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Edited by Alan M. Lambowitz, University of Texas, Austin, TX, and approved September 26, 2005 (received for review July 19, 2005)
Abstract
RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and ≈20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).
Many mitochondrial mRNAs in trypanosomatids are edited by the insertion and deletion of uridine nucleotides (Us) as directed by guide RNAs (gRNAs) (1). The key steps of RNA editing are the coordinated endonucleolytic cleavage of pre-edited mRNA (pre-mRNA); terminal uridylyl transferase or U-specific exoribonuclease-mediated insertion or deletion of Us, respectively; and ligation. These enzymatic activities are in an ≈20S multiprotein complex, the editosome, which contains at least 20 proteins (2). The proteins responsible for U insertion (KRET2), deletion (KREX1), and ligation (KREL1 and KREL2) have been identified (3–7), but the protein (or proteins) responsible for endonucleolytic cleavage remain unidentified.
The RNA editing endonuclease activity by editosome-containing extracts cleaves synthetic ATPase subunit 6 (A6) or cytochrome b (CYb) pre-mRNAs in vitro in a gRNA-directed manner (8–10). The cleavage occurs 5′ to the pre-mRNA:gRNA anchor duplex, leaving a 3′ hydroxyl and 5′ phosphate at the cleavage site. Cleavage of insertion sites is inhibited by adenosine nucleotides, whereas cleavage of deletion sites is stimulated by these nucleotides (11), suggesting that there may be distinct endonucleases. Several editosome proteins identified by mass spectrometry have motifs suggestive of nuclease function and may be RNA editing endonucleases (2).
Kinetoplastid RNA editing protein (KREP) B1, KREPB2, and KREPB3 have RNase III, U1-like Zn2+ finger, and dsRNA-binding motifs indicative of endonucleases. RNase III and dsRNA-binding motifs are typical in bacterial and eukaryotic endonucleases, and U1-like Zn2+ finger motifs imply RNA and protein interactions in a complex (12). The RNase III motifs of KREPB1, KREPB2, and KREPB3 all conserve the amino acids that are critical to endonuclease function (13). A report indicating that KREPA3 (formerly TbMP42) has endo-exoribonuclease activity in vitro suggested it might be an editing endonuclease (14). However, the observed cleavage activity is atypical for editing, and no cleavage defect using canonical substrates was shown (8–10, 14). We therefore sought to determine whether the editosome proteins with an RNase III motif were RNA editing endonucleases.
We show here that repression of KREPB3 expression inhibits cell growth in procyclic forms (PFs) and bloodstream forms (BFs) and blocks RNA editing in vivo. It also results in the loss of in vitro endonuclease cleavage of insertion but not deletion editing sites and does not affect the subsequent catalytic steps of editing. Exclusive expression of KREPB3 with point mutations in critical RNase III motif residues has similar inhibitory effects. We conclude that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and rename it KREN2 (kinetoplastid RNA editing endonuclease 2).
Materials and Methods
Plasmid Constructs and Transfections. Detailed descriptions of plasmid construction and transfections are provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Two plasmids, pZJM-KREN2 and p2T7-KREN2, were made by cloning 1,031 or 604 base pairs of KREN2 into pZJM (15) or p2T7 (16), respectively. These plasmids were transfected into PF 29-13 or BF single marker cells as described in ref. 17 to create the PF ZJM-KREN2, PF 2T7-KREN2, and BF ZJM-KREN2 RNA interference (RNAi) cell lines.
Plasmids for making the RKO-KREN2 cell line were based on published vectors pLew13, pLew90, and pLew79 (6, 17). Briefly, 303 base pairs of 5′ UTR and 323 base pairs of 3′ UTR from KREN2 flank the Neor marker of pLew13 to make pSKO-KREN2. The Neor marker is replaced with Hygr marker from pLew90 to make pDKO-KREN2. The KREN2 ORF is cloned into pLew79 to make pReg-KREN2 for tetracycline (tet)-regulatable KREN2 expression. The RKO-KREN2 cell line was generated by a series of transfections (Fig. 6, which is published as supporting information on the PNAS web site) to introduce the pSKO-KREN2, pReg-KREN2, and pDKO-KREN2 plasmids sequentially into BF (427 strain) cells. In these cells, KREN2 expression depends on the presence of tet.
