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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Sep 8;103(38):13944–13949. doi: 10.1073/pnas.0604476103

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

Xuedong Kang *, Guanghan Gao *, Kestrel Rogers *, Arnold M Falick †,‡, Sharleen Zhou †,‡, Larry Simpson *,§
PMCID: PMC1599893  PMID: 16963561

Abstract

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Keywords: Leishmania, nuclease, RNase III, L-complex

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves the participation of at least three RNA-linked multiprotein complexes, the ≈19-polypeptide ligase-containing core editing complex (L-complex), the mitochondrial RNA-binding protein (MRP) complex, and the RNA editing 3′ terminal uridylyl transferase (TUTase) (RET)1 complex(es) (1–3). L-complex proteins that have been expressed in active form and characterized include the RNA editing ligases 1 and 2 (REL1 and REL2) (4–7), the RNA editing 3′–5′ U-specific exonucleases (REX1 and REX2) (8–10), and the RET2′3′ TUTase (11–13). Characterization of the protein components of the L-complex led to several nuclease candidates: LC6A, or MP61, MP90, and MP67 contain RNase III motifs, and LC8, or MP44, has a highly diverged RNase III motif (11, 14–16). Although TbMP90 and TbMP61 were stated in recent reviews (1, 16) to also contain double-strand RNA-binding motifs, such motifs are not readily identifiable (L.S., unpublished data). In addition, the MP42 L-complex protein, which has only zinc finger (ZnF_C2H2) and single-strand RNA-binding (SSB) motifs, nevertheless exhibited both exonuclease and endonuclease activities, but the activities identified did not show the required specificity, and the role of this protein remains an open question (17).

Trotter et al. (18) and Carnes et al. (19) recently provided in vivo evidence for a specific role in cleavage at mRNA U-deletion and U-insertion editing sites for, respectively, the essential TbMP90 and TbMP61 RNase III motif-containing proteins. It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18, 19).

In this study, we provide both indirect and direct evidence for a role of the MP90 L-complex protein in the initial cleavage at preedited mRNA U-deletion editing sites, and we show that a full-round cycle of in vitro U-deletion editing can be reconstituted with just three recombinant proteins: REX1, REL1, and MP90. MP90 has been functionally renamed as RNA editing nuclease 1 (REN1). Functional names for the editing-related proteins will soon replace the current confusing nomenclatures. Table 1 provides a current listing of the various laboratory-specific and functional names to assist the reader.

Table 1.

Nomenclature of L-complex proteins

LC MP Band KREP Functional
LC1 MP81 II KREPA1
LC2 MP100 I KREPC1 REX1
LC3 MP99 KREPC2 REX2
LC4 MP63 III KREPA2
LC5 MP46 KREPB4
LC6a MP61 KREPB3 REN2
LC6b MP57 KRET2 RET2
LC7a MP52 IV KREL1 REL1
LC7b MP42 VI KREPA3
LC7c MP49 KREPB6
LC8 MP44 KREPB5
LC9 MP48 V KREL2 REL2
LC10 MP24 KREPA4
LC11 MP18 VII KREPA6
MP90 MP90 KREPB1 REN1
MP67 MP67 KREPB2
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References: LC (11), MP (40), Band (41), and KREP (1).

Results

Down-Regulation of Expression of TbMP90 Is Lethal and Specifically Affects U-Deletion-Site Cleavage Activity.

We have confirmed the results of Trotter et al. (18) that expression of TbMP90 is required for viability of procyclic Trypanosoma brucei cells. As shown in Fig. 1A, RNAi down-regulation of MP90 mRNA produced a slow-growth phenotype. However, in contrast to Trotter et al., a decrease in the S value of the L-complex in a glycerol gradient was observed after 6 days of RNAi (Fig. 1C).

Fig. 1.

Fig. 1.

