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. 2009 Feb;15(2):277–286. doi: 10.1261/rna.1353209

The MRB1 complex functions in kinetoplastid RNA processing

Nathalie Acestor 1, Aswini K Panigrahi 1, Jason Carnes 1, Alena Zíková 1, Kenneth D Stuart 1
PMCID: PMC2648719  PMID: 19096045

Abstract

Mitochondrial (mt) gene expression in Trypanosoma brucei entails multiple types of RNA processing, including polycistronic transcript cleavage, mRNA editing, gRNA oligouridylation, and mRNA polyadenylation, which are catalyzed by various multiprotein complexes. We examined the novel mitochondrial RNA-binding 1 (MRB1) complex that has 16 associated proteins, four of which have motifs suggesting RNA interaction. RNase treatment or the lack of kDNA in mutants resulted in lower MRB1 complex sedimentation in gradients, indicating that MRB1 complex associates with kDNA transcripts. RNAi knockdowns of expression of the Tb10.406.0050 (TbRGGm, RGG motif), Tb927.6.1680 (C2H2 zinc finger), and Tb11.02.5390 (no known motif) MRB1 proteins each inhibited in vitro growth of procyclic form parasites and resulted in cells with abnormal numbers of nuclei. Knockdown of TbRGGm, but not the other two proteins, disrupted the MRB1 complex, indicating that it, but perhaps not the other two, is required for complex assembly and/or stability. The knockdowns resulted in similar but nonidentical patterns of altered in vivo abundances of edited, pre-edited, and preprocessed mt mRNAs, but did not appreciably affect the abundances of mRNAs that do not get edited. These results indicate that MRB1 complex is critical to the processing of mt RNAs, and although its specific function is unknown, it appears essential to parasite viability.

Keywords: Trypanosoma brucei, RNA processing, mitochondrion, RNA binding

INTRODUCTION

Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT), or sleeping sickness, is a blood-borne pathogenic parasite transmitted by tsetse flies. It has a complex life cycle that alternates between the bloodstream forms (BFs) in the mammalian host and the procyclic forms (PFs) in the midgut of the tsetse fly vector. The mitochondrion in these organisms possesses an unusual mitochondrial (mt) DNA, termed kinetoplast DNA (kDNA), which consists of a network of intercatenated maxicircles and minicircles (Englund et al. 1982; Stuart 1983; Simpson 1986). The maxicircles encode mt rRNAs and several mRNAs that are homologous to the mt DNAs of other organisms. The minicircles encode guide RNAs (gRNAs) that specify the edited sequences of the mRNAs. There are naturally occurring and laboratory produced strains of trypanosomatids, which lack all kDNA (akinetoplastic [Ak]), or most of the kDNA sequences (dyskinetoplastic [Dk]). These mutants cannot grow as PFs but can grow as BFs as a result of other compensatory mutations or adaptations (Schnaufer et al. 2002). The polycistronic maxicircle and minicircle transcripts require post-transcriptional endonucleolytic cleavage, followed by steps, which include RNA editing, polyuridylation, and polyadenylation, to produce the mature mt mRNAs, gRNAs, and rRNAs. These RNA processing steps may be highly integrated and coordinated with mt RNA turnover processes (Blum and Simpson 1990; Koslowsky and Yahampath 1997; Militello and Read 1999; Grams et al. 2000; Stuart et al. 2002).

The RNA editing process, which is catalyzed by 20S editosomes, has been studied to a certain extent (for recent reviews, see Lukes et al. 2005; Stuart et al. 2005; Aphasizhev 2007); however, there is limited information on many aspects of RNA editing and other RNA processing in the mitochondrion of trypanosomes. The editing process involves other complexes, including the T1 complex, which appears to add 3′oligoU tails to the gRNAs, and the MRP1/MRP2 complex, which may play a matchmaking role in associating cognate gRNA with mRNA (Aphasizhev et al. 2003; Simpson et al. 2004; Schumacher et al. 2006; Zikova et al. 2008). Other complexes, which contain RBP16 or TbRGG1 (which is unrelated to TbRGGm), have roles that differentially affect the abundance of both edited and unedited mt RNAs, but their specific roles have not been determined (Pelletier and Read 2003; Vondruskova et al. 2005; Goulah et al. 2006; Hashimi et al. 2008). An uncharacterized ∼19S complex appears to function in processing of polycistronic gRNA transcripts (Grams et al. 2000). In addition, the 3′ polyadenylation of mt mRNAs may function to regulate mt mRNA stability (Ryan et al. 2003), either stabilizing or destabilizing the mRNA depending upon that mRNA's editing status (Kao and Read 2005). The mt mRNAs are differentially edited between life-cycle stages, and their abundance correlates with the metabolic differences between the stages. For example, apocytochrome b (CYb) and cytochrome oxidase subunit II (COII) mRNAs are abundant in PFs, where energy is generated through cytochrome-mediated oxidative phosphorylation, while these mRNAs are essentially absent in BFs, where energy is generated strictly through glycolysis (Stuart et al. 1997).

