Association of guide RNA binding protein gBP21 with active RNA editing complexes in Trypanosoma brucei

Abstract

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

FIG. 1

Antigen for MAb production. The in vitro RNA editing activity was enriched by subcellular fractionation of mitochondria followed by (NH₄)₂SO₄ precipitation and glycerol gradient fractionation to prepare immunogen used for the production of MAbs (see Materials and Methods). (A) In vitro editing assay (U deletion) of fractions from sequential steps of (NH₄)₂SO₄ precipitation of crude mitochondrial lysate, showing editing activity in the 45% pellet. The product formed by the deletion of three U’s from editing site 1 of the input RNA (A6short/Tag.1) is indicated. (B) In vitro RNA editing assay (U deletion) of the 20S fraction from glycerol gradient sedimentation of the 45% (NH₄)₂SO₄ precipitate showing the editing activity.

FIG. 2

Identification of target protein. (A) Western blot analysis of crude mitochondrial lysate separated by SDS-PAGE on a 12% (wt/vol) gel. MAb 56 (and five other MAbs not shown) recognized an ∼21-kDa protein. (B) Immunoprecipitation of gRNA UV-cross-linked proteins. Proteins that cross-link with uniformly labeled gRNA in crude mitochondrial lysate and immunoprecipitate with the MAbs specific to gBP21 after treatment with RNase A were separated by SDS-PAGE on a 12% (wt/vol) gel and visualized by autoradiography as described in Materials and Methods. The four MAbs shown (and two others not shown) recognize an ∼21-kDa UV-cross-linking protein. (C) Western blot analysis showing that the MAbs (MAb 56 shown) recognize recombinant gBP21. Time points refer to sampling intervals after induction of culture (see Materials and Methods). Sizes are indicated in kilodaltons at the left.

FIG. 3

Immunofluorescence with MAbs specific to gBP21. Procyclic and bloodstream forms of T. brucei were fixed and stained with MAbs against gBP21 and with DAPI (see Materials and Methods). (A) DAPI staining showing the nucleus and smaller kinetoplast of procyclic T. brucei. (B) Procyclic T. brucei after incubation with MAb 56 and development with a goat anti-mouse antibody conjugated with fluorescein isothiocyanate. The kinetoplast is evident as an intensely staining spot in the network of mitochondrial tubules. (C) Bloodstream T. brucei examined as in panel B, showing the single tubular mitochondrion.

FIG. 4

Supershift of gRNA-specific RNP complexes G1 and G2. Specific complexes that form with uniformly labeled gRNA were separated on a 4% (wt/vol) nondenaturing polyacrylamide gel and then autoradiographed. The lane marked as containing no MAb shows gRNA-specific RNP complexes that form in the absence of the MAbs. Lanes in which MAbs specific for gBP21 were added to the reaction mixture (see Materials and Methods) show a supershift of RNP complexes G1 and G2.

FIG. 5

Immunoprecipitation of in vitro editing activity. Material that immunoprecipitates with MAbs against gBP21 was tested for its ability to support in vitro RNA editing that deletes (A) or inserts (B) U’s. The right gel in panel B shows a longer separation of the RNA molecules that increases the resolution of the edited product from the input RNA. Input RNA, 3′ cleavage product, chimeras, and edited product are diagramed to the right of the corresponding RNA molecule. Editing products were identified by their comigration with known editing products. Editing activity is immunoprecipitated with magnetic beads coupled with MAbs specific for gBP21 but not with immunomagnetic beads alone (No MAb) or with immunomagnetic beads coupled to a control antibody (ODB2).

FIG. 6

Immunoprecipitation of TUTase and [α-³²P]ATP self-adenylylatable proteins (RNA ligase). (A) TUTase activity, as monitored by the addition of [α-³²P]UTP to yeast tRNA (counts per minute are shown), immunoprecipitates with the anti-gBP21 MAbs but not with immunomagnetic beads alone (No MAb) or immunomagnetic beads coupled with control MAb ODB2. (B) Anti-gBP21 MAbs immunoprecipitate the [α-³²P]ATP self-adenylylatable proteins (RNA ligase) while immunomagnetic beads and the control antibody (ODB2) do not.

FIG. 7

Immunoprecipitation of gRNA and mRNA. Northern blots of immunoprecipitated RNA were probed with oligonucleotide probes for gA6[14]gRNA (A), MURF4.1 (which is complementary to 3′ A6 mRNA sequence which does not get edited; this probe reveals preedited as well as partially edited A6 mRNA) (B), and MURF4.14 (which is complementary to 5′ edited A6 sequence; this probe reveals fully and partially edited A6 mRNA) (C).

FIG. 8

SDS-PAGE analysis of immunoprecipitated proteins. Proteins present in the material immunoprecipitated from crude mitochondrial extract with gBP21-specific MAb 56 were separated by SDS-PAGE on a 10% (wt/vol) gel and silver stained. Thirteen prominent bands are seen; sizes of standards are indicated in kilodaltons at the left.

FIG. 9

Proteins in the immunoprecipitate that UV cross-link with gRNA. Uniformly labeled gRNA was added to crude mitochondrial extract and to material immunoprecipitated with MAb 46, UV cross-linked, treated with RNase A, and separated on an SDS–12% polyacrylamide gel, followed by visualization of the proteins by autoradiography as described in Materials and Methods. The cross-linked 21-kDa protein as well as proteins of ∼55, ∼16, and ∼90 kDa are evident. Sizes are indicated in kilodaltons at the left.