The Yeast Mitochondrial Degradosome

100

In plant mitochondria, transcription proceeds well beyond the region that will become mature 3 extremities of mRNAs, and the mechanisms of 3 maturation are largely unknown. Here, we show the involvement of two exoribonucleases, AtmtPNPase and AtmtRNaseII, in the 3 processing of atp9 mRNAs in Arabidopsis thaliana mitochondria. Down-regulation of AtmtPNPase results in the accumulation of pretranscripts of several times the size of mature atp9 mRNAs, indicating that 3 processing of these transcripts is performed mainly exonucleolytically by AtmtPNPase. This enzyme is however not sufficient to completely process atp9 mRNAs, because with down-regulation of another mitochondrial exoribonuclease, AtmtRNaseII, about half of atp9 transcripts exhibit short 3 nucleotide extensions compared with mature mRNAs. These short extensions can be efficiently removed by AtmtRNaseII in vitro. Taken together, these results show that 3 processing of atp9 mRNAs in Arabidopsis mitochondria is, at least, a twostep phenomenon. First, AtmtPNPase is involved in removing 3 extensions that may reach several kilobases. Second, AtmtRNaseII degrades short nucleotidic extensions to generate the mature 3 -ends.Functional RNAs are generated through a series of complex post-transcriptional processes. The nature of these processes can vary depending on the organism or cellular compartment considered and may include maturation of 5Ј and 3Ј termini, splicing of introns, capping, editing, base modifications, or addition of nongenomically-encoded nucleotides such as polyadenylation. Processing of mRNAs is well documented for bacterial and eukaryotic nuclear mRNAs (1, 2). Our knowledge of maturation of plastid transcripts is also extensive for both Chlamydomonas and higher plants (3). However, mRNA maturation processes in plant mitochondria are far less well characterized. Two lines of evidence suggest that processing of 3Ј extremities occurs for any RNA in plant mitochondria. First, run-on experiments have demonstrated that transcription proceeds beyond the region that corresponds to mature 3Ј-ends of mRNAs and rRNAs (4). Second, it has been shown using an in vitro transcription system that inverted repeats that terminate some mitochondrial mRNAs are not recognized as transcription termination signals (5). These inverted repeats seem to be essential as processing signals in vitro (5) and in vivo (6, 7). However, the ribonucleases involved in 3Ј mRNA maturation processes remain to be identified in plant mitochondria.Escherichia coli represents one of the model organisms for which the function, mechanism, and regulation of ribonucleases (RNases) are intensively studied (for example, see Refs. 1, 8, and 9). Among the eight characterized exoribonucleases in E. coli, polynucleotide phosphorylase (PNPase) 1 and RNaseII are the main exoribonucleases involved in degrading mRNAs in a 3Ј to 5Ј direction but participate also in processing events. PNPase-like proteins are also present in human mitochondria (10) and in chloroplasts. In the latter, it is involved both ...

“…In yeast mutants lacking degradosome components, several molecular phenotypes have been described, including the accumulation of mRNA precursors (Ref. 15 and references therein).…”

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confidence: 99%

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confidence: 99%

Two Exoribonucleases Act Sequentially to Process Mature 3′-Ends of atp9 mRNAs in Arabidopsis Mitochondria

Perrin

Meyer

Zaepfel

et al. 2004

100

“…Thus the degradosome appears to play a significant role in RNA turnover and processing. Indeed, the purified degradosome functions in a stepwise fashion in the presence of a partially duplexed RNA: first, the duplex is made single stranded in an NTP-and Suv3p-dependent step followed by degradation (11).…”

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confidence: 99%

“…exon reopening reactions), which decreases expression (9). Degradation of excised group I introns is carried out by a two-protein complex called the mitochondrial degradosome or mtEXO (10,11). The mitochondrial degradosome includes Dss1p, a 3Ј 3 5Ј single-stranded exonuclease, and Suv3p, a DEXH/D protein, one of the largest family of proteins that are involved in RNA metabolism (11)(12)(13).…”