In previous work we identified the novel mt RNA-binding 1 (MRB1) complex in PF T. brucei, and identified up to 16 associated proteins in complexes using either monoclonal antibody (mAb) or tandem affinity purification-tag (TAP-tag) (Hashimi et al. 2008; Panigrahi et al. 2008). However, the exact composition of the MRB1 complex is unclear. The variable protein content in the different pull-downs indicate the complex may have distinct subcomplexes, or it is composed of one or more complexes and/or proteins that are linked via RNAs. We found that four of the 16 have a RNA binding motif; one has an RNA helicase motif; and the protein designated TbRGGm, also called TbRGG2 (Fisk et al. 2008), has a glycine-rich N-terminal region with four RGG and nine SGG tripeptides as well as a C-terminal RNA recognition motif (RRM). Despite their similar names, TbRGGm is unrelated to TbRGG1, another mt RNA binding protein. However, both have glycine-rich N-terminal regions, and TbRGG1 appears to interact via RNA with the MRB1 complex, which contains TbRRGm (Hashimi et al. 2008). In this study, we further characterized the MRB1 complex by repressing the expression of three of its associated proteins via RNAi, namely, TbRGGm (Tb10.406.0050); Tb927.6.1680, which has five C2H2 zinc finger motifs; and Tb11.02.5390, which has no known motifs. RNAi knockdowns of each of these resulted in growth inhibition, and abnormalities in the number and distribution of nuclei, and kDNA in PFs. These studies also indicate that TbRGGm, but not the other two proteins, is required for complex assembly and/or stability. Analyses using RNase treatment, or mutants, which lack substantial kDNA sequences, indicate that MRB1 complex binds kDNA transcripts. The knockdowns also differentially affected the in vivo abundances of edited, pre-edited, and preprocessed mt RNAs. Hence, the MRB1 complex has a role associated with the processing of RNAs transcribed from kDNA.

RESULTS

Association of MRB1 complex with kinetoplast transcripts

To determine whether MRB1 complex binds RNA, mt vesicles isolated from wild-type (wt) PF cells were lysed in the presence or absence of RNase A, and cleared lysates were fractionated on glycerol gradients. We monitored the complex by Western analysis of the fractions using mAb53, which is specific to Tb927.7.2570, a protein associated with the complex (Panigrahi et al. 2008). The complex has a fairly broad distribution in the gradient and sediments centered at ∼30S. Sedimentation was shifted to ∼20S upon RNase A treatment (Fig. 1A), indicating MRB1 complex is associated with RNA. Similar results were obtained when the same fractions were analyzed using mAb43 in native dot blots (Panigrahi et al. 2008; data not shown). The sedimentation profile of the editosomes in the same fractions was virtually unaffected by the RNase treatment; however, less overall signal was obtained, resulting in an apparent decrease in the amount of editosomes in fractions 11–15 (Fig. 1B).

FIGURE 1.

FIGURE 1.

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The MRB1 complex binds to kDNA transcripts. Western analysis of mt vesicles isolated from wt PF cells, lysed in the presence (+) or absence (−) of 0.1 mg/mL RNase A, and fractionated on 10%–30% glycerol gradients. Every other 30 μL fractions were probed with (A) mAb53, which is specific for the Tb912.7.2750 protein, or (B) a mix of mAbs against the KREPA1 and KREPA2 20S core editosome. Western analysis of glycerol gradients fractions from whole-cell lysates of (C) wt BF cells and T. evansi AnTat 3/3 Dk cells, or (D) BF RKO-KREN2 cells grown in the presence (KREN2 expressed) or absence (KREN2 repressed) of tet for 3 d, all examined with mAb53.