WT and mutated KREN2 genes were cloned into the pHD1344tub plasmid [a generous gift from Achim Schnaufer (Seattle Biomedical Research Institute)], creating the pWT-β, pE227V-β, and pD234A-β plasmids. Transfections of the RKO-KREN2 cell line with pWT-β, pE227V-β, or pD234A-β were performed to integrate constitutively expressed KREN2 alleles into the β-tubulin locus, making the RKO-WT-β, RKO-E227V-β, and RKO-D234A-β cell lines, respectively.
Growth Curves. For PF growth curves, cells were grown to logarithmic phase and split to 1 × 106 cells per ml every 48 h in SDM-79 supplemented with 15% FBS and appropriate antibiotics. For BF growth curves, cells were grown to logarithmic phase and split to 2 × 105 cells per ml every 24 h in HMI-9 supplemented with 10% FBS and appropriate antibiotics. Cell numbers were determined every 24 h by using a Coulter Counter. To induce expression of dsRNA for RNAi, 1 μg/ml tet was added to media. In BF cells containing the regulatable Reg-KREN2 allele, 1 μg/ml tet maintained ectopic expression. To remove tet and repress the Reg-KREN2 allele, cells were centrifuged at 1,300 × g for 10 min, resuspended in media without tet, and then recentrifuged and resuspended a second time. Equal cell numbers were then grown in the absence or presence (by readdition) of tet.
Fractionation of Cell Lysates on Glycerol Gradients. The protocol for partially purifying mitochondria from PF cells was a modified version of the method described in ref. 18, and buffer compositions are fully outlined therein. Cells (7.5 × 109) were resuspended in 35 ml of SBG buffer, centrifuged at 6,000 × g for 10 min at 4°C, and resuspended in 35 ml of DTE buffer. Cells were Dounce-homogenized on ice, and 5.9 ml of 60% sucrose was added. Samples were centrifuged at 15,800 × g for 10 min at 4°C. Supernatants were discarded, and crude mitochondrial pellets were resuspended in 6.8 ml of STM buffer. DNA was digested by incubation on ice for 1 h with 21 μl of 1M MgCl2/21 μl of 0.1M CaCl2/5 μl of RNase-free DNase I (Promega). STE buffer (6.9 ml) was added, and samples were centrifuged at 15,800 × g for 10 min at 4°C. Supernatants were discarded, and crude mitochondrial pellets were resuspended in 500 μl of lysis buffer (10 mM Tris, pH 7.2/10 mM MgCl2/100 mM KCl/1 mM Pefabloc/2 μg/ml leupeptin/1 μg/ml pepstatin/1 mM DTT). Triton X-100 was added to 1% with mixing by inversion for 15 min at 4°C. Whole-cell lysates of BF cells were prepared by resuspending 7.5 × 108 cells in 750 μl of lysis buffer and adding Triton X-100 to 1% as above. PF and BF lysates were cleared by two centrifugation steps of 17,000 × g for 15 min at 4°C. PF and BF lysates were loaded onto 10–30% glycerol gradients containing 20 mM Hepes (pH 7.9), 10 mM Mg, 50 mM KCl, and 1 mM EDTA and centrifuged at 38,000 rpm in a Beckman SW40 Ti rotor for 5 h at 4°C. Glycerol gradients were divided into 0.5-ml fractions from the top, flash-frozen on liquid nitrogen, and stored at –80°C. For each sample within an experiment, equivalent cell numbers were lysed.
Western Blots. Glycerol gradient fractions (30 μl) were separated by electrophoresis on 10% SDS/PAGE gels, transferred to Immobilon-P membranes (Fisher), and probed by using monoclonal antibodies against KREPA1 (TbMP81), KREPA2 (TbMP63), KREL1, and KREPA3 (TbMP42) as described in ref. 19.
In Vitro Enzymatic Assays. Peak glycerol gradient fractions of ≈20S complex were assayed, with amounts from 1 to 15 μl as indicated. Reactions were incubated at 28°C for 3 h. For all endonuclease and editing assays, RNAs were EtOH-precipitated, resolved on 11% polyacrylamide 7 M urea gels, and analyzed with a PhosphorImager (Molecular Dynamics).
