An Evolutionary Conserved Gene with an Expanding Repertoire of RNA Degradation Functions

Home , DCPS (gene), Degradosome, Exoribonuclease, RNase PH, RNA polymerase III

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 degradation, 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. Analysis of the hPNPaseold-35 multiple exoribonuclease performs RNA degradation promoter identified an IFN-stimulated response element in both the cytoplasm and the nucleus (Mitchell et al., that showed increased binding of ISGF3 complex on 1997; Hoof and Parker, 1999; Raijmakers et al., 2004; IFN-b treatment (Leszczyniecka et al., 2003). Mutation Buttner et al., 2006). However, PNPase has been identi- in this site abolished IFN-b induction of the promoter fied in higher animals such as mouse, rat and human indicating that hPNPaseold-35 is regulated at the level of indicating that the exosome and PNPase might serve transcription. In addition to the IFN-stimulated re- specialized functions in these species (Leszczyniecka sponse element, the hPNPaseold-35 promoter contains et al., 2002, 2003, 2004; Raijmakers et al., 2002). additional putative regulatory protein-binding sites, In all species, PNPase contains five motifs that are con- including a site for E2F transcription factor 3 (E2F3), spicuously preserved through evolution extending from a transcriptional repressor that is responsible for prokaryotes and plants to mammals (Leszczyniecka gene silencing during the G1 to S phase transition et al., 2004; Almeida et al., 2008). Two conserved (Gewartowski et al., 2006). catalytic RNase PH regions, a small domain of B250 Studies using cellular fractionation and/or immuno- a.a. residues related to the E. coli RNase PH enzyme and fluorescence showed that endogenous hPNPaseold-35 and involved primarily in the 30 processing of transfer RNA overexpressed C-terminal myc-tagged hPNPaseold-35 precursors, are present at the N-terminus of hPNPaseold-35 localize only to the mitochondria (Piwowarski et al., (Leszczyniecka et al., 2004). These RNase PH domains 2003; Chen et al., 2006). hPNPaseold-35 has a typical are separated by an a-helix that is unique to PNPase