We also analyzed the sedimentation of MRB1 complex in whole-cell extracts from wt BF and from a naturally occurring Dk strain of T. brucei, Trypanosoma evansi AnTat 3/3, which lacks kDNA maxicircles and contains one minicircle sequence class rather than hundreds (Borst et al. 1987; Songa et al. 1990; Lai et al. 2008). Western analyses showed that the MRB1 complex from wt BF cells has a similar sedimentation profile as in PF cells. However, the signal with mAb53 is shifted to 5-10S in Dk cells (Fig. 1C), suggesting that MRB1 complex is normally associated with kDNA transcripts. The sedimentation profile of MRB1 complex was also evaluated using whole-cell extracts from BF cells in which RNA editing has been inhibited by repression of kinetoplastid RNA editing endonuclease 2 (KREN2) by conditional inactivation (Carnes et al. 2005). Western analysis of those glycerol gradient fractions (Carnes et al. 2005) with mAb53 showed that the MRB1 complex sedimentation profile was not altered in cells in which KREN2 expression was repressed (Fig. 1D). Thus, fully edited RNAs are not required for the presence of MRB1 complexes with high S values. Together, these data indicate that MRB1 complex is associated with kDNA transcripts. However, MRB1 complex may be specific for RNAs other than edited mRNAs, such as pre-edited, preprocessed mRNAs, mt rRNAs, or gRNAs, although perhaps not gRNAs alone since these are present in the T. evansi Dk strain.

Complex components are critical for in vitro growth of T. brucei

To evaluate whether MRB1 complex function is essential in T. brucei, expression of three of its associated proteins, TbRGGm (Tb10.406.0050, RGG motif), Tb927.6.1680 (C2H2 Zn finger motif), and Tb11.02.5390 (no known motifs), were knocked down in PF cells using tetracycline (tet)-inducible RNAi. Northern analyses of RNA prepared from noninduced cells and from RNAi-induced cells, collected at day 2 and day 4 post-induction, revealed nearly complete loss of their mRNAs within 2 d following RNAi induction (Fig. 2). Since no antibodies specific for TbRGGm, Tb927.6.1680, or Tb11.02.5390 are available, we could not directly assess their corresponding protein levels. Nevertheless, in each case repression of gene expression resulted in reduced cell growth by day 4 and lasted up to 10 d compared with noninduced cell lines (Fig. 2A–C). Knockdown of Tb11.02.5390 had less inhibition of growth compared with the other two. The growth defects resulting from the repression of any of the three of the components of MRB1 complex implies that they are essential proteins in PF T. brucei.

FIGURE 2.

FIGURE 2.

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TbRGGm, Tb927.6.1680, and Tb11.02.5390 are important for the in vitro growth of procyclic parasites. Growth curves of a representative RNAi cell lines of (A) TbRGGm, (B) Tb927.6.1680 (C2H2 Zn finger), and (C) Tb11.02.5390 (unknown function) grown in the presence (filled squares) or absence of tet (open squares). The cumulative cell numbers were calculated by multiplying the cell densities by the dilution factor. The insets show Northern analyses of the corresponding mRNAs with the days sampled indicated, and stained gels of rRNAs in the lower panel serving as loading controls.

Repression of TbRGGm affects complex integrity

The effect of repression of MRB1 proteins by RNAi on the integrity of the complex was examined by sedimentation in glycerol gradients. In TbRGGm-depleted cells, Western analyses of the glycerol fractions using mAb53 showed that by day 3 after RNAi induction a proportion of the complex shifted to a lower S value (Fig. 3A). By day 5, most of the Western signal was detected in fractions 1–7, indicating that TbRGGm is an essential component of the complex (Fig. 3A). Western analysis of the same glycerol gradient fractions with mAbs that are specific for KREPA1 and KREPA2, two editosome proteins, showed a slight decrease in the protein amount by day 5 but overall no change in sedimentation profile upon TbRGGm repression (Fig. 3A). Neither the amount nor the sedimentation of the MRB1 complex or editosomes was altered in cells in which Tb927.6.1680 (Fig. 3B) or Tb11.02.5390 (Fig. 3C) was repressed by RNAi. Similar results were obtained when the same fractions were analyzed using mAb43 in native dot blots (Panigrahi et al. 2008; data not shown). These data suggested that TbRGGm, but not the other two proteins, is important to the structural integrity or stability of the MRB1 complex. Moreover, the diversity in sedimentation may reflect differences in the relative amount of MRB1 proteins and their associations with RNA(s).

