Significance Mitochondria generate signals that regulate nuclear gene expression via retrograde signaling, but this phenomenon is rendered more complex by the quantitative differences in the percentage of mutant and normal mtDNAs that can exist within patient cells. This study demonstrates that depending upon its relative cytoplasmic levels, a single mtDNA point mutation can cause a discrete set of cellular transcriptional responses within cells of the same nuclear background. This qualitative regulation of nuclear gene expression by quantitative changes in mtDNA mutant levels challenges the traditional “single mutation–single disease” concept and provides an alternative perspective on the molecular basis of complex metabolic and degenerative diseases, cancer, and aging.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long-standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.Trypanosome RNA editing is an unprecedented form of transcript maturation that involves many cycles of posttranscriptional deletion and insertion of uridylate (U) residues to generate essential mitochondrial mRNAs (reviewed in references 24, 43, and 47). These editing events can occur at over half the phosphodiester bonds of a pre-mRNA's coding region (15), creating over three-quarters of the codons, including translation start and stop signals. Short, trans-acting guide RNAs (gRNAs), which are complementary to mature mRNA sequence, direct the editing via mismatches in base pairing with the pre-mRNA (5). Furthermore, individual U deletion and U insertion cycles can be reproduced in vitro using a synthetic pre-mRNA segment, its cognate gRNA, and Trypanosoma brucei extract (23, 42), such as the "traditional" extract (18, 38) prepared from Percoll-enriched mitochondria.Editing cycles involve pre-mRNA cleavage, then terminal U removal or U addition, and finally rejoining of the pre-mRNA fragments (Fig. 1A) (10, 23, 35, 41). The pre-mRNA is cleaved just upstream of an anchor duplex which forms where the pre-mRNA base pairs with the 5Ј region of a gRNA. U deletion occurs when the pre-mRNA residue just upstream of this duplex is a U, and U insertion occurs when it is not a U (Fig. 1A). Notably, an adenosine di-or triphosphate is required for cleavage at U deletion sites but is inhibitory for cleavage at U insertion sites (nonhydrolyzable AMP-CP is convenient to use for in vitro studies) (8). Next, at the 3Ј end crea...

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patient's mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA-encoded MWFE polypeptide has been shown to interact with various mtDNA-encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.

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In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an ϳ20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the ϳ20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as ϳ10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the ϳ20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.