Insertion endonuclease. Cleavage of insertion substrates was assayed as previously described by using the 70-nt A6-eES1 pre-mRNA and gA6[14] gRNA (8). Buffer conditions for each 30-μl reaction were modified to 22.5 mM Hepes, pH 7.9/10 mM Mg(OAc)2/0.5 mM DTT/1 mM EDTA/50 mM KCl/100 μM UTP/2.5 mM CaCl2/8 units of RNasin/0.5 μg torula type VI RNA. ATP was omitted to optimize for the accumulation of the cleavage product over ligation.
Deletion endonuclease. Cleavage of deletion substrates was assayed as previously described by using the 73-nt A6short/TAG.1 pre-mRNA and a derivative of D33 gRNA called D34 (Table 1, which is published as supporting information on the PNAS web site) (9, 20). Buffer conditions for each 30-μl reaction were modified to 25 mM Tris, pH 7.0/10 mM Hepes, pH 7.9/10 mM Mg(OAc)2/0.5 mM DTT/1 mM EDTA/2.5 mM CaCl2/8 units of RNasin/0.5 μg torula type VI RNA/1 mM ADP to optimize for cleavage product over ligation.
Precleaved editing. Precleaved deletion and insertion editing were assayed as previously described by using 5′-labeled U5 5′CL and U5 3′CL with gA6[14]PC-del and 5′-labeled 5′CL18 and 3′CL13pp with gPCA6–2A RNAs, respectively (21, 22).
CYb endonuclease. CYb insertion cleavage assays were performed as described in refs. 23–25. To enhance detection of cleavage activity on CYb substrate, a 37-nt gRNA (CYb37g, Oligos Etc., Wilsonville, OR; Table 1) that directs insertion of two Us was used in place of the standard 27-nt gRNA (26).
Real-Time PCR. Oligonucleotide sequences are given in Table 1. RNA was isolated from ≈5 × 107 to ≈5 × 108 cells by using 5 ml of TriZol LS (Invitrogen). Ten micrograms of total RNA was DNase I-treated by using the DNAfree kit (Ambion, Austin, TX). RNA integrity was confirmed by using an RNA nanochip on a BioAnalyzer (Agilent Technologies, Palo Alto, CA). To make real-time PCR templates, 4.5 μg of RNA was converted to cDNA by using random hexamers and Taqman reverse transcription reagents (Applied Biosystems) in a 30-μl reaction. Each experiment had a reaction without reverse transcriptase as a control. The 30-μl reaction was diluted in water 2- to 10-fold, depending on the number of targets to be analyzed. For each 25-μl real-time PCR, we combined 12.5 μl of SYBR Green Master Mix (Applied Biosystems), 5 μl of 1.5 μM forward oligonucleotide, 5 μl of 1.5 μM reverse oligonucleotide, and 2.5 μl of cDNA template (or minus reverse transcriptase control) per well of a 96-well plate (Applied Biosystems). Applied Biosystems Prism 7000 thermocycler conditions for all reactions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Template was further diluted 1:50 for β-tubulin and 18S rRNA internal controls so C t values were similar to less abundant edited and pre-edited RNAs. Thermal dissociation confirmed PCR generated a single amplicon. Amplicon sizes were analyzed by 2.5% agarose gel electrophoresis to confirm amplification of the correct target. Oligonucleotides for real-time PCR were designed with Applied Biosystems primer express 2.0 software. Relative changes in target amplicons were determined by using the Pfaffl method, with PCR efficiencies calculated by linear regression using LinRegPCR (27, 28).
Results
Growth Inhibition upon Repression of KREN2 Expression. Repression of KREN2 expression by RNAi in PF cells inhibited growth but did not affect editosome sedimentation (Figs. 1A and 2A ). Two independently generated PF KREN2-specific RNAi cell lines, ZJM-KREN2 and 2T7-KREN2, were created by cloning portions of the KREN2 mRNA sequence into plasmids with opposable tet-inducible T7 promoters. The PF ZJM-KREN2 cell line had significant growth inhibition by day 5 after KREN2 repression compared with control cells (Fig. 1 A ). Neither the amount nor the sedimentation of ≈20S editosomes was altered in cells in which KREN2 was repressed by RNAi (Fig. 2 A ). Western analysis of glycerol gradient fractions with antibodies to four known editosome proteins showed that they cosedimented and primarily localized to fractions 7–11. Similar results were obtained for PF 2T7-KREN2 cell lines after KREN2 repression (data not shown). The lack of an antibody against KREN2 precluded direct assessment of KREN2 protein levels.