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1735 (Symmons et al., 2002). The RNA-binding property of Parker, 2000). Alternatively, after deadenylation, hPNPaseold-35 is conferred by two C-terminal RNA- mRNA can be degraded in a 30–50 direction by the binding domains, KH and S1 (Symmons et al., 2000; exosome complex and the oligonucleotide cap structure Raijmakers et al., 2002; Symmons et al., 2002; is hydrolyzed by the decapping enzyme DcpS (Wang Leszczyniecka et al., 2002, 2004). In addition to the and Kiledjian, 2001). In addition, mRNA can also be characteristic five motifs, plant PNPase contains an degraded by endoribonucleases and aberrant mRNA N-terminal target peptide allowing translocation to may be degraded by specialized pathways such as chloroplast and the mammalian PNPase contains an nonsense-mediated decay or nonstop decay that degrade N-terminal mitochondrial localization signal facilitating mRNA containing a premature stop codon or lacking a its subcellular localization in mitochondria (Piwowarski stop codon, respectively (Dodson and Shapiro, 2002; et al., 2003; Yehudai-Resheff et al., 2003 and Sarkar Frischmeyer et al., 2002; Cao and Parker, 2003). et al., 2005). The catalytic activity of PNPase enzyme in PNPase, as a phopsphorylase, it incorporates Pi and bacteria is located mainly in the second RNase PH adenosine diphosphate (ADP) in degradation and domain (Jarrige et al., 2002) while in spinach chloro- polymerization process, respectively (Littauer and plasts, both RNase PH domains have equal polyadeny- Grunberg-Manago (1999). Optimal degradation activity lation and exoribonuclease activity. Deletion and depends on the concentration of Pi and it varies from mutation analysis have identified the critical regions of species to species (Portnoy et al., 2005). The human bacterial PNPase activity. Deletion of the S1 or KH PNPase is active in much lower concentration of Pi RNA-binding domain reduces the enzymatic activity by compared with bacterial PNPase (Portnoy et al., 2005). 50- or 19-fold, respectively, while deletion of both S1 The specificity of the enzyme for the polymerization and KH domains results in 1% of the enzymatic activity reaction is, like that of the E. coli. PNPase, is high for (Stickney et al., 2005). These deletions do not interfere ADP, with much less activity for other nucleotide with interaction with RNase E to form the degrado- diphosphates (NDPs) and no activity for ATP or other some. Site-directed mutagenesis reveals that the catalytic nucleotide triphosphates (NTPs). More interestingly, center of PNPase is located in the second RNase PH hPNPase displays no preferential activity for polyadeny- domain around the binding site for tungstate, a phosphate lated RNA-like bacterial or chloroplast PNPase (Portnoy analogue (Jarrige et al.,2002).Interestingly,aG454D et al., 2005). mutation in the second RNase PH domain displays defective RNA binding and impairs the autogenous c-myc mRNA is the target of hPNPaseold-35 regulation of PNPase mRNA indicating that in addition In the cytoplasm, adenoviral-mediated overexpression to S1 and KH domains, the catalytic domains are also of hPNPaseold-35 could directly degrade c-myc mRNA by capable of binding to RNA (Regonesi et al., 2004). virtue of its 30-50 exoribonuclease property and this The alignment of the proteins in eukaryotic exosome degradation is specific for c-myc as compared with other clearly points to a structural similarity with bacterial mRNAs, such as c-jun, glyceraldehyde 3-phosphate PNPase. The three-dimensional structure of the PNPase dehydrogenase or GADD 34 (Sarkar et al., 2003). It is from the bacterium Streptomyces antibioticus has still not clear what confers the specificity of hPNPaseold-35 revealed that the enzyme is a ring (doughnut)- shape for c-myc mRNA. There might be a specific sequence in formed by a homotrimeric complex, with the hexameric c-myc mRNA that allows hPNPaseold-35 binding and PH-domains surrounding a central channel that degradation. In E. coli, PNPase degrades a family of can accommodate a single-stranded RNA molecule cold shock proteins that do not show any sequence (Symmons et al., 2002). Similarly, the same core similarity (Yamanaka and Inouye, 2001). Considering structure is formed in the exosome: six RNase PH this observation, the secondary structure of the mRNA homologues comprise the core while three additional rather than its primary sequence might be the determin- exosome subunits, which contain S1 RNA-binding ing factor for specificity of hPNPaseold-35 binding and domains, are positioned on the outer surface of the ring degradation of c-myc. The presence of either of the (Mitchell and Tollervey, 2000; Raijmakers et al., 2002; RNase PH domains was sufficient for degradation of Symmons et al., 2002). c-myc mRNA and induction of morphological, biochemical and gene expression changes by hPNPaseold-35 (Sarkar et al., 2005) (described in next section of this review). Although in bacteria, presence of the KH Specialized functions of hPNPaseold-35: RNA degradation and the S1 RNA-binding domains are required for functional activity of PNPase, hPNPaseold-35 still retained In eukaryotes, the poly(A) tail of mRNA first becomes its functional activity on removal of KH and S1 deadenylated by three different enzyme complexes, the domains (Sarkar et al., 2005; Stickney et al., 2005). first composed mainly of Ccr4p and Pop2p, the second containing Pan2p and Pan3p and the third is a poly(A)- specific exonuclease called PARN (poly(A) RNases) hPNPase might be involved in degradation of RNA (Parker and Song, 2004). Following deadenylation, a in mammalian mitochondria decapping enzyme consisting of Dcp1p and Dcp2p In mammals, mitochondrial RNA (mtRNA) degrada- removes the 50 cap structure thus facilitating degradation is not well defined as no RNA degrading complex tion by the 50,30 exoribonuclease Xrn1p (Tucker and has been identified. The current view is largely based on

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1736

Figure 1 Schematic model of miRNA biogenesis and stability. After synthesis by RNA polymerase II, primary transcripts of (pri) miRNA are recognized by Drosha, which excises the hairpin precursor and released precursor (pre) miRNA. From nucleus, exportin five delivers the miRNA precursor to Dicer and its RNA-binding partner in the cytoplasm for final processing to the mature 22-nt miRNAs. One strand is selected for stable association with Argonaute, in which it serves as a guide to target and regulate specific mRNAs. By executing exonuclease activity hPNPaseold-35 specifically degrades mature miRNAs. However, their substrate recognition mechanism is unknown.