FIGURE 3.

FIGURE 3.

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TbRGGm is required for MRB1 complex assembly and/or stability. Western analysis of mt lysates from whole PF RNAi cells in which (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 were either expressed or repressed for 3 or 5 d as indicated. Glycerol gradients fractions were probed with either mAb53 or a mix of mAbs specific for KREPA1 and KREPA2 core proteins of 20S editosomes.

Repression of complex components alters the in vivo abundance of mitochondrial mRNAs

To further assess the in vivo consequences of RNAi knockdown of TbRGGm, Tb927.6.1680, and Tb11.02.5390, the abundance of seven edited and corresponding pre-edited mRNAs, as well as two never-edited and three preprocessed mRNAs, was examined by quantitative real-time PCR (qRT-PCR). The abundance of these RNAs in cells, in which RNAi was induced for 4 d, was determined relative to cells in which RNAi was not induced, as previously described using 18S rRNA and β-tubulin mRNA as two independent internal controls (Carnes et al. 2005). In most cases, the knockdowns resulted in a decline of edited mRNA levels and an increase of pre-edited mRNAs, while the levels of mRNAs that do not get edited were largely unchanged (Fig. 4). However, there were transcript specific differences among the different knockdowns. TbRGGm knockdown resulted in substantial declines, particularly of the extensively edited ND7, COIII, RPS12, and A6 mRNAs (Fig. 4A), consistent with the observation by Fisk et al. (2008). The knockdown of Tb927.6.1680 resulted in somewhat lower declines of ND7, COIII, and RPS12 edited mRNAs (Fig. 4B), while that of Tb11.02.5390 resulted in even lower declines of edited COIII and A6 edited mRNAs (Fig. 4C). The levels of some pre-edited mRNAs increased several fold, but there was no consistent correlation between the decrease of edited mRNAs and increase of their respective pre-edited mRNAs. However, the levels of all of the pre-edited mRNAs were increased in knockdown of Tb11.02.5390, which had the least effect on the edited mRNAs (Fig. 4C).

FIGURE 4.

FIGURE 4.

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Effects of repression of expression of TbRGGm, Tb927.6.1680, or Tb11.02.5390 on the in vivo abundance of mt RNAs. qRT- PCR analysis of total RNA isolated from PF cells, in which expression of (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 was repressed for 4 d by RNAi. For each target amplicon, the relative change in RNA abundance was determined by using either β-tubulin mRNA (black bars) or 18S rRNA (with bars) as an internal control. 1 indicates no change in relative amount of RNA, while bars above 1 indicate an increase and bars below 1 indicate a decrease in relative RNA amount on this log-scale graph. The assays were of pre-edited (P) and edited (E) NADH dehydrogenase subunit 7 (ND7), cytochrome oxidase subunits 2 (COII) and 3 (COIII), cytochrome reductase subunit b (Cyb), ribosomal protein S12 (RPS12), maxicircle unknown reading frame 2 (Murf2) and ATPase subunit 6 (A6) mRNAs, and the never-edited COI and ND4 mRNAs. The preprocessed regions linking 9S rRNA (9S) with ND8, Cyb with A6, and RPS12 with ND5 were also assayed.

Repression of TbRGGm and Tb11.02.5390 resulted in a relative increase of polycistronic preprocessed maxicircle transcripts as shown by qRT-PCR, using primers that flank the junctions of adjacent genes (Fig. 4A,C). These primers flank the junctions between 9S/ND8, Cyb/A6 and RPS12/ND5 in the preprocessed transcripts (see Materials and Methods). There was little or no change in the levels of these preprocessed mRNAs upon repression of Tb927.6.1680 expression (Fig. 4B). Thus, overall, knockdowns of TbRGGm, Tb927.6.1680, and Tb11.02.5390 expression differentially affected the levels of specific transcripts in RNA editing and processing.

