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.