our understanding of the E. coli RNA degradosome and this recent finding it is hypothesized that the human yeast mitochondrial exosome. Similar to cytoplasmic suppressor of Var1 3-hPNPase complex might be mRNAs, mtRNAs also require long poly (A) tails for involved in removing truncated RNA species through recruitment of poly (A)-binding proteins for mainte- hPNPase-mediated degradation. Further experimenta- nance of stability (Temperley et al., 2003). Human tion is required to validate this possibility. mitochondria-specific poly (A) polymerase has been shown to synthesize mtRNA poly(A) tails. Knocking down poly (A) polymerase by small interfering RNA Modulation of miRNAs by hPNPaseold-35 decreases the length of poly (A) tail of mitochondrial MicroRNAs (miRNAs), a subset of non-coding RNAs, mRNA and decreases their steady-state levels (Nagaike are 22–25-nt long endogenously initiated short RNA et al., 2005). The mitochondrial membrane potential molecules that regulate gene expression at the post- (Dc) and oxygen consumption also decreased in poly transcriptional level and have important roles in a (A) polymerase small interfering RNA-treated cells. In multiplicity of biological functions, including cell contrast, knocking down of hPNPase showed signifi- differentiation, tumorigenesis, apoptosis and metabo- cantly extended poly(A) tails of mtRNA although it did lism (Ambros, 2004). An individual miRNA is able to not affect the steady-state levels of these mRNAs or control the expression of more than one target mRNA their translational products (Temperley et al., 2003; and each mRNA may be regulated by multiple Nagaike et al., 2005). In these studies, only the levels of miRNAs. The interaction between miRNA and mRNA full-length mtRNAs were quantified without consider- are usually restricted to the ‘seed’—6–8-nt at the 50 ing the truncated and polyadenylated mtRNAs that region of miRNA and partially complementary sites in normally occur in human mitochondria (Slomovic et al., the 30 UTRs of target mRNAs, resulting in either 2005). In normal mammalian mitochondria, truncated translational repression or target degradation (Kim, and polyadenylated transcripts do not accumulate and 2005). miRNAs are initially transcribed principally by are rapidly degraded (See and Fitt, 1972). Recent studies either RNA polymerase II or RNA polymerase III as suggest that knocking down human suppressor of Var1 long primary transcripts of miRNAs, which are further 3 helicase leads to an accumulation of shortened processed by the nuclear RNase Drosha and cyto- polyadenylated mtRNA species (Khidr et al., 2008). plasmic RNase Dicer to produce precursor miRNAs Human suppressor of Var1 3 makes a heteropentameric and mature miRNAs, respectively (Calin and Croce, complex with hPNPase at a 2:3 molar ratio in a 2006) (Figure 1). In principle, miRNA abundance coordinated manner to degrade double-stranded RNA could be controlled by developmental and tissue specific (dsRNA) substrates (Wang et al., 2009). Considering signaling (Landgraf et al., 2007). The steady-state levels

