Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides.

Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.

SP1 and NFY Regulate the Expression of PNPT1, a Gene Encoding a Mitochondrial Protein Involved in Cancer.

The Polyribonucleotide nucleotidyltransferase 1 gene (PNPT1) encodes polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease involved in mitochondrial RNA degradation and surveillance and RNA import into the mitochondrion. Here, we have characterized the PNPT1 promoter by in silico analysis, luciferase reporter assays, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), siRNA-based mRNA silencing and RT-qPCR. We show that the Specificity protein 1 (SP1) transcription factor and Nuclear transcription factor Y (NFY) bind the PNPT1 promoter, and have a relevant role regulating the promoter activity, PNPT1 expression, and mitochondrial activity. We also found in Kaplan-Meier survival curves that a high expression of either PNPase, SP1 or NFY subunit A (NFYA) is associated with a poor prognosis in liver cancer. In summary, our results show the relevance of SP1 and NFY in PNPT1 expression, and point to SP1/NFY and PNPase as possible targets in anti-cancer therapy.

Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E.

RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5'-sensor pocket that renders enzyme activity maximal on 5'-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself ('recessive resurrection'). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5'-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5'-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH's endonucleolytic action.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability.

R loops are nucleic acid structures comprising an DNA-RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.

Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized.

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.

Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence.

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.

Oocyte Factors Suppress Mitochondrial Polynucleotide Phosphorylase to Remodel the Metabolome and Enhance Reprogramming.

Oocyte factors not only drive somatic cell nuclear transfer reprogramming but also augment the efficiency and quality of induced pluripotent stem cell (iPSC) reprogramming. Here, we show that the oocyte-enriched factors Tcl1 and Tcl1b1 significantly enhance reprogramming efficiency. Clonal analysis of pluripotency biomarkers further show that the Tcl1 oocyte factors improve the quality of reprogramming. Mechanistically, we find that the enhancement effect of Tcl1b1 depends on Akt, one of its putative targets. In contrast, Tcl1 suppresses the mitochondrial polynucleotide phosphorylase (PnPase) to promote reprogramming. Knockdown of PnPase rescues the inhibitory effect from Tcl1 knockdown during reprogramming, whereas PnPase overexpression abrogates the enhancement from Tcl1 overexpression. We further demonstrate that Tcl1 suppresses PnPase's mitochondrial localization to inhibit mitochondrial biogenesis and oxidation phosphorylation, thus remodeling the metabolome. Hence, we identified the Tcl1-PnPase pathway as a critical mitochondrial switch during reprogramming.

A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Šaway from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.

Streptomyces coelicolor polynucleotide phosphorylase can polymerize nucleoside diphosphates under phosphorolysis conditions, with implications for the degradation of structured RNAs.

We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The kcat/Km value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the kcat/Km for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3' ends of the products of those reactions using [(32)P]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3' tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3' tails to a substrate that already possessed an unstructured 3' tail. We propose a model in which the transient synthesis of 3' tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.

Human polynucleotide phosphorylase (hPNPaseold-35): should I eat you or not--that is the question?

RNA degradation plays a fundamental role in maintaining cellular homeostasis whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3'-5' exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel "overlapping pathway screening" approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. The objective of this review is to provide an up-to-date perspective of the recent discoveries made relating to hPNPaseold-35 and the impact they continue to have on our comprehension of its expanding and diverse array of functions.

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3'-untranslated region stability, processing, and degradation.

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3'-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3'-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3'-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3'-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3'-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3'-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3'-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3'-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3'-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.

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.

Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni.

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.

Involvement of pnp in survival of UV radiation in Escherichia coli K-12.

Polynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3'→5' exoribonuclease or exoDNase in the presence of inorganic phosphate (P(i)), and extends a 3'-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H(2)O(2)- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H(2)O(2)-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.

Human polynucleotide phosphorylase (hPNPase(old-35)): an evolutionary conserved gene with an expanding repertoire of RNA degradation functions.

Human polynucleotide phosphorylase (hPNPase(old-35)) is an evolutionary conserved RNA-processing enzyme with expanding roles in regulating cellular physiology. hPNPase(old-35) was cloned using an innovative 'overlapping pathway screening' strategy designed to identify genes coordinately regulated during the processes of cellular differentiation and senescence. Although hPNPase(old-35) structurally and biochemically resembles PNPase of other species, overexpression and inhibition studies reveal that hPNPase(old-35) has evolved to serve more specialized and diversified functions in humans. Targeting specific mRNA or non-coding small microRNA, hPNPase(old-35) modulates gene expression that in turn has a pivotal role in regulating normal physiological and pathological processes. In these contexts, targeted overexpression of hPNPase(old-35) represents a novel strategy to selectively downregulate RNA expression and consequently intervene in a variety of pathophysiological conditions.