Fig. 1.
Repression of KREN2 inhibits growth in PFs and BFs. (A) Growth of the PF ZJM-KREN2 cell line in the presence of KREN2 expression (without tet, circles) and after repression of KREN2 expression by RNAi (with tet, squares). (B) Growth of the BF RKO-KREN2 cell line in the presence of KREN2 expression (with tet, circles) and after repression of KREN2 expression (without tet, squares). Recovery of KREN2 expression (triangles) by the addition of tet to repressed cells at day 4 (arrow) is also shown.
Fig. 2.
Editosome sedimentation is unaltered by repression of KREN2 in PFs and BFs. (A) PF ZJM-KREN2 cells grown in the absence (KREN2 expressed) or presence (KREN2 repressed) of tet for 4 days were lysed, fractionated on glycerol gradients, and analyzed by Western blot. Preload lane is cell lysate before gradient fractionation. Western blots were simultaneously probed with monoclonal antibodies against editosome proteins KREPA1, KREPA2, KREL1, and KREPA3. (B) Similar analysis of BF RKO-KREN2 cells grown in the presence (KREN2 expressed) or absence (KREN2 repressed) of tet for 3 days.
Conditional repression of KREN2 expression in the BF RKO-KREN2 cell line also inhibited growth and was eventually lethal (Fig. 1B ). The RKO-KREN2 BF cell line was made by elimination of both endogenous KREN2 alleles and insertion of a tet-inducible Reg-KREN2 allele into the rDNA intergenic locus. Attempts to make KREN2 double knockouts in the absence of Reg-KREN2 expression were unsuccessful. The genotype of the cells was confirmed by PCR and Southern analyses (data not shown). tet withdrawal, which halts Reg-KREN2 expression, resulted in cessation of cell growth by day 4 (Fig. 2 A ). No living cells were observed after 9 days. When tet was reintroduced 4 days after its withdrawal, cell growth resumed after a lag of 2 days. Western analysis of glycerol gradient fractions of whole-cell lysates showed that sedimentation of editosomes was unchanged after KREN2 repression, although their abundance was slightly reduced (Fig. 2B ). Repression of KREN2 by RNAi in BF cells was also examined by creating the BF ZJM-KREN2 cell line. KREN2 RNAi also inhibited growth in BF cells after 3 days, although the effect was less severe (data not shown). Expression of KREN2 is thus essential for survival of BF Trypanosoma brucei and probably in PFs, given the lower repression by RNAi.
Loss of RNA Editing in Vivo upon Repression of KREN2. The levels of several edited and pre-edited mRNAs were assayed simultaneously by real-time PCR to determine the effect of KREN2 repression on editing in vivo. Primers were designed to be specific for each edited or pre-edited amplicon and were directed to the 5′ end of edited targets or the 3′ end of pre-edited targets (29). Data were normalized to two independent internal controls, β-tubulin and 18S rRNA, to prevent internal control-specific data skewing. Serial dilutions of templates showed that each primer pair produced a dose response in real-time PCRs (data not shown).
A reduction of edited RNAs generally paralleled the reduction of KREN2 mRNA after its repression (Fig. 3). In PF ZJM-KREN2 cells, the level of KREN2 mRNA was decreased by ≈50% in comparison to cells with unaltered KREN2 expression (Fig. 3A ). Loss of KREN2 mRNA was also seen by using Northern analysis (data not shown). Edited RPS12, ND3, COIII, and CYb mRNAs were reduced by a similar amount. The levels of pre-edited RNAs in cells with repressed KREN2 expression were similar or somewhat elevated compared with cells with nonrepressed KREN2 expression. The mRNAs for KREN1 and KREPB2, editosome proteins that also contain an RNase III motif, were essentially unchanged by KREN2 RNAi, as was the case for ND4 mRNA, which does not get edited. In the BF RKO-KREN2 cell line, KREN2 mRNA was reduced to <7% of the level in cells expressing KREN2 (Fig. 3B ). Edited mRNAs were reduced by a similar amount, except for COII mRNA (≈45% of control) and, to a lesser extent, ND7 mRNA (≈17% of control). The abundance of pre-mRNAs was either unchanged or increased in cells in which KREN2 was repressed. Pre-edited RPS12 and ND3 mRNAs were significantly more abundant after KREN2 repression, with levels of ≈220% and ≈215% of the controls, respectively. Unedited ND4 mRNA was slightly decreased, perhaps reflecting minor secondary effects. Thus, repression of KREN2 expression resulted in a proportional reduction in the accumulation of edited mRNAs in both PF and BF cells.