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1737 of miRNAs, crucial for its profound impact on a hPNPaseold-35 is an exoribonulease and in vivo immuno- wide array of biological processes (Tsuchiya et al., pricipitation assays clearly demonstrate specific binding 2006; Filipowicz et al., 2008) are presumably regulated of mature miRNAs to hPNPaseold-35, a pre-requisite step by the opposing activities of miRNA biogenesis and for enzymatic activity indicating that miRNAs undergo degradation. enzymatic degradation. In the biogenesis process, miRNAs might be regulated both transcriptionally and post-transcriptionally. Nu- hPNPaseold-35 preferentially degrades miR-221. Although merous Pol II-associated transcription factors such as the overexpression of hPNPaseold-35 selectively downregu- myogenin and MYOD1 are involved in transcriptional lates specific miRNAs, the preferences to miRNAs varied control of miR-1 and miR-133 genes during myogenesis significantly. In melanoma cells, degradation of miR-221 (Rao et al., 2006; Chen et al., 2006). Some miRNAs are by hPNPaseold-35 wasmoreprofoundcomparedwithother under the control of tumor suppressive p53 (reviewed by miRNAs tested. This preferential activity might be at- He et al., 2007) or the oncogenic transcription factor tributed to high binding affinity of hPNPaseold-35 to the c-myc (He et al., 2005; Chang et al., 2008). Epigenetic mature miR-221, a hypothesis that needs to be tested control also contributes to miRNA gene regulation (Figure 3). (Bueno et al., 2008). Several miRNAs expressions are also regulated at the post-transcriptional level. The Role of hPNPaseold-35-mediated miRNA degradation in primary transcript of let-7 (pri-let-7) is expressed in both IFN-b-mediated growth inhibition. hPNPaseold-35 is an undifferentiated and differentiated ES cells, whereas IFN-b-inducible early response gene and treating a mature let-7 is detected only in differentiated cells, panel of melanoma cells with IFN-b also downregulates indicating that let-7a might be post-transcriptionally hPNPaseold-35-target miRNAs. This effect was abrogated controlled (Suh et al., 2004; Thomson et al., 2006; by knocking down hPNPaseold-35, thus establishing Wulczyn et al., 2007). Excluding this activity, turnover of miRNA also might contribute in maintaining its functional and mechanistic links between IFN-b-mediated hPNPaseold-35 induction and miRNA downregulation. level; however, this remains a largely unexplored area. IFN-b is a potent growth inhibitor and one of the In Caenorhabditis elegans (Kennedy et al., 2004) an exoribonuclease ERI1 (also known as THEX1) was growth inhibiting mechanisms in melanoma is the induction of hPNPaseold-35, which in turn raises the level previously shown to degrade small interfering RNAs. of p27Kip1, a cyclin-dependent kinase inhibitor protein, Another exoribonuclease named small RNA degrading by degrading c-myc mRNA (Sarkar et al., 2006). nuclease proteins were reported to affect the stability of However, forced degradation of c-myc by small inter- miRNAs in plants (Ramachandran and Chen, 2008). fering RNA did not completely block IFN-b-mediated Very recently, we demonstrated a role of hPNPaseold-35 in growth arrest indicating the existence of additional human miRNAs degradation (Das et al., 2010). This novel way of regulating miRNA levels is being actively pathways potentially regulating this effect (Barnes and Karin, 1997; Obaya et al., 1999; O’Hagan et al., 2000). explored by our research group. As miR-221 directly targets p27Kip1 mRNA (Fornari et al., 2008), IFN-b-mediated hPNPaseold-35-dependent hPNPaseold-35 downregulates specific miRNAs. miRNA miR-221 downregulation might also be involved in microarray analysis between Ad.hPNPaseold-35- and inducing growth arrest. Ad.vec-infected human melanoma cells identified specific miRNAs differentially regulated by hPNPaseold-35, which have been subsequently validated by qPCR and Other biological functions of hPNPaseold-35 protein Northern blot analyses. Of interest, robust downregulation of several miRNAs (for example, miR-221, miR- hPNPaseold-35 and cellular senescence 222 and miR-106b) by hPNPaseold-35 was observed while Senescence is a state of irreversible growth arrest a number of miRNAs were not affected (miR-184, miR- induced spontaneously in primary cells after a finite let7a) suggesting specificity and selectivity of this number of population doublings (replicative senescence) enzyme-mediated miRNA downregulation. Moreover, or induced by endogenous and exogenous acute and an inverse correlation between these miRNAs and chronic stress signals (stress- or aberrant-signaling- hPNPaseold-35 in primary melanocytes and different induced senescence (Hayflick, 1976; Serrano et al., melanoma cell lines support the biological relevance of 1997). hPNPaseold-35 was first described as an induced this protein in regulating specific miRNAs (Figure 2). gene during terminal differentiation and cellular senescence two end-stage processes sharing several over- Post-transcriptional modification of miRNA biogenesis by lapping features including irreversible growth arrest, hPNPaseold-35. miRNA genes are initially transcribed as marked inhibition of DNA synthesis and modulation of long primary transcripts of miRNAs, which are further telomerase activity (Sarkar et al., 2006). Overexpression processed to produce precursor miRNAs and mature of full-length hPNPaseold-35 or either one of its RNase miRNAs (Figure 1) (Calin and Croce, 2008). Ad. PH domains in human melanoma cells and melanocytes hPNPaseold-35 did not show any effect on primary induces distinctive changes associated with senescent transcripts of miRNA and precursor miRNA. Only phenotypes (Sarkar et al., 2003, 2005) including induc- old-35 mature miRNAs were downregulated by hPNPase . tion of SA-b-gal activity, cell cycle arrest in G1 phase

