3'-overhang, assuming the 5'-cleavage fragment remains in the editing complex. The trimming is either U-specific (67 ) or terminates at the adjacent upstream base pair. The gRNA essentially functions as a `splint' and facilitates ligase-mediated joining of the two fragments when there is no bulge (62 ). The presence of a gRNA-dependent ladder of 1-13 Us is attributed to ligation occurring prior to complete trimming or after excessive trimming (60 ). One could argue that the gRNA-dependent ladder is an artifact of this in vitro system, insofar as the assay is indirect and the system is inefficient. However, direct evidence for a non-templated 3'-terminal addition of Us to the 5'-cleavage fragment was obtained in the T.brucei A6 mRNA in vitro system; 5'-cleavage fragments terminating in one to four Us were visualized even though the added gRNA only templated two U insertions (62 ). This suggests that the L.tarentolae in vitro data is meaningful.

The idea that multiple Us are added to the 5'-cleavage fragment and then the Us are trimmed back is the major conceptual modification of the original enzyme cascade model, which proposed the addition of a single U per cycle (16 ). It is an important concept since it ties together U deletions and U insertions into one mechanism and, in addition, predicts the production of a certain proportion of misedited sequences as a normal consequence of the editing process. The effective UTP concentration at the editing site appears critical in this model, since U insertions are completely dependent on the presence of at least 30-50 [mu]M UTP (E.Byrne and L.Simpson, unpublished results). In the case of a U deletion site the data from Cruz-Reyes et al. (67 ) suggests that UTP may inhibit exonuclease trimming at a U deletion site. It is possible that the effective concentration of UTP at an editing site is somehow regulated, perhaps by UTP binding proteins (L.Simpson, unpublished results). Another possibility is that the 3'-exonuclease activity in vivo has a specificity for single-stranded 3'-oligo(U) overhangs, whereas the in vivo U insertion activity prefers to add Us to a nick in a duplex RNA.

The requirement for exogenous UTP, the apparent requirement for [alpha]-[beta] bond ATP hydrolysis, the observed patterns of pre-mRNA cleavage and U insertion and the lack of inhibition of the gRNA-mediated U insertion process by chemical blockage of the 3'-end of the gRNA are all in agreement with a protein-mediated process. The chemistry of the process is in question, as the possibility of a protein-mediated transesterification mechanism, as suggested by Cech (27 ), is still open. In fact, the possible involvement of an RNA ligase in editing implies that at least part of the editing chemistry will include a transesterification step, since most RNA ligases studied to date proceed by a transesterification mechanism (68 ).

Regardless of which model proves to be the correct one, the various editing models have been pivotal in providing hypotheses for probing the mechanism of editing in vitro. Ultimately, it is careful biochemical studies in conjunction with genetic analysis that will conclusively answer the remaining questions of the actual chemistry of the editing process.

THE ACTIVITIES

Evidence for a protein-mediated editing mechanism, albeit circumstantial, has also been provided by the detection of various enzymatic activities in mitochondrial extracts from trypanosomatids. Initially, a TUTase (23 ), an endoribonuclease (25 ,26 ) and an RNA ligase activity (23 ) were reported. All of these activities seem to possess some sort of specificity for pre-edited mRNA and/or gRNA and this specificity suggested some involvement of these activities in RNA editing. In fact, the presence of these activities provided the initial stimulus for the development of the enzyme cascade model of editing.

These activities have been fractionated to different extents. Initially, Pollard et al. (69 ) found that the three activities of T.brucei extracts as well as the mRNA and gRNA could be separated into two distinct peaks by glycerol gradient sedimentation. One peak sedimenting at 19S contained TUTase, RNA ligase and gRNAs, while mRNA and also gRNA were found in a second peak in the 35-40S region of the gradient. Since then, co-fractionating putative editing activities have been obtained in several laboratories, both with T.brucei and with L.tarentolae extracts (70 -74 ). Peris et al. (75 ) recently reported the separation of potential editing activities from both species into two peaks; a major peak sedimenting at 10S, which contains TUTase and gRNAs, and a minor peak sedimenting at 20S, which contains the RNA ligase and some TUTase. The TUTase-containing and ligase-containing complexes could be visualized on native gels and were termed the T-IV complex and the ligase complex. In addition, a heterodisperse series of gRNA- and mRNA-containing complexes were found throughout the gradient. The main difference between the T.brucei and L.tarentolae extracts is that the 20S peak of T.brucei contains as much TUTase activity as the 10S peak. Both the gRNA-independent U insertion activity in L.tarentolae (70 ) and the gRNA-dependent U deletion and U insertion activities from T.brucei (67 ,71 ) were found to sediment at ~20S, together with the ligase complex, suggesting that this complex may represent a minimal editing enzymatic unit (76 ).

