The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine-subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R-like enzyme, which possesses both processive exo-and endonuclease activities, whereas the latter is a distributive RNase D-like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs-hDIS3 and hDIS3L-with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.
We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.
RNA decay is usually mediated by protein complexes and can occur in specific foci such as P-bodies in the cytoplasm of eukaryotes. In human mitochondria nothing is known about the spatial organization of the RNA decay machinery, and the ribonuclease responsible for RNA degradation has not been identified. We demonstrate that silencing of human polynucleotide phosphorylase (PNPase) causes accumulation of RNA decay intermediates and increases the half-life of mitochondrial transcripts. A combination of fluorescence lifetime imaging microscopy with Förster resonance energy transfer and bimolecular fluorescence complementation (BiFC) experiments prove that PNPase and hSuv3 helicase (Suv3, hSuv3p and SUPV3L1) form the RNA-degrading complex in vivo in human mitochondria. This complex, referred to as the degradosome, is formed only in specific foci (named D-foci), which co-localize with mitochondrial RNA and nucleoids. Notably, interaction between PNPase and hSuv3 is essential for efficient mitochondrial RNA degradation. This provides indirect evidence that degradosome-dependent mitochondrial RNA decay takes place in foci.
Accurate tRNA processing is crucial for human mitochondrial genome expression, but the mechanisms of mt-tRNA cleavage and the key enzymes involved in this process are poorly characterized. At least two activities are required for proper mt-tRNA maturation: RNase P cleaving precursor molecules at the 5' end and tRNase Z at the 3' end. In human mitochondria only RNase P has been identified so far. Using RT-PCR and northern blot analyses we found that silencing of the human ELAC2 gene results in impaired 3' end of mt-tRNAs. We demonstrate this for several mitochondrial tRNAs, encoded on both mtDNA strands, including tRNA (Val) , tRNA (Lys) , tRNA (Arg) , tRNA (Gly) , tRNA (Leu(UUR)) and tRNA (Glu) . The silencing of the MRPP1 gene that encodes a subunit of mtRNase P resulted in inhibition of both 5' and 3' processing. We also demonstrate the double mitochondrial/nuclear localization of the ELAC2 protein using immunofluorescence. Our results indicate that ELAC2 functions as a tRNase Z in human mitochondria and suggest that mt-tRNase Z preferentially cleaves molecules already processed by the proteinaceous mtRNase P.
The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3′-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.
The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in human mitochondria. In vitro reconstitution experiments show that GRSF1 promotes G4 melting that facilitates degradosome-mediated decay. Among degradosome and GRSF1 regulated transcripts we identified one that undergoes post-transcriptional modification. We show that GRSF1 proteins form a distinct qRRM group found only in vertebrates. The appearance of GRSF1 coincided with changes in the mitochondrial genome, which allows the emergence of G4-containing RNAs. We propose that GRSF1 appearance is an evolutionary adaptation enabling control of G4 RNA.
Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclearmitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G -* T transversion that creates a Val --Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.The formation of the 3' ends of most yeast mitochondrial mRNAs occurs by posttranscriptional processing at a highly conserved dodecamer sequence (1-3). The requirements for processing at a dodecamer to generate a functional mRNA became apparent with the discovery of a mutant, PZ206, in which the dodecamer and 195 base pairs (bp) of flanking 3' noncoding sequences at the end of the varl gene were deleted (4, 5). This deletion resulted in the formation of varl transcripts with aberrant 3' ends such that those RNAs were not functional for translation of the varl protein. Since varl is required for assembly of the 37S mitochondrial ribosomal subunit (6, 7), PZ206 cells are respiratory-deficient and thus unable to grow on glycerol.This laboratory has described (8, 9) the properties of a dominant suppressor allele (SUV3-J) of a nuclear gene (suv3) that allows the aberrant var1 transcripts in PZ206 to be translated, restoring respiratory function to those cells. In those studies, it was found, unexpectedly, that in cells containing a wild-type mitochondrial genome, the suppressor SUV3-1 allele has pleiotropic effects on mitochondrial RNA metabolism. For example, there is a dramatic overaccumulation of the excised w intron in SUV3-1 cells and an overaccumulation of six of seven other excised group I introns tested; most of these other excised introns are undetectable in wild-type (suv3) cells. Analysis of mRNA abundance in SUV3-1 cells shows that it also causes a 2-to 5-fold reduction in the mRNA levels of the COB gene, and a 20-fold reduction in the levels of COXI mRNA; the lowered abundance of the COXI mRNA is due to a decrease in the splicing of COXI intron Sf. Despite the reduction in the abundance of those mRNAs, protein labeling experiments indicate that the amounts of cytochrome b and coxI proteins are about t...
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