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1738

Figure 2 hPNPaseold-35 target miRNAs. HO-1 cells were either infected with Ad.vec or Ad.hPNPaseold-35 at a multiplicity of infection (m.o.i.) of 5000 vp/cells for 3 days and subjected to miRNA microarrays and potential target miRNAs miR-106b, miR-25, miR-221, miR-222, miR-let7a and miR-184 were validated for differential expression by using primary- and mature miRNA-speciﬁc Taqman probes with qPCR. (b) Northern blotting was performed to detect mature miRNAs and its precursor species by using speciﬁc probes. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), miR-RNU44 and U6 RNA were used to normalize the pri- and mature miRNA in qPCR and Northern blotting data, respectively. (c, d) The basal level of different miRNAs (c) and mRNA for hPNPaseold-35 (d) were evaluated by qPCR in different cell lines, including normal human epidermal melanocytes (NHEM), normal immortal human melanocytes FM-516-SV (referred to as FM-516), radial growth phase primary melanoma WM-35, vertical growth phase primary melanoma WM-278 and metastatic melanoma HO-1, C8161.9, and MeWo. (Taken from Das et al., 2010).

p27kip1 and a decrease in p21CIP1/WAF-/MDA-6. The increase in p27kip1 is most likely secondary to the decrease in c-myc that controls p27kip1 expression by multiple mechanisms (Barnes and Karin, 1997; Obaya et al., 1999; O’Hagan et al., 2000). However, it is also a consequence of downregulation of miR-221 that specifically targets the mRNA of p27kip1. As oxidative stress mediates cellular senescence (Colavitti and Finkel, 2005; Passos et al., 2006) the induction of reactive oxygen species (ROS) following upregulation of hPNPaseold-35 could also be involved in induction of the senescent Figure 3 hPNPaseold-35 preferentially degrades miR-221. (a) Comparative degradation between miR-221, miR-222 and miR- phenotype. Further studies are necessary to investigate 106b at a 2 h time point using in vitro degradation assays. P-value the role of the interactive network of c-myc, miR-221, was calculated using Student’s t-test by comparing the specific p27kip1 and ROS in regulating the senescence process. miRNA normalized by miR-RNU-44. The data represent mean±s.d. of two independent experiments each done in triplicate. (b) A direct interaction between targeted miRNAs with in vitro old-35 translated hPNPaseold-35 was confirmed by immunoprecipitation hPNPase and aging-associated inflammation and Northern blotting. Aging represents a conundrum in the evolution of higher organisms that is associated with progressive with inhibition of DNA synthesis followed by induction degenerative diseases the underlying pathology of of apoptosis, and inhibition of telomerase activity. In which is often chronic inflammation (Kiecolt-Glaser total, these findings indicate that hPNPaseold-35 might et al., 2003). Oxidative stress has an important role in have an essential role in senescence- and differentiation- induction of chronic inflammation. ROS generated by associated growth inhibition. Mechanistically, hPNPaseold-35- mitochondria induce oxidative stress in DNA, protein induced senescence is associated with an increase in and lipid and this activity is more prominent in tissues