Cytological effects

Cells in which expression of the MRB1 proteins had been knocked down by RNAi had cytological anomalies that were evident from fluorescence microscopic analysis of DAPI stained cells. Cells in which TbRGGm was knocked down displayed a variety of cells with an aberrant nuclear content. The two most dominant anomalies were cells with no nucleus and one kinetoplast (0N1K), i.e., zoids (Ploubidou et al. 1999), and cells with two nuclei and one kinetoplast (2N1K) (Fig. 5). These comprised up to 25% of the total cell population. Cells with more than two nuclei were also observed. Similar results were obtained with knockdowns of Tb927.6.1680 and Tb11.02.5390 (data not shown). FACS analyses showed that parasites with less than the haploid DNA content (i.e., <1C) were evident 3 d after RNAi knockdown of TbRGGm expression, and these accumulated in larger numbers as did cells with 4C DNA content by day 5 (Fig. 6A). The number of zoids observed by immunofluorescence accounted for the peak of <1C DNA content observed by flow cytometry. The pattern upon knockdown of Tb927.6.1680 or Tb11.02.5390 expression was similar to that with TbRGGm but was more moderate (Fig. 6B,C, respectively). Thus, loss of TbRGGm, Tb927.6.1680, or Tb11.02.5390 resulted in aberrant cytokinesis in PF T. brucei.

FIGURE 5.

FIGURE 5.

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Microscopy of TbRGGm-depleted procyclic T. brucei. Phase and DAPI Immunofluorescence micrographs of TbRGGm-depleted cells 5 d after induction of RNAi showing (A) the three major cell cycle stages and (B) representative images of abnormal TbRGGm depleted cells. DAPI staining of nuclear and kinetoplast DNA are blue. K Indicates kDNA; N, nucleus.

FIGURE 6.

FIGURE 6.

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FACS analysis of T. brucei cells following RNAi knockdown of (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 expression. The times following RNAi induction are indicated and the DNA amounts, where C is haploid amount DNA content, are indicated by the arrows over each peak of the flow cytometry profiles.

DISCUSSION

The results reported here indicate that MRB1 complex binds mt RNA in vivo, its function affects the cellular abundance of edited, unedited, and preprocessed mt RNAs, and it is critical for PF cell growth and survival. The RNA binding function, which was implied by the RNA binding, Zinc finger, RNA helicase, and RGG motifs in some of the MRB1-associated proteins, is supported by the substantial S value shifts after RNase treatment as well as analysis of a Dk mutant, which lacks most mt transcripts due to the absence of kDNA maxicircles. The dramatic effects on mt RNAs, upon knockdowns of expression of each of three of the MRB1-associated proteins, point to a role in mt RNA processing, but its specific function is currently unclear. Nevertheless, MRB1 complex function appears essential since knockdowns of expression of each of three of its associated proteins inhibit growth and result in cells with abnormal numbers of nuclei and kDNA networks, which must be nonviable. The loss of MRB1 complex function may result in a cascade of events that disable numerous mt processes, and result in the dramatic cellular phenotype observed.

MRB1 complex composition

The stable composition of MRB1 complex is incompletely understood, reflecting its recent discovery (Hashimi et al. 2008; Panigrahi et al. 2008), limited study, and the few available tools. Complexes isolated using either mAbs or TAP-tag identified up to 16 associated proteins. The complex that was monitored using mAb53 sediments at ∼30S when derived from enriched PF mt or wt BF cells, at ∼20S upon RNase treatment, and at 5-10S when from Dk BF cells. Hence, mt RNA is associated with MRB1 complex, and some proteins may be associated with MRB1 complex through interaction with RNA. TbRGGm has been shown to bind to RNA in vitro with a preference for poly (U) (Fisk et al. 2008). Therefore, the shift of the MRB1 complex to a lower S value, in RNase-treated cells, may reflect the loss of interaction between TbRGGm and RNA. The sedimentation profiles of MRB1 from RNase-treated lysates and from TbRGGm lysates of RNAi induced cells are similar, suggesting that the interaction between TbRGGm and MRB1 complex is mediated by RNA.