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Conditional RNAi down-regulation of TbMP90 expression in T. brucei procyclic cells. (A Upper) Growth curve of noninduced and tet-induced cells. The cultures were diluted daily to maintain log-phase growth. Tetracycline (1 μg/ml) was added to initiate RNAi. (A Lower) RT-PCR of TbMP90 mRNA from cells at days 3, 6, and 9. D, day. (B) Full-round editing and cleavage activities of the 20S gradient fraction from cells down-regulated for TbMP90 expression for 6 days and 9 days by using 5′-end-labeled U-deletion substrate. Numbers below the lanes show percent of editing. (C) Glycerol gradients of mitochondrial lysates from the RNAi cells at days 0, 6, and 9. (Upper) Fractions were autoadenylated to show the location of the REL1 and REL2 ligases. The L-complex-containing fractions are circled. (Lower) Western blot analysis of the same fractions using antibodies against MP81, MP63, and MP42. (D) Diagrams of the U-deletion substrates (20). The nt 22 and nt 25 cleavage sites are indicated in S1 and the nt 11 cleavage site in S2. The 32P-label is indicated with an asterisk.

The down-regulation of TbMP90 expression also affected full-round −3U-deletion editing activity of the mitochondrial 20S gradient fraction (Fig. 1B Upper), apparently by inhibiting the endonuclease cleavage activity (Fig. 1B Lower). The model U-deletion mRNA/guide RNA (gRNA) substrate (S1) (20) used for this assay is shown in Fig. 1D. The observed cleavage site of the 5′-end-labeled mRNA at nt 22 was due to a cleavage at nt 25, followed by a presumed rapid 3′–5′ exonuclease digestion.

Inhibition of RNA ligase activity by the addition of inorganic pyrophosphate (PPi) (21) increased the amount of the cleavage product while eliminating the production of fully edited product (Fig. 2A). The cleavage was stimulated by nucleoside adenosine α/β-methylene diphosphate (AMP-CP) (22) (Fig. 2B Upper) and required the annealed cognate gRNA (Fig. 2B Lower).

Fig. 2.

Fig. 2.

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Requirement for gRNA and stimulatory effect of AMP-CP on cleavage at the U-deletion site during full-round editing in vitro. (A) The 20S gradient fraction from noninduced cells was assayed for full-round U-deletion editing and cleavage activity by using 5′-end-labeled U-deletion substrate. The arrows indicate the −3U edited product and the cleavage fragment. The size marker lane contains partially hydrolyzed 5′-labeled RNA. Addition of PPi to the reaction to inhibit ligation (right lane) prevented formation of the edited product and slightly increased the yield of cleavage fragments. (B) The 20S gradient fraction from noninduced cells was assayed for cleavage activity in the presence of PPi to inhibit ligase activity. (Upper) AMP-CP stimulates cleavage activity. The percent cleavage is shown below each lane. (Lower) Cleavage requires the presence of annealed cognate gRNA.

Deletion of the LmMP90 RNase III Domain Eliminates the Cleavage Activity and Affects Stability of the L-Complex.

LmMP90-tandem affinity purification (TAP) was episomally expressed in Leishmania tarentolae cells. The protein was targeted to the mitochondrion (Fig. 3A Left) and incorporated into the L-complex, as shown by cosedimentation with L-complex markers and detection by Western blot analysis (Fig. 3B) (11). A mutant Leishmania major MP90 protein with the RNase III domain deleted (LmMP90-D-TAP) was also expressed in L. tarentolae. There was a definite effect on the stability of the L-complex as shown by the gradient in Fig. 3B Left. Western blot analysis of the protein composition of these complexes (fraction 11 from WT gradient and fraction 9 from −D gradient) revealed a decrease in the abundance of LC1, LC4, and LC7B, again suggesting a partial breakdown. PAP analysis of these fractions revealed the presence of the tagged proteins (Fig. 3B Right). Some degradation, especially of the −D protein, can be seen.

Fig. 3.

Fig. 3.

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Expression of TAP-tagged LmMP90 in L. tarentolae. (A) Western blot analysis using PAP reagent of expressed LmMP90-TAP in cytosol (Cyto) and mitochondrial (Mito) fractions. (B Left) Sedimentation of mitochondrial lysates from transfected L. tarentolae. Fractions were assayed for the L-complex by autoadenylation of REL1 and REL2. The ≈20S L-complex fractions are circled. (B Center) Western blot analysis of fractions 11 and 9 from both gradients, using antibodies against LC1, LC4, and LC7B. (B Right) Western blot analysis of the same fractions using the PAP reagent. Arrows indicate intact LmMP90 and LmMP90-D bands. (C Left) Comparison of cleavage activities of peak fractions from gradients in B. Control lanes have input RNA. PPi and AMP-CP were present. The same amount of protein was used for each reaction. (C Center) Stimulation of cleavage activity of purified LmMP90-TAP pull-down by AMP-CP. (C Right) Comparison of cleavage activities of pull-downs using LmMP90-TAP and LmMP90-D-TAP cells. The numbers below the lanes show relative ratios of cleavage.

