Piotr P. Stepien

The Yeast Mitochondrial Degradosome ITS COMPOSITION, INTERPLAY BETWEEN RNA HELICASE AND RNase ACTIVITIES AND THE ROLE IN MITOCHONDRIAL RNA METABOLISM

The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism. Previous purifications suggested that it was composed of three subunits. Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes. We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3′- and 5′-ends. The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing. We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.

Involvement of human ELAC2 gene product in 3' end processing of mitochondrial tRNAs

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 tRNAVal, tRNALys, tRNAArg, tRNAGly, tRNALeu(UUR) and tRNAGlu. 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.

Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

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.

Human Polynucleotide Phosphorylase, hPNPase, is Localized in Mitochondria

The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation. Here, we demonstrate that the hPNPase is localized in mitochondria. Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.

Human mitochondrial RNA decay mediated by PNPase–hSuv3 complex takes place in distinct foci

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.

Overexpressed yeast mitochondrial putative RNA helicase Mss116 partially restores proper mtRNA metabolism in strains lacking the Suv3 mtRNA helicase.

RNA helicase, encoded by the Saccharomyces cerevisiae nuclear gene SUV3, is a subunit of the mitochondrial (mt) degradosome: an enzyme complex that takes part in turnover of mtRNAs. Deletion of the SUV3 gene leads to a variety of disturbances in mtRNA metabolism and results in respiratory incompetence of yeast cells. Here we show that the nuclear gene MSS116, which codes for a mitochondrial putative RNA helicase necessary for splicing of several mt introns, can suppress the lack of the SUV3 gene. Overexpression of the Mss116 putative helicase from a multicopy plasmid present in the SUV3‐deleted strains partially restores respiratory competence, brings the steady‐state levels of COB and ATP6/8 mRNA back almost to normal and lowers the accumulation of 21S rRNA and ATP6/8 RNA precursors to the wild‐type levels. To the best of our knowledge, this is the first reported case of a substitution of one RNA helicase by another, belonging to a different class of RNA helicases. Copyright

Genetic and biochemical approaches for analysis of mitochondrial degradosome from Saccharomyces cerevisiae.

Publisher Summary Regulation of RNA turnover is one of the major mechanisms of controlling gene expression in yeast mitochondria. RNA degradation is carried out by the yeast mitochondrial degradosome (also known as mtEXO); a multiprotein enzyme complex showing NTP-dependent 3′ → 5′-exoribonuclease activity in vitro . The enzyme is composed of three subunits with molecular masses of 75, 84, and 111xa0kDa, all of which are encoded in the nuclear genome and after translation in the cytoplasm are imported into the mitochondria. The SUV3 and DSSI genes have been identified as encoding the 84- and 111-kDa subunits, respectively. Despite intensive research, many questions about the yeast mitochondrial degradosome remain unanswered. Efforts to identify the third gene encoding the degradosome subunit are currently in progress in the laboratory. Successful purification of relatively large amounts of the active enzyme should enable us to study its interactions with RNA and other proteins. A combination of genetical and biochemical approaches will increase the understanding of how the system of mtRNA turnover is regulated.

Assignment1 of SUPV3L1 to human chromosome band 10q22.1 by in situ hybridization

Diseases affecting mitochondrial function are an increasingly popular area of research (Wallace, 1994). Many of them are due to mutations in nuclear DNA, and few of the responsible genes have been mapped. In contrast, in baker’s yeast Saccharomyces cerevisiae hundreds of nuclear genes are known which control mitochondrial function. We have been working on such a yeast gene, SUV3 encoding a DEAD box RNA helicase whose product is essential for maintenance of functional mitochondria (Stepien et al., 1992). A partial cDNA clone of the human homologue of SUV3 was described by Adams et al. (1995) and we have used this clone to map the position of the human homologue called SUPV3L1. Materials and methods

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

RNA Degradation in Yeast and Human Mitochondria

RNA turnover in yeast mitochondria is controlled by the complex called degradosome, which consists of two nuclear-encoded proteins: the SUV3 gene codes for an RNA helicase and the DSS1 gene codes for an RNase. In contrast to yeast, much less is known about RNA degradation in human mitochondria. We suggest that the key enzyme involved in this process is nuclear-encoded polynucleotide phosphorylase, hPNPase.