No abstract

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth inhibition. It also resulted in the loss of both proteins, even when only one of the MRP mRNAs was reduced, indicating a mutual dependence for stability. Elimination of the MRPs gave rise to substantially reduced levels of edited CyB and RPS12 mRNAs but little or no reduction of the level of edited Cox2, Cox3, and A6 mRNAs as measured by poisoned primer extension analyses. In contrast, edited NADH-dehydrogenase (ND) subunit 7 mRNA was increased 5-fold in MRP1؉2 double knockdown cells. Furthermore, MRP elimination resulted in reduced levels of Cox1, ND4, and ND5 mRNAs, which are never edited, whereas mitoribosomal 12 S rRNA levels were not affected. These data indicate that MRP1 and MRP2 are not essential for RNA editing per se but, rather, play a regulatory role in the editing of specific transcripts and other RNA processing activities.Kinetoplastida are early diverged flagellates that differ from other eukaryotes by a number of features. They contain a remarkable single mitochondrion, within which is a large mass of circular DNA molecules that are intercatenated in a unique arrangement (1). Moreover, their mitochondrial RNA processing is also highly unusual. The majority of mitochondrial mRNAs are extensively changed by RNA editing, which is the extensive insertion and less frequent deletion of uridines (Us) at multiple sites. Small guide RNA (gRNA) 1 molecules direct the pattern of U insertions and deletions by base pairing between the pre-edited mRNA and gRNA. The editing process occurs via a series of "cut-and-paste" steps, and several of the enzymes that catalyze this process, including RNA ligases and terminal uridylyl transferases, have now been identified (for recent reviews see Refs. 2-5).

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid ␤-oxidation pathway. Here, we asked whether fatty acid ␤-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5⌬/pex5⌬ mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2⌬/ fox2⌬ mutant, which lacks the second enzyme of the ␤-oxidation pathway. Both mutant strains had strongly reduced ␤-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2⌬/fox2⌬ mutant, and not the pex5⌬/pex5⌬ mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2⌬/fox2⌬ mutant was comparable to that of the icl1⌬/icl1⌬ mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid ␤-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2⌬/fox2⌬ mutant is largely due to a dysfunctional glyoxylate cycle.

In mitochondria of Kinetoplastida belonging to the suborder Trypanosomatina, the nucleotide sequence of transcripts is post‐transcriptionally edited via insertion and deletion of uridylate residues. In order to shed more light on the evolutionary history of this process we have searched for editing in mitochondrial RNAs of Trypanoplasma borreli, an organism belonging to the suborder Bodonina. We have cloned and sequenced a 5.3 kb fragment derived from a 37 kb mitochondrial DNA molecule which does not appear to be a part of a network structure and have found genes encoding cytochrome c oxidase (cox) subunit 1, cox 2 and apocytochrome (cyt) b, and genes encoding the small and large subunit mitoribosomal RNAs. The order in which these genes occur is completely different from that of trypanosomatid maxicircle genes. The 5′ and 3′ termini of both the cytb and cox1 gene are cryptic, the protein coding sequences being created by extensive insertion/deletion of Us in the corresponding mRNA sections. Phylogenetic analyses of the protein and ribosomal RNA sequences demonstrated that the separation between T.borreli and Trypanosomatina was an early event, implying that U‐insertion/deletion processes are ancient. Different patterns of editing have persisted in different lineages, however, since editing of cox1 RNA and of relatively small 3′‐terminal RNA sections is not found in trypanosomatids. In contrast, cox2 RNA which is edited in trypanosomatids by the insertion of four Us, is unedited in T.borreli.

The glyoxylate cycle, a metabolic pathway required for generating C 4 units from C 2 compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5D/D strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2D/D strain that is completely deficient in fatty acid boxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2D/D strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2D/D strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2D/D mutant.

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid ␤-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C 2 carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid ␤-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid ␤-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

We have determined the nucleotide sequence of a maxi-circle segment from the insect trypanosome Crithidia fasciculata mitochondrial DNA, on which the genes for the major maxicircle transcripts of 9S and 12S are localized. The 5'-terminal sequences of these RNAs were determined by wandering spot analysis. The map coordinates of the 9S and 12S RNAs from Trypanosoma brucei were adjusted with respect to a previous report with the aid of primer extension analysis with reverse transcriptase. This approach allowed us to align the corresponding genes from both organisms which show an overall sequence homology of 77%. The 9S and 12S RNA genes from the two trypanosome species contain sequences, closely related to some of the regions that are universally conserved among ribosomal RNAs from members of the three primary kingdoms and their organelles, even though the overall level of sequence homology is extremely low. These universal sequences occur at positions in the 9S and 12S RNAs that are analogous to those occupied by their counterparts in authentic ribosomal RNAs. The characteristic secondary structure elements flanking these universal sequences in genuine ribosomal RNAs can also be formed in the trypanosomal 9S and 12S RNAs. These results provide unequivocal evidence for a ribosomal function of the 9S and 12S RNAs of trypanosomal mitochondria, notwithstanding their extremely small size (estimated to be 612 and 1141 nucleotides in C. fasciculata, 611 and 1150 nucleotides in T. brucei) and their unusual base composition (83% A+U).