The AMP-CP-stimulated U-deletion-site cleavage activity of fraction 9 from the LmMP90-D-TAP gradient was also significantly decreased (Fig. 3C Left). LmMP90-TAP pull-down also showed an AMP-CP-stimulated cleavage activity with the U-deletion substrate (Fig. 3C Center). The LmMP90-D-TAP pull-down showed a decrease in this activity (Fig. 3C Right), in agreement with the above results using gradient fractions.

Recombinant MP90 Has Cleavage Activity That Is Specific for a Model U-Deletion Site, and Mutations of the RNase III Domain Affect the Activity.

TbMP90-TAP and TbMP90-D-TAP proteins were expressed by using baculovirus-infected insect cells and were affinity-purified. As shown in Fig. 4A, the final protein fractions showed three major bands. Mass spectrometry and Western blot analysis showed that the upper bands in lanes 1 and 2 are TbMP90-calmodulin-binding protein (CBP) and TbMP90-D-CBP, respectively. The two lower bands in both lanes are the common contaminants, heat-shock protein (hsp70) and β-tubulin.

Fig. 4.

Fig. 4.

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U-deletion cleavage activity of rTbMP90-CBP. (A) Stained gels and Western blots of the purified rTbMP90-CBP (wt) and rTbMP90-D-CBP (D) proteins. (B) Cleavage of −3U-deletion (Fig. 1D, S2) and +3U-insertion (Fig. 4E) substrates using rTbMP90-CBP. Marker is 3′-end-labeled partially hydrolyzed RNA. The slight lack of correspondence between marker bands and fragment bands is a known artifact (39). rTb20S, peak L-complex gradient fraction. U-deletion cleavage reactions were performed in the presence of PPi and AMP-CP, and U-insertion cleavage reactions were performed in the presence of PPi and UTP. (C) Comparison of cleavage activities of rTbMP90-CBP and rTbMP90-D-CBP. Percent cleavage is indicated below each lane. Effects of AMP-CP and proteinase K treatment are also shown. (D) Titration of Mg2+ requirement for cleavage of U-deletion substrate. (E) Sequence of 3′-end-labeled +3U-insertion substrate used in B Right.

Incubation of the recombinant TbMP90-CBP protein with the 3′-end-labeled model U-deletion RNA substrate shown in Fig. 1D (S2) (20) and the 3′-end-labeled model U-insertion substrate (23) shown in Fig. 4E yielded a cleavage of the U-deletion substrate that coincided with that produced by the L-complex gradient fraction but no detectable cleavage of the U-insertion substrate (Fig. 4B). The U-deletion-site cleavage activity of the rTbMP90-CBP protein was destroyed by digestion with proteinase K (Fig. 4C). The rTbMP90-D-CBP protein showed little detectable cleavage activity (Fig. 4C). All reactions were performed in the linear range of activity of the rTbMP90 enzyme (data not shown) under optimal Mg2+ concentration (Fig. 4D). It is interesting that the recombinant enzyme showed no detectable stimulation of cleavage activity by AMP-CP (Fig. 4C), possibly suggesting that the allosteric regulation of TbMP90 enzyme activity in isolated L-complex by adenine nucleotides (22) may involve other interacting proteins.

Because the specific activity of the recombinant TbMP90-CBP protein expressed in insect cells was fairly low (Fig. 4B Left shows 3% of input cleaved by incubation for 3 h with 30 nM enzyme), we decided to attempt to improve the activity by expression of TAP-tagged LmMP90 in a homologous expression system, L. tarentolae, with the putative N-terminal mitochondrial target signal deleted to limit expression to the cytosol. This recombinant protein proved to be more difficult to affinity-isolate than the insect cell-expressed TbMP90-TAP protein. After two consecutive IgG Sepharose and calmodulin agarose columns, the final preparations still had two major contaminant bands (Fig. 5A). These contaminants were shown by MS to be the 50-kDa β-tubulin protein and the 70-kDa tobacco-etch virus (TEV)-MBP protease used to release the tagged protein from the IgG column. The rLmMP90-CBP protein band, present as a minor stained band, was identified by Western blot analysis (Fig. 5A Right). A mutant LmMP90-CBP with a highly conserved residue in the RNase III motif involved in protein dimerization (24) (Fig. 5D) E378, changed to K378 (LmMP90-E/K), was also expressed in L. tarentolae and isolated by affinity chromatography (Fig. 5A).