Fig. 3.
Repression of KREN2 reduces editing in vivo. Real-time PCR analysis of total RNA isolated from PF ZJM-KREN2 (day 4) and BF RKO-KREN2 (day 3) cell lines. Analysis was performed in triplicate. For each target amplicon, the relative change in RNA abundance was determined by using either β-tubulin (left bar in each pair) or 18S rRNA (right bar in each pair) as an internal control. Open bars denote pre-mRNA, dark gray bars denote edited mRNA, black bars denote RNase III motif-containing mRNA, and light gray bars denote never-edited mRNA ND4. (A) Editing is reduced after repression of KREN2 by RNAi in PF cells. (B) Editing is reduced in BF RKO-KREN2 cells after repression of KREN2. Gray line marked by arrow represents no change in target RNA level; bars above or below this line represent an increase or decrease in RNA, respectively.
Loss of Insertion Endonuclease Activity in Vitro. Repression of KREN2 led to a specific deficiency in endonuclease activity on insertion editing substrates. Peak ≈20S glycerol gradient fractions from PF ZJM-KREN2 cells in which KREN2 expression was repressed had an ≈93% reduction of endonuclease activity with the A6 insertion editing substrate RNA compared with fractions from cells in which KREN2 was expressed (Fig. 4A ). In contrast, cleavage of the A6 deletion editing substrate was unaffected by KREN2 repression (Fig. 4B ). The other steps of in vitro RNA editing were similarly unaffected by KREN2 repression, as determined by precleaved insertion and deletion editing assays. In precleaved editing assays, three RNAs (a radiolabeled 5′ mRNA fragment, a 3′ mRNA fragment, and a gRNA) mimic the editing substrate after cleavage of the pre-mRNA by endonuclease. For precleaved insertion, gRNA directs addition of two Us by terminal uridylyl transferase to the radiolabeled 5′ mRNA, which is then ligated to a 3′ mRNA by ligase. For precleaved deletion, the gRNA directs removal of four Us by U-specific exoribonuclease from the radiolabeled 5′ mRNA, which is then ligated to a 3′ mRNA by ligase. Repression of KREN2 did not alter the ability of peak gradient fractions to add Us and ligate edited RNA in precleaved insertion assays (Fig. 4C ) or the ability to remove Us and ligate edited RNA in precleaved deletion assays (Fig. 4D ). Similar results were observed for the PF 2T7-KREN2 cell line (data not shown). The loss of insertion site-specific endonuclease activity after KREN2 repression was more dramatic in BF RKO-KREN2 cells. Cleavage of an insertion editing substrate was essentially eliminated in peak ≈20S glycerol gradient fractions from cells in which KREN2 was repressed (Fig. 4E ), but the ability to cleave a deletion editing substrate remained unaffected (Fig. 4F ). Precleaved insertion and deletion assays showed that the ability of these fractions to catalyze the U addition, U removal, and ligation steps of editing were also unaltered (Fig. 4 G and H ). Similarly, a reduction in insertion editing cleavage but not the other activities of editing was observed with fractions from the BF ZJM-KREN2 cell line, although the reduction was less severe than with the RKO-KREN2 cell line (data not shown). The observed loss of insertion endonuclease activity was not limited to A6 substrate. A decrease in insertion cleavage activity with a CYb-derived substrate was observed in peak ≈20S glycerol gradient fractions from both PF and BF cells in which KREN2 was repressed (Fig. 7, which is published as supporting information on the PNAS web site). The cleavage of CYb substrate RNA was decreased by 92–96% in fractions from PF ZJM-KREN2 RNAi cells and by 95–100% in fractions from repressed BF RKO-KREN2 cells. Reductions in insertion site cleavage were also observed for PF 2T7-KREN2 and BF ZJM-KREN2 RNAi cell lines (data not shown). Thus, repression of KREN2 specifically impaired cleavage of an insertion substrate, but other catalytic steps were unaffected.
