Oncogene (2011) 30, 1733–1743 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW Human polynucleotide phosphorylase (hPNPaseold-35): an evolutionary conserved gene with an expanding repertoire of RNA degradation functions SK Das1, SK Bhutia1, UK Sokhi1, R Dash1, B Azab1, D Sarkar1,2,3 and PB Fisher1,2,3 1Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA; 2VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA and 3VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA Human polynucleotide phosphorylase (hPNPaseold-35)is eukaryotic cells, a variety of ribonucleases(RNases) act an evolutionary conserved RNA-processing enzyme cooperatively, initially shortening the 30 poly (A) tail of with expanding roles in regulating cellular physiology. an mRNA by deadenylases followed by the removal of hPNPaseold-35 was cloned using an innovative ‘overlapping the 50 cap structure by a decapping enzyme, which pathway screening’ strategy designed to identify genes enables the degradation of the transcript by a 50-30 coordinately regulated during the processes of cellular exoribonuclease. Alternatively, the mRNA might be differentiation and senescence. Although hPNPaseold-35 degraded from the 30 end by the cytoplasmic exosome, a structurally and biochemically resembles PNPase of other multiprotein complex of diverse 30-50 exoribo- species, overexpression and inhibition studies reveal that nucleases. Polynucleotide phosphorylase (PNPase) is a hPNPaseold-35 has evolved to serve more specialized and 30-50 exoribonuclease that uses the phosphorolytic diversified functions in humans. Targeting specific mRNA mechanism to degrade RNA (Mohanty and Kushner, or non-coding small microRNA, hPNPaseold-35 modulates 2000; Yehudai-Resheff et al., 2001; Sarkar et al., 2006). gene expression that in turn has a pivotal role in regula- It is conserved evolutionarily and is expressed in ting normal physiological and pathological processes. In different species including bacteria, plants, worms, flies, these contexts, targeted overexpression of hPNPaseold-35 mice and humans. Our group cloned the human represents a novel strategy to selectively downregulate homolog of polynucleotide phosphorylase (hPNPaseold-35) RNA expression and consequently intervene in a variety of in the unique contexts of differentiation and senescence, pathophysiological conditions. using an ‘overlapping pathway screening’ scheme. We Oncogene (2011) 30, 1733–1743; doi:10.1038/onc.2010.572; documented that hPNPaseold-35 has a key role in published online 13 December 2010 regulating both of these fundamental physiological processes (Leszczyniecka et al., 2002, 2003, 2004; Sarkar Keywords: hPNPaseold-35; senescence; RNA degradation; et al., 2003, 2004, 2005, 2006, 2007; Sarkar and Fisher, c-myc; miRNA 2006). We presently review recent advances in our understanding of hPNPaseold-35-mediated RNA degrada- tion, in particular its ability to target different classes of RNAs. Introduction RNA degradation and/turnover are major processes Cloning, expression and localization of hPNPaseold-35 controlling RNA levels and are important regulators of physiological and pathological processes (Parker and hPNPaseold-35 was cloned by using an overlapping Song, 2004). Labile messenger RNAs to more stable pathway screening approach (Leszczyniecka et al., non-coding RNAs (mostly ribosomal RNA and transfer 2002) during a screen for genes upregulated in the RNA, but also the expanding class of small regula- process of terminal cellular differentiation and senes- tory RNAs) are eventually degraded by a complex cence. Although terminal cell differentiation and cellular process involving the simultaneous or sequential inter- senescence represent two discrete phenomena, these play of multiple proteins. Many of these proteins are processes have several common characteristics. Both evolutionary conserved extending from prokaryotes to are distinguished by irreversible growth arrest associated higher mammals and serving comparable functions. In with marked inhibition of DNA synthesis, inhibition of telomerase activity and modulation of discrete programs Correspondence: Dr PB Fisher, Department of Human and Molecular of gene expression, especially upregulation of cyclin- Genetics, VCU Institute of Molecular Medicine, VCU Massey Cancer dependent kinase inhibitors (Fisher et al., 1985, 1986; Center, Virginia Commonwealth University, School of Medicine, 1101 Campisi, 1992). Combined treatment of metastatic East Marshall Street, Sanger Hall Building, Room 11-015, Richmond, human melanoma cells HO-1 with recombinant human VA 23298-0033, USA. E-mail: pbfi[email protected] fibroblast interferon (IFN)-b and the protein