Fig. 5.

Fig. 5.

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U-deletion cleavage activity of rLmMP90-CBP and mutant LmMP90 (E/K)-CBP obtained from expression in L. tarentolae. (A) Isolation of rLmMP90-CBP. (Left) Sypro-stained gels of the final preparations of LmMP90-CBP and LmMP90-CBP (E/K). (Right) Western blot analysis with anti-CBP antibody. The location of the rLmMP90-CBP band is indicated. (B) U-deletion cleavage activity of rLmMP90-CBP and rLmMP90-CBP (E/K). Equal amounts of protein (30 nM) were used. The percent cleavage is shown below each lane. (C) Stimulation of cleavage activity by 3 mM AMP-CP. The relative ratio of cleavage is shown below each lane. (D) Alignment of RNase III motifs from E. coli, S. cerevisiae, LmMP90, and TbMP90. Absolutely conserved residues are in dark gray. E378 in LmMP90 is indicated with an arrow.

The recombinant LmMP90-CBP protein preparation showed higher specific activity of U-deletion-site cleavage than that obtained from insect-cell expression of TbMP90-TAP (Fig. 5B). The E/K mutant protein showed a significantly decreased cleavage activity (Fig. 5B). The recombinant LmMP90-CBP showed a stimulation of cleavage activity by AMP-CP (Fig. 5C), unlike the situation with the less active TbMP90-CBP isolated from insect cells. This may be a species difference or be due to the presence of other L-complex components contaminating the preparation. This problem requires further work to resolve.

Reconstitution of gRNA-Mediated in Vitro Full-Round U-Deletion Editing with Recombinant LmMP90, LmREX1, and LtREL1.

We showed that in vitro precleaved U-deletion editing could be reconstituted previously with two recombinant enzymes, LmREX1 and LtREL1 (9). The addition of recombinant LmMP90-CBP allowed reconstitution of full-round gRNA-mediated −3U editing by the putative scheme diagrammed in Fig. 6A. The −3U-edited product produced by incubation of 3′-end-labeled U-deletion preedited substrate with the three recombinant proteins is shown in lane 1 of Fig. 6B. The efficiency is low (4% conversion) but is equivalent to that previously reported with gradient- or column-purified fractions (25, 26). A small band of edited product can be seen in the rLm90 alone lane (≈1% conversion), which may be a result of expression in a homologous system and copurification of a small amount of editing proteins.

Fig. 6.

Fig. 6.

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Reconstitution of full-round editing with three recombinant proteins. The 3′-end-labeled U-deletion substrate was incubated with a mixture of recombinant LtREL1, LmREX1, and LmMP90 proteins. (A) Diagram of proposed reaction steps involving cleavage by LmMP90, deletion of 3′ Us by REX1, and ligation by REL1. (B) Full-round −3U deletion editing. The positions of the edited products containing no Us (−3), one U (−2), and two Us (−1) are indicated. The percent edited product is indicated below each lane.

An effect of the adjacent upstream nucleotides in the gRNA and/or mRNA on U-deletion-site cleavage activity has been shown for L-complex fractions isolated from mitochondrial lysate (20, 27, 28). To test the effect of changing the adjacent upstream nucleotide in the mRNA from a U to a C on the activity of the recombinant LmMP90 nuclease, the U-deletion substrates shown in Fig. 7A were assayed for full-round editing activity by using a mixture of rLmMP90, rLmREX1, and rLtREL1 and, for cleavage activity, by using rLmMP90 alone. There was a significant inhibition of full-round U-deletion editing by substitution of a C for a U at the cleavage site for both the peak L-complex gradient fraction (F10) and the mixture of recombinant proteins (Fig. 7B Upper). Cleavage activity was assayed in the absence of ATP to inhibit ligase activity and in the presence of AMP-CP. The inhibitory effect on the cleavage was more pronounced for the L-complex gradient fraction than for the rLmMP90-CBP (Fig. 7B Lower).

