Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex

Abstract

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

FIG. 1

Fractionation of RNA editing complex from T. brucei mitochondria. In vitro deletion RNA editing was used as the functional assay to monitor purification of the complex. (A) Cleared mitochondrial lysate prepared with 0.5% Triton X-100 was fractionated on an SP Sepharose column. (B and C) Fractions containing editing activity, as indicated by the dark lines below each panel, were sequentially fractionated on Q Sepharose (B) and Superose 6 (C) columns. (D) The complex was further purified by sedimentation on a 10-to-30% glycerol gradient, with fraction 1 being the top of the gradient. Diamonds, deletion editing; dotted line, absorbance at 280 nm; dashed line, KCl gradient profile (the KCl concentration [molar units] is the value on the righthand y axis divided by 10).

FIG. 2

SDS-PAGE profile of fractions from complex purification. A sample from each step of purification was separated by SDS-PAGE and stained with silver nitrate. Results for protein size standards (M), cleared mitochondrial lysate (Cr), and pooled editing activity-positive fractions from SP Sepharose (SP), Q Sepharose (Q), and Superose 6 (S6) columns are presented. Glycerol gradient fractions 1 to 10 (fraction 1 is at the top) are shown (fractions 11 to 23 are not shown since essentially no protein was detected in these fractions). The numbers on left indicate the sizes of molecular mass markers, in kilodaltons. The most purified editing activity-positive fraction from the glycerol gradient (7) shows 20 major polypeptide bands.

FIG. 3

Immunoanalysis of the editing complex using a MAb. (A) MAb P3C1-G2 raised against the purified complex reacts with an ∼49-kDa protein in Western analysis of a partially purified complex. (B) MAb P3C1-G2 specifically immunoprecipitates editing activity from the 20S mitochondrial fraction (see Materials and Methods for details) (lanes 3 and 4). Edited RNA, chimeras, and 3′ cleavage products and the input RNA from which they are derived are indicated. MAb 58 (1), the negative control, did not immunoprecipitate these activities (lane 2). Editing activity immunoprecipitated with 200 and 400 mM KCl (lanes 3 and 4, respectively). The positive deletion editing control using the mitochondrial 20S fraction is also shown (lane 1). (C) MAb P3C1-G2 immunoprecipitates both 50- and 44-kDa adenylylatable proteins. MAb 58, which was used as a negative control, essentially did not immunoprecipitate the adenylylation activity. The control (+ve) for these proteins using the mitochondrial 20S fraction is shown. (D) MAb P3C1-G2 also immunoprecipitated both the 50- and 44-kDa proteins from the mitochondrial 20S fraction in a buffer containing 400 mM KCl following their adenylylation. (E) Western analysis of glycerol gradient-fractionated cleared mitochondrial lysate using MAb P3C1-G2.

FIG. 4

Identification of TbMP52. (A) Sample tandem mass spectrum derived by CID of a peptide precursor ion, m/z 1135.0 (bottom), and the peptide sequence predicted by SEQUEST (top) showing b- and y-type ions (above and below the sequence, respectively). (B) Amino acid sequence of the complete ORF identified by 11 tryptic peptide matches with CID spectra. The identified peptides are underlined (at two different positions, two and three peptides were contiguous). The dashed underline indicates the probable N-terminal peptide in the mature protein that is nontryptic, and the double underline indicates the peptide with the highest correlation score (Table 1). The predicted mitochondrial targeting signal is italicized.

FIG. 5

Immunofluorescence with MAb P3C1-G2, which is specific for TbMP52. Procyclic T. brucei cells were stained with MAb (A) and DAPI (B), showing the nucleus and smaller kinetoplast.

FIG. 6

(A) Alignment of predicted amino acid sequences of TbMP52 and related proteins TbMP48 and L5701.8. Potential N-terminal amino acids are in bold. The TbMP48 gene sequence is from contig TRYP1.0.7383 of chromosome I (Sanger Center T. brucei database) and the L5701.8 ORF is from L. major chromosome 1 (20). The alignment indicates amino acids that are conserved (∗), semiconserved (:), and partially conserved (.) among all these proteins. (B) Predicted amino acid sequence of TbMP48 showing the 12 tryptic peptides (two of which were contiguous) that were identified by mass spectrometric analysis (underlined). The first N-terminal peptide (dashed underline) is nontryptic, and the 17 amino acids at the N terminus (italicized) are a predicted mitochondrial targeting signal. (C) CID spectrum of the likely N-terminal peptide of TbMP48.

FIG. 7

Expression of rTbMP48 and rTbMP52 with an N-terminal six-His tag. (A) Coomassie blue-stained gel of total E. coli lysates separated on SDS-PAGE showing protein size standards (M; sizes [in kilodaltons] are on the left), uninduced cells (U), and cells 3 h after induction with 1 mM IPTG (I). (B) Western analysis showing the reaction of MAb P3C1-G2 with rTbMP52 (lane 2). E. coli cells expressing rTbMP48 (lane 1) were used as a negative control.