kinase C Received 1 September 2010; revised 20 October 2010; accepted 30 activator mezerein induces irreversible growth arrest October 2010; published online 13 December 2010 accompanied by morphological, biochemical, antigenic Human polynucleotide phosphorylase and RNA degradation SK Das et al 1734 and gene expression changes culminating in a state of mitochondrial localization signal at the NH2-terminal ‘terminal differentiation (Fisher and Grant, 1985; Fisher and it is imported into the mitochondria by i-AAA et al., 1985, 1986; Guarini et al., 1989, 1992; Jiang et al., (ATPases associated with several diverse cellular acti- 1993, 1995). Screening of a temporal complementary vities) protease Yme1, localized into mitochondrial DNA library generated from terminally differentiated intermembrane space and maintains mitochondrial HO-1 melanoma cells with complementary DNAs from homeostasis (Chen et al., 2006). However, our studies senescent progeriod fibroblasts identified 75 genes, document that overexpressed C-terminal HA-tagged termed old-1 to -75, which were upregulated during hPNPaseold-35 localizes both in cytosol and mitochondria both terminal differentiation and senescence. Sequence (Sarkar et al., 2005) indicating that hPNPaseold-35 might analysis of one particular clone, old-35, confirmed its reside within or outside mitochondria. In these contexts, identity to the PNPase gene, resulting in the gene being the targets and the consequences of hPNPaseold-35 renamed hPNPaseold-35 (Leszczyniecka et al., 2002). expression in different cellular compartments may be The hPNPaseold-35 gene consists of 28 exons and 27 distinct and diverse, thereby expanding the repertoire of introns spanning 54 kb in chromosome 2p15–2p16.1 activities of this interesting enzyme. (Leszczyniecka et al., 2003). Of interest, this unstable genomic region is prone to cytogenetic alterations in human cancers and in various genetic disorders RNA degradation machinery: PNPase and exosome (Kirschner et al., 1999) such as B-cell lymphoma (Fukuhara et al., 2006), type I hereditary nonpolyposis RNases are enzymes that are master regulators of colorectal cancer, familial male precocious puberty, stability and decay of RNA (Deutscher, 1993a, b; Carney complex, Doyne’s honeycomb retinal dystrophy Allmang et al., 1999; Deutscher and Li, 2001). Depend- and DYX-3, a form of familial dyslexia (Kirschner et al., ing on their degradative properties, RNases are divided 1999). hPNPaseold-35 mRNA expression could be into two functional classes, endoribonucleases that detected in all normal tissues analyzed with the cleave RNA molecules internally and exoribonucleases highest expression being detected in heart and brain that act at the end of RNA chains (Deutscher, 1993b). (Leszczyniecka et al., 2002). However, to date, there is RNA decay pathways in two of the most comprehen- no evidence linking expression and function of hPNPase sively studied model systems, the prokaroyte Escherichia to any of the aforementioned pathological processes. coli and the eukaryote Saccharomyces cerevisiae, are In bacteria, PNPase autogenously regulates its ex- different. In eukaryotes exoribonucleases can degrade pression by promoting the decay of PNPase mRNA by RNA both at 50–30 and 30–50 directions (Deutscher and binding to the 50-untranslated leader region of an RNase Li, 2001), whereas in prokaryotes RNA degradation III-processed form of this transcript (Jarrige et al., takes place only in the 30–50direction. However, an interest- 2001). To date the only known regulators of hPNPaseold-35 ing aspect of RNA decay in both prokaryotes and transcription are type I IFN (IFN-a and IFN-b) in both eukaryotes is the presence of multiprotein complexes normal and cancer cells with diverse backgrounds known as the degradosome and the exosome, respectively. irrespective of their p53 and Rb status (Leszczyniecka In E. coli, PNPase is associated with the endonuclease et al., 2002). Double-stranded RNA and poly(I) RNase E, RNA helicase and the glycolytic enzyme poly(C), a known inducer of IFN-a and IFN-b, also enolase to form the degradosome and executes its stimulate hPNPaseold-35 expression while IFN-g and processive 30–50 phosphorolysis or degradation of tumor necrosis factor-a have minimal or no effect, RNA species on endonucleolytic cleavage by RNase E. respectively. hPNPaseold-35 is an early IFN response gene PNPase acts as an integral component of degradosome and its induction depends on the Janus-activated kinase/ like RNA-helicase and enolase. In contrast, in yeast, signal transducers and activators of transcription signal PNPase is absent and the exosome, a complex of transduction pathways.