Fig. 7.

Fig. 7.

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Effect of adjacent upstream nucleotide on full-round editing and cleavage at U-deletion site. (A) Diagrams of RNA substrates used. The standard substrate has a U upstream of the cleavage site, and the other has a C. (B Upper) full-round U-deletion editing. The control lanes have the input RNA. F10 indicates the peak L-complex fraction from a gradient of mitochondrial lysate from WT cells. U indicates the upstream U RNA and C the upstream C RNA. The reactions in the final two lanes contained a mixture of rLtREL1, rLmREX1, and rLmMP90. The edited product is indicated. (B Lower) Cleavage activity. The reactions were performed in the presence of AMP-CP and the absence of ATP to inhibit ligase activity. The cleavage product is indicated.

Discussion

We have firmly established that the L-complex RNase III-containing MP90 protein is an endoribonuclease specific for the initial cleavage of the mRNA at U-deletion editing sites in mRNA/gRNA hybrids. In line with suggestions for nomenclature of editing proteins (2, 18), the MP90 endoribonuclease is designated REN1. The L-complex 20S gradient-fraction cleavage activity was eliminated by RNAi down-regulation of expression of TbREN1 in procyclic T. brucei. The TbREN1-down-regulated L-complex also decreased in S value, indicating an effect on stability. The difference from Trotter et al. (18), who reported that there was no effect on L-complex stability by down-regulation, may be due to allowing more time for down-regulation.

Recombinant TbREN1-CBP protein expressed in insect cells showed cleavage at a gRNA-determined U-deletion site in the model substrate and had no detectable cleavage activity with a model U-insertion substrate. A requirement for the REN1-RNase III motif for cleavage activity was confirmed by showing that TbREN1-CBP protein with the entire RNase III motif deleted (TbREN1-D) showed no activity, and the LmREN1-CBP protein with an E378/K378 substitution showed only 30% of the activity of WT enzyme (24). Recombinant LmREN1-CBP isolated from expression in L. tarentolae cell cytosol showed at least 15-fold higher U-deletion endonuclease activity than the rTbREN1-CBP protein isolated from insect-cell expression. In both cases, the recombinant proteins were not purified to homogeneity, but the identified major contaminants (β-tubulin, TEV protease, and hsp70) should not have contributed to the nuclease activity. The higher level of activity may be due to expression in a homologous system producing properly modified and folded protein.

The reported allosteric stimulatory effect of adenine nucleotides on cleavage at U-deletion sites (22) was confirmed for gradient-isolated 20S fractions and mitochondrial TAP pull-downs. However, the rTbREN1-CBP protein expressed in insect cells did not appear to show stimulation by AMP-CP, whereas the rLmREN1-CBP protein isolated from L. tarentolae cytosol did show a stimulatory effect. The reason remains to be investigated.

Carnes et al. (19) provided indirect evidence for a role of the MP61 or TbREN2 L-complex protein in cleavage of preedited mRNA at U-insertion sites, but the roles of the MP67 and LC8 (MP44) RNase III L-complex proteins are still uncertain, especially in view of the lack of any phenotype of MP67-down-regulated cells. And, of course, the precise topology and stoichiometry of the various L-complex proteins, including the REL1 and REL2 subcomplexes and the internal core L-complex proteins and their interaction with mRNAs and gRNAs (29), remain open questions that are critical for understanding this type of RNA editing in detail (30). In addition, the roles of the RNA-dependent interacting complexes such as the mitochondrial RNA-binding protein complex (MRP)1/2 RNA-binding complex and the RET1 3′ terminal uridylyl transferase complex(es) that, together with the L-complex and perhaps other complexes, constitute the “editosome,” are also outstanding important questions.

The ability to achieve a full round of gRNA-mediated U-deletion editing with recombinant REN1 nuclease, REL1 RNA ligase, and REX1 U-specific 3′–5′ exonuclease opens the door to a detailed investigation of this reaction and the role of additional L-complex factors as they become available.

Materials and Methods

Glycerol Gradient Sedimentation of Mitochondrial Lysate.