The lack of an effect on MRB1 complex sedimentation upon RNAi knockdowns of Tb927.6.1680 or Tb11.02.5390 implies that they are not essential to the structural integrity of the complex identified by the mAb53. This resembles the situation for some editosome proteins (Carnes et al. 2005, 2008; Trotter et al. 2005). Alternatively, TbRGGm may be a core protein in the MRB1 complex, while the two other proteins may be “peripherally” or transiently associated with the core complex via protein–protein and/or protein–RNA interactions. However, while their knockdowns were substantial, they were incomplete. Thus, we cannot exclude the possibility that their protein levels may not have been sufficiently limiting to affect MRB1 complex integrity. Nevertheless, the similar effects of their RNAi knockdowns to that of TbRGGm indicate that they have a functional, if not physical, association with the MRB1 complex. Additionally, TbRGGm was identified in complexes isolated via TAP-tagged TbRGG1 (Hashimi et al. 2008), and TbRGG1 plus five MRB1-associated proteins (Tb927.7.2570, Tb927.3.3800, Tb927.4.4150, Tb927.1.3010, and Tb11.02.5390) were identified in mt polyadenylation complexes isolated via TAP-tagged KPAP1 (Etheridge et al. 2008). While RNAi knockdown studies showed that TbRGG1 and KPAP1 are critical for PF cell growth and that repression of their expression affects the levels of in vivo edited mRNAs, we have not detected TbRGG1 or KPAP1 in TAP-tagged or mAb precipitated MRB1 complexes. These protein associations may reflect RNA binding by these proteins, which occurs during complex purification rather than in vivo functional associations. Alternatively, they may reflect dynamic interactions among proteins and the different complexes that perform mt RNA processing.

Cell viability

MRB1 complex function is essential, as demonstrated by the severe growth and cytological defects resulting from repression of TbRGGm, Tb927.6.1680, or Tb11.02.5390 expression. In a recent study, TbRGGm has been reported to be critical for viability of both PF and BF cells (Fisk et al. 2008). The effects on edited RNA are sufficient to account for the growth and viability effects. The generation of zoids, binucleate cells, as well as the appearance of a small proportion of multinucleate cells implies blockage of nuclear segregation during cytokinesis. It seems unlikely that the function of a mt complex such as MRB1 complex would be directly involved in the processes of nuclear segregation. Similar cytological phenotypes result from blocking various processes. These include knockdowns of a mitotic cyclin or subunits of the transcription factor IIH (TFIIH) homolog in PF T. brucei (Hammarton et al. 2003; Tu et al. 2006; Lecordier et al. 2007). Repression of a PHO80 homolog (CycE1/CYC2) results in cell cycle arrest at the G1/S checkpoint and generates slender zoids of T. brucei, while repression of a B-type cyclin homolog (CycB2) results in cell cycle arrest at the G2/M transition and generates stumpy zoids (Li and Wang 2003). Zoids are also generated in many cells upon inhibition of DNA replication or microtubule function (Robinson et al. 1995; Ploubidou et al. 1999). It seems that loss of editing, as a result of MRB1 complex function, minimally compromises mt ATP synthesis with consequent pleiotropic effects on other mt and cellular processes, including nuclear segregation. However, the coordination of nuclear and kDNA replication and the physical proximity of the kinetoplast with the flagellar basal body, which has a key role in cytokinesis in T. brucei (Kohl and Bastin 2005), leaves open the possibility that MRB1 complex function may have a more direct role in nuclear segregation. The functions of the individual MRB1 proteins are unknown, and while they are in the mitochondria, all are encoded in nuclear DNA and translated in the cytoplasm, and dual cellular locations cannot be excluded.

Association with RNA

The broad distribution of MRB1 complex in the gradients may reflect its association with various sizes and types of RNA and/or variable protein composition, due to proteins associating and dissociating with the complex. The lower S value of MRB1 complex from Dk mutants compared to RNase treatment may be due to differences in protein content or bound RNA. The Dk mutants lack maxicircle transcripts, and hence the mRNA/gRNA duplexes, which normally form during editing. Thus, the MRB1 complexes from Dk mutants may lack bound cellular RNA, possibly due to the absence of a substrate for the RNA binding domain of TbRGGm. In contrast, mt RNA is associated with MRB1 complex from wt cells, and some RNA may be protected from removal by RNase. Alternatively, the MRB1 complexes from Dk cells may lack some proteins since kDNA is no longer required (Schnaufer et al. 2002), thus likely removing selective pressure for retention of MRB1 complex function.

Function in RNA processing

Despite the effect on the levels of edited RNAs, MRB1 complex does not appear to be stably associated with editosomes. The editosomes do not shift in S value upon TbRGGm knockdown or upon RNase treatment or in Dk cells (Domingo et al. 2003), whereas all affect the S value of the MRB1 complex. Reciprocally, inactivation of editing by conditional activation of KREN2 expression had no effect on MRB1 complex sedimentation. Thus, the MRB1 complex is not essential for RNA editing per se, and the effect on RNA editing is likely to be indirect. One of its components, TbRGGm, has been proposed to be an editing accessory factor (Fisk et al. 2008). However, the variable effects of knockdowns of the MRB1 components on different edited mRNAs, pre-mRNAs, and preprocessed transcripts as seen in this study imply that MRB1 complex may have functions that precede RNA editing, such as those associated with precursor processing or RNA trafficking.

