This study reports that MAbs specific for the gRNA binding protein gBP21 can immunoprecipitate in vitro RNA editing activity and its component activities of gRNA-specific endoribonuclease, TUTase, and RNA ligase (protein self-adenylylation). In addition, gRNA and preedited, partially edited, and fully edited mRNAs are also immunoprecipitated. This finding suggests that the antibodies immunoprecipitate functional editing complexes by virtue of their association with the gBP21 protein. The MAbs are specific for gBP21 since they reveal a single ~21-kDa protein in Western blot analyses of samples from isolated T. brucei mitochondria (Fig. A) and from Escherichia coli expressing recombinant gBP21 (Fig. C) and since they immunoprecipitate a ~21-kDa protein that specifically cross-links with gRNA (Fig. B). The recovery of six of 81 MAbs specific for gBP21 shows that this protein is present in the 20S fraction from the 30 to 45% (NH4)2SO4 precipitate and suggests that it may be abundant in this fraction and/or highly immunogenic.
The immunoprecipitation of editing activity and its associated activities appears to be specific but does not definitively demonstrate a functional association of gBP21 with the editing complex. The specificity is evident from the results that MAbs specific for other mitochondrial components or non-T. brucei antigens as well as immunomagnetic beads to which additional antibodies are not coupled do not precipitate the activities. The inability to immunoprecipitate the activities after treatment of mitochondrial extract with (and subsequent to inactivation of) micrococcal nuclease suggests a functional association between gBP21 and the editing complex which involves RNA. However, although the addition of EGTA does not inhibit the editing activity (and other activities), the editing activity is not detectable in either the immunoprecipitate or supernatant after micrococcal nuclease treatment and inactivation. In contrast, TUTase and the self-adenylylatable proteins are seen in the supernatant after treatment. The loss of in vitro RNA editing activity may have been the consequence of the manipulations, dilution, or dissociation. Thus, a more direct assessment is needed to ascertain the functional association of gBP21 with the complex. Nevertheless, the immunoprecipitation of RNA editing activities that insert and delete U’s by the anti-gBP21 MAbs indicates that gBP21 is associated with complexes that perform both types of RNA editing, which implies that they may be similar.
The coprecipitation of in vitro RNA editing activities that insert and delete U’s, the component activities of endoribonuclease, TUTase, and RNA ligase, as well as gRNA and mRNA, suggests that all of these activities and molecules might be assembled within a multicomponent macromolecular complex. This is consistent with the current view that editing occurs by a series of enzyme catalyzed steps (4
). According to this model, editing is initiated by an endoribonucleolytic cleavage at a site that is directed by the gRNA (25
). This gRNA-dependent endoribonuclease sediments at ~20S, as do RNA editing complexes (8
), while gRNA-independent endoribonuclease activity remains at the top of glycerol gradients. The relationship between the two endoribonuclease activities is unclear; they may be distinct enzymes or simply differ in their association with the complex (17
). Nevertheless, gRNA-dependent endoribonuclease is immunoprecipitated by anti-gBP21 MAbs (Fig. ). The MAbs also immunoprecipitate activities that add or remove U’s at the 3′ end of the 5′ cleavage product of pre-mRNA, since RNA edited by U insertion or deletion is produced by the immunoprecipitate. TUTase activity, detected as the addition of U’s to yeast tRNA, is also immunoprecipitated by the anti-gBP21 MAbs (Fig. A) and may be responsible for the U insertion. It is unclear if this enzyme is also responsible for the posttranscriptional addition of U’s to the 3′ end of gRNA or if there are multiple U-addition enzymes. Removal of U’s during RNA editing may conceivably occur by the reverse activity of the same enzyme that adds U’s. However, one study (21
) suggests that, at least for TUTase, this may not be the case since UMP and not UTP is released upon U removal, although these studies did not examine authentic sites that are edited. The anti-gBP21 MAbs also immunoprecipitate the 57- and (to a lesser extent) 50-kDa self-adenylylatable proteins (Fig. B). These proteins are probably RNA ligases since they accumulate when incubated with nonligatable substrates but deadenylylate and release AMP upon incubation with ligatable substrates (22
). Thus, they may be responsible for the RNA ligase activity that catalyzes the final step in a single round of RNA editing. The requirement for ATP α-β bond hydrolysis for in vitro RNA editing may reflect the ATP requirement of RNA ligase, although other steps in editing may also require ATP hydrolysis (22