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells.

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3'-5' exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase(old-35) identified miRNAs differentially and specifically regulated by hPNPase(old-35). One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27(kip1), displayed robust down-regulation with ensuing up-regulation of p27(kip1) by expression of hPNPase(old-35), which also occurred in multiple human melanoma cells upon IFN-beta treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase(old-35). Inhibition of hPNPase(old-35) by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-beta-mediated growth inhibition, accentuating the importance of hPNPase(old-35) induction and miR-221 down-regulation in mediating IFN-beta action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase(old-35) might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.

Regulation of Escherichia coli polynucleotide phosphorylase by ATP.

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.

Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases.

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. Not only homopolymeric poly(A)-tails, comprised exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. Polynucleotide phosphorylase (PNPase) and the archaeal exosome, which bear strong similarities to one another, both functionally and structurally, were found to polymerize the heteropolymeric tails in bacteria, spinach chloroplasts, and archaea. As phosphorylases, these enzymes use diphosphate nucleotides as substrates and can reversibly polymerize or degrade RNA, depending on the relative concentrations of nucleotides and inorganic phosphate. A possible scenario, illustrating the evolution of RNA polyadenylation and its related functions, is presented, in which PNPase (or the archaeal exosome) was the first polyadenylating enzyme to evolve and the heteropolymeric tails that it produced, functioned in a polyadenylation-stimulated RNA degradation pathway. Only at a later stage in evolution, did the poly(A)-polymerases that use only ATP as a substrate, hence producing homopolymeric adenosine extensions, arise. Following the appearance of homopolymeric tails, a new role for polyadenylation evolved; RNA stability. This was accomplished by utilizing stable poly(A)-tails associated with the mature 3' ends of transcripts. Today, stable polyadenylation coexists with unstable heteropolymeric and homopolymeric tails. Therefore, the heteropolymeric poly(A)-rich tails, observed in bacteria, organelles, archaea, and human cells, represent an ancestral stage in the evolution of polyadenylation.

Progression elevated gene-3 promoter (PEG-Prom) confers cancer cell selectivity to human polynucleotide phosphorylase (hPNPase(old-35))-mediated growth suppression.

The poor prognosis of pancreatic cancer patients using currently available therapies mandates novel therapeutics that combine anti-neoplastic potency with toxicity-minimizing cancer specificity. Employing an overlapping pathway screen to identify genes exhibiting coordinated expression as a consequence of terminal cell differentiation and replicative senescence, we identified human polynucleotide phosphorylase (hPNPase(old-35)), a 3',5'-exoribonuclease that exhibits robust growth-suppressing effects in a wide spectrum of human cancers. A limitation to the anti-neoplastic efficacy of hPNPase(old-35) relates to its lack of cancer specificity. The promoter of Progression Elevated Gene-3 (PEG-Prom), discovered in our laboratory via subtraction hybridization in a transformation progression rodent tumor model functions selectively in a diverse array of human cancer cells, with limited activity in normal cells. An adenovirus constructed with the PEG-Prom driving expression of hPNPase(old-35) containing a C-terminal Hemaglutinin (HA)-tag (Ad.PEG.hPNPase(old-35)) was shown to induce robust transgene expression, growth suppression, apoptosis, and cell-cycle arrest in a broad panel of pancreatic cancer cells, with minimal effects in normal immortalized pancreatic cells. hPNPase(old-35) expression correlated with arrest in the G(2)/M phase of the cell cycle and up-regulation of the cyclin-dependent kinase inhibitors (CDKI) p21(CIP1/WAF-1/MDA-6) and p27(KIP1). In a nude mouse xenograft model, Ad.PEG.hPNPase(old-35) injections effectively inhibited growth of human pancreatic cancer cells in vivo. These findings support the potential efficacy of combining a cancer-specific promoter, such as the PEG-Prom, with a novel anti-neoplastic agent, such as hPNPase(old-35), to create a potent, targeted cancer therapeutic, especially for a devastating disease like pancreatic cancer.