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1739 from aged individuals or aged experimental animals for maintaining homeostasis comes from both loss-of- than their young counterparts (Harman, 1957; Hagen function and gain-of-function studies, although contra- et al., 1997; Chen, 2000; Finkel and Holbrook, 2000). A dictory results also indicate that knocking down prominent mechanism by which ROS modulates diverse hPNPaseold-35 does not affect mitochondrial morphology intracellular molecular processes is by turning on the or the rate of oxygen consumption (Nagaike et al., expression of proinflammatory cytokines (Baldwin, 2005). Knocking down of hPNPaseold-35 reduced mito- 1996) and through regulating the activity of nuclear chondrial DC and enzymatic activities of couple factor (NF)-kB (Schreck et al., 1992). hPNPaseold-35 is respiratory complexes compared with control cells localized in mitochondria and induces ROS that resulting in mitochondrial dysfunction such as lactate subsequently leads to NF-kB activation, which is accumulation and reduction of steady-state ATP levels inhibited by anti-oxidant N-acetyl-L-cysteine. Activation (Chen et al., 2006a, b, c). Concurrently, overexpression of NF-kB leads to increased production of pro- results in increased ROS accumulation over time inflammatory cytokines such as interleukin-6, interleukin-8, confirming a role for hPNPaseold-35 in mitochondrial RANTES and matrix metalloproteinase-3 (MMP-3), homeostasis (Sarkar et al., 2004). However, it is not which could also be inhibited by treatment with understood how mitochondrial homeostasis is precisely N-acetyl-L-cysteine suggesting the involvement of regulated by hPNPaseold-35, as no known substrates are hPNPaseold-35 in producing pathological changes asso- located in the intermembrane space. Accordingly, one ciated with aging by generating pro-inflammatory must also consider potential non-enzymatic functions of cytokines via ROS and NF-kB (Sarkar et al., 2004; hPNPaseold-35 in maintenance of mitochondrial home- Sarkar and Fisher, 2006). ostasis. It is possible that hPNPaseold-35 affects oxidative phosphorylation directly by impacting on components of the respiratory complexes or indirectly through Overexpression of hPNPaseold-35-induced growth interference in mitochondrial fusion. Consistent with inhibition in different cancer cells and its molecular this possibility, a very recent study (Wang et al., 2010) mechanism showed that components of the electron transport On the basis of cell line studies it is observed that slow chains were reduced at both RNA and protein levels and sustained overexpression of hPNPaseold-35 induces in PNPase RNA interference-transfected HEK293 cells growth arrest ultimately culminating in apoptosis, as compared with controls. A similar result was also whereas rapid overexpression of hPNPaseold-35 directly observed in liver mitochondria from a liver-specific promotes apoptosis without cell cycle changes. These PNPase knockout mouse model suggesting that the observations imply that inhibition of cell cycle progres- decrease in functional electron transport chain com- sion and induction of apoptosis by hPNPaseold-35 plexes was responsible for decreased respiration. involves multiple intracellular targets and signaling Furthermore, the ultrastructure of liver mitochondria pathways. In the context of cell cycle, hPNPaseold-35 from liver-specific knockout mice displayed disordered overexpression induces growth arrest at both G /S and 1 circular and smooth inner membrane criste, similar to G /M phase depending on the cell types, although the 2 mitochondria having impaired components of oxidative molecular mechanism is quite similar (Sarkar et al., phosphorylation pathways. Citrate synthase activity, 2004; Chan et al., 2008; Van Maerken et al., 2009). In all routinely used as a marker of aerobic capacity, also cases, c-myc is downregulated by hPNPaseold-35 accom- decreased in the liver of PNPase knockout mice panied with upregulation of cyclin-dependent kinase compared with the wild-type mice establishing a pivotal inhibitor, p21CIP1/WAF-1/MDA-6 and p27KIP1 that have role of PNPase in mitochondrial morphogenesis and essential roles in cell cycle arrest either in the G or 1 respiration in vivo. Additionally, a number of studies G /M phase. Apoptosis-inducing activity of hPNPaseold-35 2 indicate that hPNPaseold-35 potentially maintains high- is mediated by activation of double-stranded RNA- fidelity translation by sequestering oxidative stress- dependent protein kinase (Sarkar et al., 2007). Activation damaged RNA (Hayakawa and Sekiguchi, 2006; Wu of RNA-dependent protein kinase by hPNPaseold-35 and Li, 2008) supporting potential exoribonuclease- precedes phosphorylation of eukaryotic initiation factor- independent activity of this enzyme in maintaining 2A and induction of growth arrest and DNA damage- normal mitochondrial function. inducible gene 153 (GADD153) that culminates in the shutdown of protein synthesis and apoptosis. Activation of RNA-dependent protein kinase by hPNPaseold-35 Role of PNPase in translocation of RNA in mitochondria also nitiates downregulation of the anti-apoptotic protein Although the molecular mechanism of mitochondrial Bcl-xL. All of these studies elucidate a novel pathway by import of nuclear-encoded proteins is well defined, little which an evolutionary conserved RNA-metabolizing is known about the factors that control mitochondrial enzyme, hPNPaseold-35, regulates cell growth and viability. RNA import. Wang et al. (2010) have now demonstrated a direct involvement of PNPase in regulating Role of hPNPaseold-35 in maintaining mitochondrial specific cytosolic RNA import into the mitochondrial homeostasis matrix. This function of PNPase is independent of its On the basis of the localization in the mitochondrial RNA-processing function, as inactivation of RNA intermembrane space, hPNPaseold-35 is predicted to have processing by mutation did not affect RNA import. a major role in mitochondrial bioenergetics. Evidence Mammalian RNase P localizes in mitochondria and