Purified mitochondria (31) were lysed with 0.5% Nonidet P-40 in 50 mM Hepes (pH 8.1), 10 mM MgCl2, and 60 mM KCl in the presence of Complete Protease Inhibitor Mixture (Roche Diagnostics, Indianapolis, IN). The clarified extract was centrifuged in a 10–30% glycerol gradient in an SW41 rotor (Beckman Coulter, Fullerton, CA) for 20 h at 111,132 × g (30,000 rpm).

In Vitro Editing Assay.

The following chemically synthesized RNAs (Integrated DNA Technologies, Coralville, IA) were used for the U-deletion and U-insertion in vitro editing assays (20, 23): U-deletion, mRNA 5′-GAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG-3′ or 5′-UAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG-3′, gRNA 5′-GGAUAUAUACUAUAACUCCACCCUAUAACUUUCCC-3′ (20); U-insertion, mRNA 5′-UAGGGGGAGGAGAGAAGAAAGGGAAAGUACUGAUUGGAGUUAUAG-3′, gRNA 5′-GGAUAUACUAUAACUCCAAUAAACGAAGUUUUCCCUUUCUUU-3′. The peak L-complex gradient fractions of T. brucei mitochondrial lysate or TAP-purified L-complex, rTbMP90-CBP, and rLmMP90-CBP were incubated with synthetic RNAs. The mRNAs were either 5′-end-labeled by using T4 polynucleotide kinase (Invitrogen, Carlsbad, CA) and [γ-32P]ATP or 3′-end-labeled by using T4 ligase and [α-32P]pCp. Reactions were performed in 20 mM Hepes (pH 7.9)/10 mM MgCl2/10 mM KCl/1 mM DTT in the presence of RNase inhibitor at 27°C for 120 min, and the products were analyzed on an 8 M urea/15% polyacrylamide gel.

Down-Regulation of TbMP90 Expression in T. brucei Procyclics.

Primers for RNAi of T. brucei TbMP90 were designed to amplify a 500-bp sequence from the 3′ end of the gene. The PCR primer sets are as follows: 5′-CAATTGTAAGGAGTGCTAATATGCGCC-3′ and 5′-TCTAGATAAGGAGTGCTAATATGCGCC-3′; and 5′-AAGCTTATGGGGCTACATTGGCCCCTGG-3′ and 5′-GGATCCATGGGGCTACATTGGCCCCTGG-3′. Added restriction sites are shown in bold. The sequences were amplified from T. brucei genomic DNA and cloned into the pCR2.1-TOPO vector (Invitrogen). The inserts were released from the vector with HindIII and XbaI digestion and with MunI and BamHI digestion and were inserted into the pLEW-HX-100GFP (32) vector to form a head-to-head RNAi plasmid under PARP promoter control. The plasmid was transformed into T. brucei 29-13 procyclic cells that contain integrated T7 RNA polymerase and tetracycline repressor (33). The cells were grown at 27°C in SDM-79 medium as described (34). RNAi was induced by the addition of 1 μg/ml tetracycline. Total RNA from the T. brucei RNAi cell line was purified by the acid guanidium isothiocyanate method (35). RT-PCR of T. brucei TbMP90 mRNA was performed with SuperScript II reverse transcriptase according to the commercial protocol (Invitrogen) by using the primers 5′-CCTGGCGCGTAGTTCCAACT-3′ and 5′-TGTCACTACGCCCGCAGAAG-3′. RT-PCR of T. brucei TbMP67 mRNA was performed by using the primers 5′-GTCGACTCAGCCGGCCGATTGTGCTAGCGACG-3′ and 5′-AAGCTTAACTTCCTTCTCTGCTCACTGCGGC-3′.

Overexpression of L. major MP90 in L. tarentolae Mitochondria and Cytosol and TAP Isolation of the L-Complex and Recombinant LmMP90.

LmMP90 was PCR amplified from L. major genomic DNA with the primers 5′-TCTAGACTAGCGAGGCAGTGACGCCTCACC-3′ and 5′-CCCGGGATGCATTCCTCCGCAACGCACAG-3′ (restriction sites added are in bold), and the PCR products were cloned into pCR2.1-TOPO vector (Invitrogen) and recloned into the pFastBac HTA vector (Invitrogen). Then the stop codon was deleted by using the primer 5′-GCGTCACTGCCTCGCTCTAGACCCGGGAGTCTCGAGGCATGC-3′ and the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA). After digestion with Xma and XbaI, the LmMP90 gene was inserted into the corresponding sites of the pX-TAP vector. The entire RNase III motif in the pX-TAP-LmMP90 vector was deleted by using the QuikChange Mutagenesis kit. The following oligonucleotides were 5′-phosphorylated and simultaneously used in mutagenesis reactions: 5′-CGCCGCGTCGCCTTTCATATGAACGACTGGAGTAC-3′ and 5′-GCACCTGCACCTCTACCATATGGTGCCTAAGCTG-3′. After digestion with NdeI to delete the whole RNase III motif, the vector was gel-isolated and self-ligated. Colonies were selected and screened for the desired mutations.