The basis for the differential effects on the various edited, pre-edited, and preprocessed transcripts, together with the common lack of an effect on never-edited mRNAs, is unclear. Perhaps, the simplest possibility is that MRB1 complex plays a chaperone-like role associated with the processing of the kDNA transcripts. This would account for the effects on the three different types of RNA and perhaps for the preferential effect on extensively edited mRNAs and the accumulation of some pre-edited and preprocessed precursor RNAs. Transcription of maxicircle genes and minicircle gRNAs creates polycistronic RNAs, which are subsequently processed to form the mature rRNAs, pre-mRNAs, and gRNAs (Michelotti et al. 1992; Read et al. 1992). Differences in the levels of RNAi knockdowns and, possibly, differences in protein levels as well as function of specific proteins, and stoichiometry in the complex, may account for the differences seen among TbRGGm, Tb927.6.1680, and Tb11.02.5390 RNAi knockdowns. Alternatively, since mt mRNAs are differentially edited between the life cycle stages (Stuart et al. 1997; Schnaufer et al. 2002), MRB1 proteins may be involved in this regulated development process. Hence, individual MRB1 proteins may have distinct roles on specific transcripts.

MATERIALS AND METHODS

Trypanosome cell growth

PF T. brucei strain 427 was grown in vitro at 27°C in SDM-79 medium supplemented with 10% FBS and hemin (7.5 mg/mL). PF T. brucei strain 29.13 (Wirtz et al. 1999), which contains integrated genes for T7 polymerase and the tet repressor, was grown in the presence of G418 (15 μg/mL) and hygromycin (25 μg/mL). The cells were harvested at a density of 1–2 × 107 cells/mL. T. brucei BF cells strain 427 and the naturally occurring Dk strain T. evansi AnTat 3/3 were cultured in vitro at 37°C in HM1-9 medium supplemented with 10% FBS. The cells were harvested at a density of 1–2 × 106 cells/mL.

Glycerol gradient fractionation

The mt vesicles were isolated from PF cells as described (Harris et al. 1990; Panigrahi et al. 2008). The enriched mitochondria were lysed in 1 mL of the lysis buffer (10 mM Tris at pH 7.2; 10 mM MgCl2; 100 mM KCl; 1 mM DTT; 1 μg/mL pepstatin; 2 μg/mL leupeptin; 1 mM pefabloc) containing 1% Triton X-100 for 15 min at 4°C. Samples that were RNase A–treated were incubated with 0.1 mg/mL RNase A (Sigma) during lysis. The mt lysate was clarified by centrifugation at full speed in a microcentrifuge for 30 min at 4°C. The cleared supernatant was collected and fractionated on 10%–30% glycerol gradients at 38,000 rpm for 5h at 4°C (SW40 Ti rotor, Beckman), and fractions were collected from the top (Panigrahi et al. 2001).

Immunoblotting

Alternate glycerol gradient fractions (30 μL) were resolved by 10% SDS-PAGE (Bio-Rad) and transferred onto a methanol-treated PVDF membrane. Western blots were performed as described previously (Panigrahi et al. 2001, 2008) with a 1:25 dilution of the mAb KREPA1, a 1:50 dilution of the mAb KREPA2, or a 1:25 dilution of the mAb53 followed by a 1:2000 dilution of goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad). The ECL enhanced chemiluminescence (Pierce) was used to visualize antigens.