Although the data from various laboratories seems to differ in the number and/or size of the putative editing complexes, this might simply reflect subtle differences due to variations in the fractionation procedures and/or the species. However, given the high hydrophobicity of many mitochondrial proteins, it is probable that the observed apparent sizes of the editing complexes may be influenced by interaction with proteins that bind editing complexes non-specifically. Thus the actual size of the editing complexes under non-denaturing conditions could easily be overestimated. However, from the glycerol gradient fractionation data it is clear that RNA editing does take place within the realm of a multiprotein complex. It is expected that for complex assembly not only catalytic components of editing will be required, but also a set of structural proteins to provide a scaffold within which the actual catalysis occurs. The ultimate answer to the question of the size and/or nature of the editing complex will be through the establishment of a highly sensitive and quantitative in vitro editing assay, purification of the individual components and reconstitution of the active editing complex from the individual parts.

Studies on the individual components of the editing complexes are beginning to accumulate. An earlier report of a putative editing endoribonuclease failed to show a gRNA dependence of cleavage. The first experimental evidence for gRNA-dependent cleavage came in the in vitro system described by Seiwert et al. (61 ), Cruz-Reyes et al. (67 ) and Piller et al. (77 ) for U deletion editing. Piller et al. (77 ) were able to resolve three endoribonuclease activities from the T.brucei mitochondrial extract, by sensitivity to DTT, cleavage specificity and a requirement for gRNA. One activity cleaves Cyb mRNA within the pre-edited region and the specificity is unaffected by addition of cognate gRNA. A second activity is single-strand specific and cleaves Cyb mRNA in the 3'-end of the editing domain. A third activity has all the features of the predicted gRNA-dependent editing nuclease. It cleaves immediately 5' of a duplex but is also specific for the gRNA-directed editing site; digestion yields a 5'-P and a 3'-OH, consistent with the cleavage fragments being substrates for RNA ligase. This activity also sediments at ~20S. Isolation and characterization of purified nucleases, however, is necessary to confirm these assignments of activities to different enzymes. Alfonzo and Simpson (unpublished results) have recently purified to homogeneity, cloned and expressed a 22 kDa nuclease from L.tarentolae extracts with some specificity towards pre-edited RNAs and which appears to be gRNA independent; this probably corresponds to the nuclease activity previously described by Simpson et al. (25 ).

Two proteins of 50 and 45 kDa (reported as 57 and 50 kDa from T.brucei) have been identified as putative RNA ligase intermediates, since they can be covalently charged with AMP (24 ,75 ). The involvement of a particular RNA ligase in editing will require further purification and characterization. So far, the only evidence that this RNA ligase might be involved in editing is the finding that it co-fractionates in glycerol gradients with other putative editing components.

If indeed editing happens within a ribonucleoprotein complex, the process will require structural components that will not be directly involved in catalysis. It is presumed these structural factors should have specificity and high affinity for either the mRNA and/or gRNA. It is appealing to think that some interactions between editing components will help in assembly of an active complex and also help stabilize the complex once formed. The best candidate so far for a structural editing factor is a gBP21 protein purified from T.brucei by Köller et al. (78 ). gBP21 was identified by UV crosslinking to gRNA. Further analysis showed that gBP21 could bind gRNAs strongly in vitro (K_d = 5-10 nM). Although no biological significance has been proven for the strong interaction between gRNA and gBP21, in view of the low dissociation constant it may play some role in RNA editing. However, the specific role of gBP21 in editing remains an open question.

Another activity which was predicted to explain opening of the duplex formed by the 5'-most gRNA in an editing domain and the fully edited mRNA, to allow interaction with ribosomes, is an RNA helicase. This activity could also be invoked, in addition to breathing of the less thermodynamically stable portion of the duplex (51 ), to explain the melting of downstream gRNA-edited mRNA duplexes to allow formation of the anchor duplex with the adjacent upstream gRNA. A putative RNA helicase of the DEAD box family was isolated from mitochondrial extracts of T.brucei (79 ,80 ). Again, the specific role if any of this helicase in RNA editing remains an open question.

CONCLUSIONS

The overall evidence strongly indicates that U insertion/deletion RNA editing in trypanosomatids involves RNA-protein complexes and involves a series of successive specific gRNA-directed cleavages, 3'-terminal U addition, 3'-exonuclease trimming and re-ligation, almost exactly as predicted by the original enzyme cascade model (16 ). The field has progressed to the stage of biochemical characterization of individual components and reconstitution of editing activities. Major advances have been made in obtaining in vitro systems in which a complete cycle of U deletion and U insertion editing occurs, but the systems are extremely inefficient and do not progress past a single cycle of editing. There is clearly a need to optimize and reconstitute the in vitro systems with recombinant proteins and obtain progressive editing in multiple sites. Antisera against purified putative editing components should be generated and tested for immunodepletion activity and immunoprecipitation of editing complexes. Finally, gene disruption data must be obtained to confirm a role of each component in the editing process.

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*To whom correspondence should be addressed. Tel: +1 310 825 4215; Fax: +1 310 206 8967; Email: simpson@hhmi.ucla.edu
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