For cytosol expression, the putative N-terminal mitochondria signal sequence of the LmMP90 gene in the pX-TAP vector was deleted by using the QuikChange Mutagenesis kit. The primer, 5′-CGCCGCGTCGCCTTTAAACGACTGGAGTACA-3′, was used for generating a point mutation of E to K in the RNase III motif. L. tarentolae cells were transfected and selected for G418 resistance. Mitochondria were isolated as described (31) from late log-phase cell cultures (108 cells per ml) grown in brain–heart infusion medium with 10 μg/ml hemin and 100 μg/ml geneticin. The TAP-affinity isolation of the tagged LmMP90 protein was performed as described (11). A recombinant TEV protease–maltose-binding peptide-fusion protein, expressed from MBP-TEV(S219V)-Arg-5 fusion plasmid-transfected Escherichia coli, was used in some isolations (36, 37).

Expression and Purification of Recombinant TbMP90.

Oligonucleotides used for PCR of TbMP90 (added restriction sites are in bold) are as follows: 5′-CGGTCCGATGGGGCTACATTGGCCCCTGG-3′ and 5′-CCCGGGCGCACCAACCGAGATGCC-3′. The full-length TbMP90 gene was cloned into pCR2.1-TOPO (Invitrogen) and then digested and inserted into the pFastBac1 vector (Invitrogen) with a C-terminal TAP-Tag for baculovirus expression in Sf-9 insect cells as described by the company. The protein was purified by the TAP procedure (11).

The QuikChange Multisite-Directed Mutagenesis kit (Stratagene) was used for the deletion of the entire RNase III motif in the pFastBac1-TbMP90-TAP vector. The following oligonucleotides were 5′-phosphorylated and simultaneously used in mutagenesis reactions: 5′-CGGCGTGTGGCGTTCGGGCCCGAAAGATTAGAATAT-3′; 5′-GTGACATTGTGGGGGCCCCTTGAGCCGCAGCTG-3′. After digestion with ApaI to delete the whole RNase III motif, the DNA was gel-isolated, self-ligated, and used to transform E. coli DH5α cells. Colonies were screened for the desired mutations.

MS.

Protein bands excised from gels were crushed, washed in 25 mM ammonium bicarbonate/50% acetonitrile, dried, reduced, derivatized with iodoacetamide, and digested with trypsin as described (38). Recovered peptides were adsorbed onto uC18 ZipTips (Millipore, Bedford, MA), washed with 0.1% TFA, and then eluted with 3 μl of 50% acetonitrile (ACN)/0.1% TFA. MS analysis (MS and tandem MS) and gene identifications were performed as described (9).

Acknowledgments

We thank all members of the Simpson laboratory for advice and discussion; Ken Stuart (Seattle Biomedical Research Institute, Seattle, WA) for the gift of the monoclonal antibodies against MP81, MP63, and MP42; David Waugh (National Cancer Institute, Bethesda, MD) for the expression plasmid for TEV-MBP protease; and Jorge Cruz-Reyes for advice and discussion on construction of substrates. This work was supported in part by National Institutes of Health Grant AI09102 (to L.S.).

Abbreviations

CBP

calmodulin-binding protein

gRNA

guide RNA

PPi

inorganic pyrophosphate

TAP

tandem affinity purification

TEV

tobacco-etch virus

U

uridine.

Footnotes

Author contributions: X.K. and L.S. designed research; X.K., A.M.F., and S.Z. performed research; G.G. and K.R. contributed new reagents/analytic tools; X.K., G.G., K.R., and L.S. analyzed data; and X.K. and L.S. wrote the paper.

The authors declare no conflict of interest.

This paper was submitted directly (Track II) to the PNAS office.

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