Plasmid constructs, transfections, and induction

RNAi constructs were prepared in the pZJM vector, which harbors opposing tet-inducible T7 promoters flanking the cloning site (Wang et al. 2000; Morris et al. 2001). As inserts we used the sequences of the following ORFs (nucleotides indicated in parentheses): TbRGGm, Tb10.406.0050 (202–789), Tb927.6.1680 (421–1158), Tb11.02.5390 (898–1879). TbRGGm ORF was PCR amplified from genomic DNA of T. brucei strain Lister 427 using the sense primer (5′-ATACTCGAGGGCTGGGGCTCTGGTGGA-3′) and antisense primer (5′-ACAAAGCTTCTCCCTATCCTCCCGCAGA-3′). Similarly, the Tb927.6.1680 ORF was amplified with the sense primer (5′-ATACTCGAGCGCGTCCTGGATTTTTTGCC-3′) and antisense primer (5′-ACAAAGCTTAAAGTGTCCATCCAATGCAGC-3′), and the Tb11.02.5390 ORF was amplified with the sense primer (5′-ATACTCGAGGTGCGCAATACTCTAGGCCA-3′) and antisense primer (5′-ACAAAGCTTTCCTGACGCAGAGCAATCC-3′). The restriction enzyme sites XhoI and HindIII, which were used for subsequent cloning, are underlined. The resulting PCR product was cloned into pZJM plasmid.

RNAi cell lines were generated by transfection with 10 μg of NotI-linearized pZJM construct as described (Schnaufer et al. 2001). Three independent clones were selected and growth curves were generated in the absence or presence of 1 μg/mL tet, which induces expression of double-stranded RNA (dsRNA). The induced and noninduced cultures were maintained between 2 × 106 and 2 × 107 cells/mL, and cell density was monitored daily using a particle counter (Beckman).

Northern blot hybridization

Total RNA was isolated from 1 × 108 exponentially growing noninduced and RNAi-induced cells by extraction with TRIzol reagent (Invitrogen) as described by the manufacturer. The α-32P dCTP-labeled probes were prepared by random priming of the PCR products used as inserts in the pZJM vector. Electrophoresis, blotting, and hybridization were as described by Maniatis et al. (1982). Signal was visualized using Molecular Dynamics PhosphorImager screens.

Quantitative real-time PCR

qRT-PCR was carried out to assess pre-edited, edited, and never-edited mRNA levels as described (Carnes and Stuart 2007). The primer pairs used to detect mt transcripts were described previously (Carnes et al. 2005), as are those amplifying 18S rRNA and β-tubulin cDNAs, which were used as reference genes. Primers that flank the junctions of 9S-ND8, Cyb-A6, and RPS12-ND5 adjacent genes are as follows: primers complementary to the 3′end of 9S (5′-AAAAGGTATTGTTGCCCACCAA-3′) and to the 5′end of ND8 (5′-CAACCAAAACTTAAAATTATTAAATTGATTC-3′), primers complementary to the 3′end of Cyb (5′-CCAATATGAATGGAATTACAATACTGAGT-3′) and to the 5′end of A6 (5′-TCCGCCCAAAATTCCTCTTT-3′), and primers complementary to the 3′end of RPS12 (5′-GGGAACCCTTTGTTTTGGTTAAAG-3′) and to the 5′end of ND5 (5′-TTCCTACCAAACATAAATGAACCTGAT-3′). The average of three cycle threshold (Ct) values for each target was used in calculations. Analysis was carried out by using the Pfaffl method, with PCR efficiencies calculated by linear regression with LinRegPCR software (Pfaffl 2001; Ramakers et al. 2003). Data were normalized to 18S rRNA and β-tubulin and relative changes in mRNA abundance after RNAi induction were expressed as fold-changes relative to noninduced control cells.

Immunofluorescence microscopy

For 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, ∼5 × 105 noninduced and RNAi-induced cells were pelleted by centrifugation, washed, fixed with 4% formaldehyde, and treated with 50 μL of 1 μg/mL DAPI to visualize DNA. Phase contrast image of the cells and their fluorescence was captured with a Nikon fluorescence microscope equipped with camera and the appropriate filters.

Fluorescence-activated cell sorter analysis

For fluorescence-activated cell sorter (FACS) analysis, ∼1 × 107 noninduced and RNAi-induced cells were suspended in 70% methanol, 30% PBS and left at 4°C for 8 d. Cells were washed once and resuspended in PBS supplemented with 20 μg/mL RNase A and 50 μg/mL propidium iodide and incubated for 30 min at 37°C. FACS was performed with a Beckman Counter Epics XL-MCL flow cytometer, and FL3-A fluorescence was recorded on 50,000 events gated according to forward and side scatter and analyzed using FlowJo software.

ACKNOWLEDGMENTS

We thank the members of the Stuart laboratory for their helpful advice and discussions. This research was conducted using equipment made possible by the Economic Development Administration–U.S. Department of Commerce and the M.J. Murdock Charitable Trust. This work was supported by NIH grant AI065935 to K.D.S.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1353209.

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