Fig. 4.
Repression of KREN2 in PF and BF decreases endonuclease activity on insertion substrates but does not alter other editing activities. All assays used peak ≈20S fractions from glycerol gradient sedimentation of lysates from PF ZJM-KREN2 RNAi cells grown for 4 days in the presence (repressed) or absence (expressed) of tet (A–D) or from BF RKO-KREN2 cells grown for 3 days in the presence (expressed) or absence (repressed) of tet (E–H). The amount (in μl) of peak fraction used in each reaction is shown above each lane. RNA substrates and products are shown in schematics; asterisk denotes radiolabel, and wedge denotes cleavage site. A6 insertion site cleavage product (black arrows) was significantly diminished by KREN2 repression (gray arrows) in PFs (A) and BFs (E). Cleavage is due to RNA editing endonuclease activity, as indicated by the requirement for gRNA (-guide). T1-digested substrate RNA was used as a marker to determine where substrate RNA was cleaved. A6 deletion site cleavage was unaltered by repression of KREN2 in PF (B) and BF (F). Cleavage product (arrows) is absent in reactions without gRNA (-guide). Precleaved insertion editing is unaltered by KREN2 repression in PFs (C) and BFs (G). The input lane has only radiolabeled substrate. Precleaved deletion editing is unaltered by KREN2 repression in PFs (D) and BFs (H).
Point Mutations in the RNase III Motif Result in Loss of Insertion Endonuclease Activity. Amino acids in the RNase III motif that may be critical for either catalysis or substrate binding are conserved in KREN2. Mutations of two of these residues (E227V or D234A) were independently introduced into KREN2 (Fig. 8, which is published as supporting information on the PNAS web site). Both WT and mutant versions of KREN2 were integrated into the β-tubulin locus of the previously described BF RKO-KREN2 cells, resulting in the RKO-WT-β, RKO-E227V-β, and RKO-D234A-β cell lines. These WT or mutant alleles are constitutively expressed, whereas the WT Reg-KREN2 allele requires tet for expression. Correct integrations of the alleles in the β-tubulin locus were shown by PCR, and expression of the KREN2 alleles was observed by real-time PCR (data not shown). Each cell line was used to test the ability of the WT or mutant KREN2 alleles in the β-tubulin locus to complement the loss of tet-induced expression of the KREN2 allele in the rDNA locus. Cells with either WT or mutant alleles grew continuously in the presence of tet at similar rates (Fig. 5A ). Withdrawal of tet, which repressed the Reg-KREN2 allele, did not alter growth of the RKO-WT-β cell line. Therefore, expression of the WT KREN2 allele in the β-tubulin locus alone is sufficient to sustain a normal rate of growth. In contrast, growth of the RKO-E227V-β mutant cell line was inhibited 3 days after withdrawal of tet, and growth of the RKO-D234A-β mutant cell line was inhibited 2 days after tet withdrawal. Both mutant cell lines ceased growth a day later. The results indicate that the conserved residues within the RNase III motif of KREN2 are essential for cell viability.
Fig. 5.
Point mutations in the RNase III signature motif of KREN2 inhibit growth and reduce insertion endonuclease activity. (A) Growth of RKO-WT-β, RKO-E227V-β, and RKO-D234A-β cell lines with the Reg-KREN2 allele expressed (E) or repressed (R), as indicated in Inset.(B) A6 insertion site cleavage in RKO-WT-β, RKO-E227V-β, RKO-D234A-β, and parental RKO-KREN2 (RKO) cell lines. Arrow indicates cleavage product. Assays were performed by using peak ≈20S fractions from each cell line in which WT Reg-KREN2 allele was expressed (E) or repressed (R). (C) The same fractions assayed for insertion cleavage were also assayed for A6 deletion site cleavage. Arrow indicates cleavage product. For both endonuclease assays, cleavage was guide-dependent (-guide) and absent when water was substituted for gradient fraction (water). T1-digested substrate RNA acted as a marker to identify the cleavage product.