The in vitro RNA editing activity as well as the associated gRNA-dependent endoribonuclease and RNA editing-associated U addition and deletion activities sediment with a broad profile centered at ~20S in glycerol gradients (8
). While TUTase and the 50- and 57-kDa self-adenylylatable proteins also sediment with a peak in this region of the gradient, a second peak of TUTase and the self-adenylylatable proteins, which is more prominent in some strains and studies, is centered at ~40S (8
). In addition, gRNA and preedited mRNA sediment near 30S, while partially and fully edited mRNAs sediment with a broad profile nearer 40S (8
). Perhaps the greater apparent sedimentation coefficient reflects the increased mRNA size due to RNA editing and/or gRNA or protein accumulation during editing. One possibility, which we favor, is that complexes sedimenting at ~20S can edit exogenously added RNAs whereas those sedimenting down to ~40S are associated with endogenous RNA and thus do not edit exogenous RNA (30
). An alternative suggestion is that the ~20S and ~40S peaks represent different complexes (18
). Western blot analysis shows that most gBP21 protein is well above the 20S fraction in glycerol gradients, indicating that the majority of it is not (or not stably) associated with the editing complexes (data not shown). However, G1 and G2 gRNA-specific complexes that contain gBP21 which form in vitro (19
) can be quantitatively supershifted by the anti-gBP21 MAbs, while the G3 complexes that contain a 90-kDa protein but not gBP21 are not (Fig. ). Thus, gBP21 may be in substantial excess relative to editing complexes and/or it does not remain associated with the editing complex during the entire editing process. One possibility is that gBP21 plays a role in the association of the gRNA with the editing complex. Perhaps G1 and G2 represent early steps in the association of gRNA with a complex that associates the gRNA with the editing complex, and the G3 (and perhaps G4) complex represents a later step in the assembly of the complex. The localization of gBP21 throughout the mitochondrion with a greater concentration in the kinetoplast might imply a functional partitioning (Fig. ). Perhaps the kinetoplast, which is the site of preedited RNA and gRNA transcription, is also a site of assembly of the editing complexes.
Studies of gRNA structure (12
) indicate that gRNAs assume a double stem-loop structure with the anchor sequence contained within the 5′ stem and the 3′ U tail extending from and not included within the 3′ stem. In vitro footprint analysis shows that gBP21 protects a substantial part of the 3′ stem-loop structure (12
). This might leave the 5′ region free to form a duplex with the pre-mRNA. UV cross-linking to gRNA with the 90-kDa protein, which is U tail dependent (19
), sediments at 10S to 20S on glycerol gradients after incubation with mitochondrial extract, as does UV-cross-linked gBP21 (8
). However, the UV-cross-linked 90-kDa protein is primarily seen in the ~40S fraction, while most UV-cross-linked 21-kDa protein is in a 10S fraction if the extract is fractionated before UV cross-linking. Perhaps there is a greater affinity or stability of the association of the gRNA with the editing complex than for gBP21, and most 90-kDa protein remains associated with a complex whereas most 21-kDa protein is free in solution. Elucidation of the roles of these proteins and the other proteins associated with the editing complex will await other studies, including gene knockout experiments.
The composition of the editing complex(es) is uncertain. In addition to gRNA and pre-mRNA, the complex(es) likely contains multiple proteins, and the possibility that other RNAs are present has not yet been excluded. UV cross-linking has identified numerous potential protein constituents of the editing complex, including gBP21 and the 90-kDa U-tail binding protein which become conspicuous in competition experiments (19
) and 55- and 16-kDa proteins revealed upon anti-gBP21 MAb enrichment (Fig. ). Complexes enriched by immunoaffinity purification consistently contain proteins with approximate apparent molecular masses of 18, 24, 25, 28, 30, 32, 45, 47, 50, 52, 64, 65, and 69 kDa (Fig. ). These may include the proteins of 16, 21, 55, and 90 kDa, given the inaccuracy of measurements, especially in comparisons of silver-stained proteins with those radiolabeled by RNA cross-linking and with the variation in silver staining intensities seen between proteins. Complexes with RNA editing and associated activities prepared by biochemical techniques have fewer prominent proteins. Those prepared by the Sollner-Webb group (21
) have major proteins with apparent molecular masses of 21, 45, 50, 55, 58, 66, 90, and 95 kDa. These protein profiles are quite similar although not identical. In any event, a demonstration of a specific association between one or more of the proteins is needed before it can be concluded that these proteins are components of the editing complex.