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confidence: 99%

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

Turk

Caprara

2010

Yeast Suv3p is a member of the DEXH/D box family of RNA helicases and is a critical component of the mitochondrial degradosome, which also includes a 3 3 5 exonuclease, Dss1p. Defects in the degradosome result in accumulation of aberrant transcripts, unprocessed transcripts, and excised group I introns. In addition, defects in SUV3 result in decreased splicing of the aI5␤ and bI3 group I introns. Whereas a role for Suv3p in RNA degradation is well established, the function of Suv3p in splicing of group I introns has remained elusive. It has been particularly challenging to determine if Suv3p effects group I intron splicing through RNA degradation as part of the degradosome, or has a direct role in splicing as a chaperone, because nearly all perturbations of SUV3 or DSS1 result in loss of the mitochondrial genome. Here we utilized the suv3-1 allele, which is defective in RNA metabolism and yet maintains a stable mitochondrial genome, to investigate the role of Suv3p in splicing of the aI5␤ group I intron. We provide genetic evidence that Mrs1p is a limiting cofactor for aI5␤ splicing, and this evidence also suggests that Suv3p activity is required to recycle the excised aI5␤ ribonucleoprotein. We also show that Suv3p acts indirectly as a component of the degradosome to promote aI5␤ splicing. We present a model whereby defects in Suv3p result in accumulation of stable, excised group I intron ribonucleoproteins, which result in sequestration of Mrs1p, and a concomitant reduction in splicing of aI5␤.Mitochondrial gene expression is largely controlled by posttranscriptional mechanisms because of the relatively simple nature of mitochondrial transcription (1-5). One critical control point is RNA degradation, which is involved in the extensive processing required of mitochondrial transcripts (6, 7). In Saccharomyces cerevisiae, intergenic regions are removed from polycistronic transcripts and in the case of two mRNAs, encoding the cytochrome oxidase I subunit (COX1) 2 and apocytochrome b subunit (COB), group I and group II catalytic introns are also removed. Splicing of these introns requires trans-acting factors to facilitate the RNA-catalyzed intron excision and ligation of flanking exons (8).Rapid turnover of excised group I introns is essential for mitochondrial gene expression because these RNAs act as endonucleases that cleave their parent, processed RNAs at the site of exon ligation (e.g. exon reopening reactions), which decreases expression (9). Degradation of excised group I introns is carried out by a two-protein complex called the mitochondrial degradosome or mtEXO (10, 11). The mitochondrial degradosome includes Dss1p, a 3Ј 3 5Ј single-stranded exonuclease, and Suv3p, a DEXH/D protein, one of the largest family of proteins that are involved in RNA metabolism (11-13). Indeed, some members show ATP-dependent unwinding of RNA helices. Suv3p is a member of the SKI2 subfamily, individuals of which are thought to couple ATP binding and hydrolysis cycles with translocation along an RNA single strand and disruption...

“…Mss116 has RNA helicase activity (50) and is required for splicing of all group I and II mitochondrial introns (12). The helicase Suv3 functions in the degradosome to remove spliced introns and in the process release limiting splicing factors, but has an additional role in aI5␤ processing (51).…”

Section: Discussionmentioning

confidence: 99%

Mne1 Is a Novel Component of the Mitochondrial Splicing Apparatus Responsible for Processing of a COX1 Group I Intron in Yeast

Watts

Khalimonchuk

Wolf

et al. 2011

Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc 1 complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5␤ intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5␤ intron is similarly impaired in mne1⌬ and mrs1⌬ cells and overexpression of Mrs1 partially restores the respiratory function of mne1⌬ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1⌬ cells is splicing of the aI5␤ intron in COX1.Cytochrome c oxidase (CcO) 3 biogenesis requires the expression and interaction of subunits encoded by mitochondrial and nuclear genomes. A myriad of nuclear-encoded assembly factors mediate CcO biogenesis (1, 2). These factors function in the processing of mitochondrial CcO subunit mRNAs, their translation and insertion into the inner membrane, and formation of metal and heme cofactor centers. Assembly is initiated with the synthesis and maturation of the Cox1 subunit, one of the three catalytic core components (3, 4). COX1 is the one CcO gene that is universally present in the mitochondrial genome. The COX1 mRNA requires processing prior to translation on mitochondrial ribosomes. Cox1 contains three of the redox centers of the enzyme with heme a, a 3 , and Cu B cofactors (5). The last redox center, the binuclear Cu A center, exists within the Cox2 subunit.Mitochondrial genomes of non-metazoan species tend to be larger due to the presence of non-coding regions including introns and additional genes not present in animals (6 -8). In Saccharomyces cerevisiae, introns are commonly found in COX1 as well as COB (the cytochrome b subunit of the bc 1 complex) and the large ribosomal RNA gene (9). Two types of introns, groups I and II, exist in COX1. Some of these introns contain open reading frames encoding maturases, related to DNA endonucleases for group I introns and reverse transcriptases for group II introns (8). Group I intron-containing COX1 alleles have been generated numerous times during evolution due to the invasive nature of the introns containing a DNA homing endonuclease (10). Most commonly used laboratory yeast strains contain up to seven introns within COX1 and in such multiple-intron strains, exons may be as short as 24 -37 bases. No compelling rationale is known why introns are maintained in COX1 and COB.The generation of mature COX1 mRNA transcript requir...