Human Polynucleotide Phosphorylase, hPNPase, is Localized in Mitochondria

Piwowarski, Jan; Grzechnik, Pawel; Dziembowski, Andrzej; Dmochowska, Aleksandra; Minczuk, Michal; Stępień, Piotr P.

doi:10.1016/s0022-2836(03)00528-x

Cited by 79 publications

(67 citation statements)

References 26 publications

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“…Sequence inspection of the N terminus suggests that endogenous PNPase resides in the mitochondria (36). In agreement with this, both endogenous PNPase and C-terminally tagged PNPase localize to mitochondria in HeLa cells (35). These data suggest that the subcellular localization of PNPase is influenced by an unblocked N terminus, the level of exogenous overexpression, or the specific cell type examined.…”

Section: Resultssupporting

confidence: 62%

“…Our data showed that PNPase localized to mitochondria in multiple cell types, specifically to the IMS in a peripheral membrane protein complex. With a classical mitochondrial targeting sequence and prokaryotic and chloroplastic stroma counterparts, PNPase was expected to regulate mtRNA levels from within the matrix (35,52). However, PNPase was surprisingly localized to the IMS, and mtRNAs were almost completely unaffected by PNPase reduction at its nadir.…”

Section: Discussionmentioning

confidence: 99%

“…To identify potential TCL1-interacting proteins, TCL1 immunoprecipitation followed by mass spectrometry analysis from human pre-B cells was performed, and polynucleotide phosphorylase (PNPase) was identified as a potential binding partner (10). Immediately, our focus shifted to studies of PNPase to determine if this interaction was possible, because TCL1 was reportedly excluded from mitochondria under standard growth conditions (11), whereas PNPase is a nucleus-encoded mitochondrial exoribonuclease (35).…”

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confidence: 99%

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Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Chen¹,

Rainey²,

Balatoni³

et al. 2006

Molecular and Cellular Biology

135

150

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We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

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Section: Resultssupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Chen¹,

Rainey²,

Balatoni³

et al. 2006

Molecular and Cellular Biology

135

150

View full text Add to dashboard Cite

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“…Nucleotide triphosphate can also be used as a substrate for polymerization activity (32,35). Because hPNPase is localized in mitochondria (36), it may be possible that hPNPase can also polyadenylate human mt mRNAs. To estimate the efficacy of the siRNAmediated knockdown, RT-PCR for the target mRNAs was carried out, and the PCR products were detected by ethidium bromide staining.…”

Section: Retrieval Of a Candidate Gene Encoding Human Mt Pap Inmentioning

confidence: 99%

Human Mitochondrial mRNAs Are Stabilized with Polyadenylation Regulated by Mitochondria-specific Poly(A) Polymerase and Polynucleotide Phosphorylase

Nagaike

Suzuki

Katoh

et al. 2005

Journal of Biological Chemistry

171

197

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Mammalian mitochondrial (mt) mRNAs have short poly(A) tails at their 3 termini that are post-transcriptionally synthesized by mt poly(A) polymerase (PAP). The polyadenylation of mt mRNAs is known to be a key process needed to create UAA stop codons that are not encoded in mtDNA. In some cases, polyadenylation is required for the tRNA maturation by editing of its 3 terminus. However, little is known about the functional roles the poly(A) tail of mt mRNAs plays in mt translation and RNA turnover. Here we show human mt PAP (hmtPAP) and human polynucleotide phosphorylase (hPNPase) control poly(A) synthesis in human mitochondria. Partial inactivation of hmtPAP by RNA interference using small interfering RNA in HeLa cells resulted in shortened poly(A) tails and decreased steady state levels of some mt mRNAs as well as their translational products. Moreover, knocking down hmtPAP generated markedly defective mt membrane potentials and reduced oxygen consumption. In contrast, knocking down hPNPase showed significantly extended poly(A) tails of mt mRNAs. These results demonstrate that the poly(A) length of human mt mRNAs is controlled by polyadenylation by hmtPAP and deadenylation by hPNPase, and polyadenylation is required for the stability of mt mRNAs.

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“…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 degradation and maturation of several RNAs (11)(12)(13)(14).…”

mentioning

confidence: 99%

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

Perrin

Meyer

Zaepfel

et al. 2004

Journal of Biological Chemistry

100

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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 ...

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Human Polynucleotide Phosphorylase, hPNPase, is Localized in Mitochondria

Cited by 79 publications

References 26 publications

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Human Mitochondrial mRNAs Are Stabilized with Polyadenylation Regulated by Mitochondria-specific Poly(A) Polymerase and Polynucleotide Phosphorylase

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

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