The effect of RNase III motif mutations on endonuclease activity was tested by using peak ≈20S glycerol gradient fractions from cells expressing the various KREN2 alleles in the β-tubulin locus. After Reg-KREN2 repression, the RKO-WT-β, RKO-E227V-β, and parental RKO-KREN2 cell lines were harvested before growth defects were apparent (at day 3); the RKO-D234A-β cell line was harvested at day 2 because of its earlier onset growth defect. Fractions from RKO-WT-β cells expressing WT KREN2 from the β-tubulin locus catalyzed cleavage of A6 insertion substrate in both the presence and absence of Reg-KREN2 expression, although cleavage was reduced by ≈12% after Reg-KREN2 repression (Fig. 5B ). In fractions from cells with the E227V allele, insertion cleavage was inhibited by 31% in the presence Reg-KREN2 expression and by 86% in its absence, compared with RKO-WT-β cells expressing both WT alleles. Moreover, insertion cleavage was reduced by 79% in fractions from cells with the D234A mutant allele in the presence of Reg-KREN2 expression and was essentially abolished in its absence. In contrast, deletion cleavage was similar in peak fractions from cells with a WT, E227V, or D234A allele regardless of Reg-KREN2 expression (Fig. 5C ). Precleaved insertion and deletion editing activities were also unaffected by the expression of the mutant KREN2 alleles in cells with or without Reg-KREN2 expression (data not shown). Thus, the WT KREN2 allele in the β-tubulin locus complemented the loss of Reg-KREN2 expression, whereas the E227V allele complemented poorly, and the D234A allele failed to complement. Therefore, exclusive expression of mutant KREN2 proteins specifically diminished or eliminated the capacity for insertion site cleavage.
Discussion
We conclude that the ≈20S editosome component KREPB3 (formerly TbMP61) is an endoribonuclease with specificity for insertion editing sites. We have found that the larger KREPB1 has the complementary deletion-specific endonuclease activity (J.R.T., N.L.E., J.C., B. Panicucci, and K.S., unpublished work), and, consequently, KREPB1 and KREPB3 are renamed kinetoplastid RNA editing endonucleases 1 and 2 (KREN1 and KREN2), respectively, according to size (1). KREN2 function is indicated by the specific loss of insertion site, but not deletion site, cleavage in vitro by ≈20S fractions from both PF and BF cells upon repression of KREN2 gene expression. These ≈20S editosomes retain not only deletion endonuclease activity but also the U-specific exoribonuclease, terminal uridylyl transferase, and RNA ligase activities for the subsequent steps of RNA editing. The RNase III-like domain is essential for the insertion site endonucleolytic activity, because point mutations in this motif block this activity. The loss of edited RNA in vivo and death of BF cells upon repression of KREN2 expression show that it is essential for RNA editing and also confirm that editing is normally essential in BF T. brucei (6, 30).
The endonuclease activity of KREN2 is likely to employ a catalytic mechanism that resembles that of other RNase IIIs, as indicated by the loss of function that results from mutation of highly conserved residues in the RNase III motif. The Aquifex aeolicus RNase III structure places conserved amino acids Aa D44 and E110 (equivalent to KREN2 D234 and E301) in proximity at the catalytic site coordinating a divalent cation (31). These residues are essential for catalysis in both Escherichia coli RNase III and human Dicer recombinant proteins (32). The D234A mutation to KREN2 eliminates cleavage of insertion editing substrates, implying that this residue is in the catalytic site. The E. coli residue equivalent to Aa E37 (equivalent to KREN2 E227) is required for efficient RNase III activity in vivo (31, 33). This residue is not essential for cleavage in vitro, but its mutation increases K m, reducing catalytic efficiency (32). The lesser effect of the E227V mutation suggests that, as observed for the equivalent residue in E. coli, this residue is also important for catalysis, perhaps because of substrate binding. Editing endonucleases appear to only cleave mRNA. Most RNase IIIs cleave both strands of a dsRNA substrate, although some also can cleave only one strand (34–37). Both biochemical and crystal structure data indicate that these RNase IIIs function as dimers (31, 38). KREN2 may function as a dimer, possibly a homodimer restricted to cleaving a single strand in the context of the editosome or perhaps a heterodimer with other editosome proteins that have an RNase III-like motif, such as KREPB4 or KREPB5, where the divergence of their RNase III motifs limits cleavage to mRNA.
The identification of KREN1 and KREN2 as deletion and insertion editing site endonucleases, respectively, leaves the role for KREPA3 uncertain. Western analysis indicates that KREPA3 is present in extracts that lack either deletion or insertion endonuclease activity due to KREN1 or KREN2 repression (J.R.T., N.L.E., J.C., B. Panicucci, and K.S., unpublished work; Fig. 2). Repression of KREPA3 results in loss of in vivo editing as well as loss of precleaved insertion editing in vitro. Preparations of recombinant KREPA3 protein have endo-exoribonuclease activity that localizes to the C-terminal region with an OB fold motif, although cleavage is atypical for editing (8–10, 14). However, these data did not rule out nonspecific cleavage due to Zn2+ hydrolysis (39, 40). Therefore, KREPA3 may have an indirect role in cleavage or an alternative function in vivo.
The identification of KREN2 as an insertion editing site-specific endonuclease complements the identification of KREN1 as a deletion editing site-specific endonuclease (J.R.T., N.L.E., J.C., B. Panicucci, and K.S., unpublished work). Separate enzymes for insertion and deletion cleavage provide a basis for the observed differential effects of adenosine nucleotides on these editing endonuclease activities (11). Furthermore, mass spectrometry of affinity-purified editosomes from cells expressing tandem affinity purification (TAP)-tagged KREN1, KREPB2, and KREN2 indicates that these proteins are mutually exclusive in editosomes and that editosomes containing KREN1 specifically cleave deletion substrates, whereas editosomes containing KREN2 specifically cleave insertion substrates (A. Panigrahi, N.L.E., G. Domingo, M. Fleck, R. Salavati, and K.S., unpublished work). These data suggest not only that the insertion and deletion endonuclease activities are due to distinct proteins, but that these proteins may also be in distinct editing complexes. The distinct complexes may be stably present or may result from exchange of different endonucleases within a stable “core complex.” The slight decrease in the amount of editosomes in peak ≈20S fractions after KREN2 repression could reflect either a loss of a KREN2 editosome or a shift in the exchange equilibrium. The endonuclease protein shuttling or higher order associations of complexes with insertion and deletion site endonucleases could accommodate the editing of pre-mRNA regions that have both insertion and deletion editing sites.
The means by which KREN2 specifically recognizes and cleaves insertion editing sites and how its activity is coordinated with the activity that cleaves deletion editing sites remains unclear. The difference in gRNA/mRNA structure between an insertion site and a deletion site is likely critical for some aspect of recognition (41, 42). Other components of the ≈20S editosome might play a role in coordinating endonuclease-substrate recognition, and understanding these interactions may help elucidate how insertion and deletion editing sites are discriminated.
The discovery that KREN2 is essential for survival reinforces the essential nature of RNA editing in BF T. brucei. Previously, conditional repression of editosome proteins KREL1 (an RNA ligase) and KREPB5 (critical for editosome integrity) indicated that they were essential in BF cells (6, 30). The addition of KREN2 to this list underscores the normal requirement for RNA editing in the pathogenic stage of this parasite, suggesting that this process may be a potential drug target.
Acknowledgments
We thank Achim Schnaufer for helpful suggestions on introducing mutant alleles and the gift of pHD1344tub, Reza Salavati for help with CYb assays, and members of K.S.'s laboratory for discussions. This work was supported by National Institutes of Health (NIH) Grant GM042188 (to K.S.), which extended work supported by NIH Grant AI014102 (to K.S.). J.C. was supported by NIH Grant AI007509 (Pathobiology Training grant, University of Washington). The Economic Development Administration (U.S. Department of Commerce) and the M. J. Murdock Charitable Trust provided funding for equipment.
Footnotes
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↵ ‡ To whom correspondence should be addressed at: Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109. E-mail: kenneth.stuart{at}sbri.org.
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Author contributions: J.C., J.R.T., N.L.E., and K.S. designed research; J.C., J.R.T., N.L.E., and A.S. performed research; J.C. contributed new reagents/analytic tools; J.C. and K.S. analyzed data; and J.C. and K.S. wrote the paper.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: KREP, kinetoplastid RNA editing protein; KREN, kinetoplastid RNA editing endonuclease; BF, bloodstream form; PF, procyclic form; gRNA, guide RNA; pre-mRNA, pre-edited mRNA; RNAi, RNA interference; CYb, cytochrome b; A6, ATPase subunit 6; tet, tetracycline.
- Copyright © 2005, The National Academy of Sciences
References
An essential RNase III insertion editing endonuclease in Trypanosoma brucei
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