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1740

Figure 4 Schematic representation of the diversified functions of hPNPaseold-35. On type I IFNs binding with its corresponding receptor, hPNPaseold-35 transcription is promoted through activation of the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Once transcribed, hPNPaseold-35 is either imported into the mitochondria intermembrane space (IMS) or mobilized into the cytosol by unknown mechanism(s). In the cytoplasm, by executing exoribonuclease activity it specifically targets mRNA/miRNAs thereby significantly affecting various biological functions, for example, induction of cell cycle arrest by targeting c-myc. In addition to maintaining mitochondrial homeostasis, hPNPaseold-35 actively participates to block the translation of oxidative stress-mediated damaged RNA by sequestering it. In addition, although not evident, hPNPaseold-35 might also be involved in removal of truncated mtRNA from mitochondria.

functions in the processing of transfer RNAs during the therapy. miRNAs have recently been shown to be maturation of mitochondrial transcripts (Chang and involved in developing drug resistance and thus combi- Clayton, 1987). RNase P is encoded in the nucleus and natorial therapy of hPNPaseold-35 and appropriate ther- PNPase is directly involved in translocation of this apeutic drug might be an effective approach for enzyme. However, the exact mechanism by which combating cancer. The lack of expression in different PNPase augments RNA import is not yet known. It is cancer cells while the ubiquitious expression in multiple hypothesized that PNPase imports RNAs from the organ/tissues raises the relevant question whether hPNPa- cytosol into the intermembrane space and then passes seold-35 serves any specialized function in specific organs? this RNA to another protein or complex that facilitates Animal models with either conditional overexpression or passage of the RNAs through the innermembrane into knockdown of PNPase expression will be extremely the matrix. Interestingly PNPase showed specificity valuable in answering these questions. Importantly, these toward import of cytosolic RNAs based on their models will facilitate studies on senescence and IFN secondary structure. Consistent with in vitro findings, action, including its anti-viral properties and role in mitochondrial RNA import was severely compromised inflammatory disease processes as well as maintaining in the liver mitochondria from liver-specific PNPase mitochondrial homeostasis. Strategies using natural com- (pnpt1) knockout mice (Wang et al., 2010). pounds and/or small molecules to block the enzymatic functions of hPNPaseold-35 might provide effective therapeutic benefit for inflammation or even provide a means Summary of retarding the aging process and enhancing longevity. Overall, delineation of the nuances of how The gradual unraveling of the multifaceted functions of hPNPaseold-35 induces its diverse effects in different old-35 hPNPase illustrates its prominent role in regulating organisms offers significant potential to comprehend diverse physiological and pathological processes the evolutionary relevance of this molecule and this will old-35 (Figure 4). As hPNPase displays substrate specifi- help delineate its quintessential role in both normal city, either mRNA or miRNA, the elucidation of how it physiology and in disease pathology. elicits such specificity will significantly improve our understanding of its RNA-processing functions. The preferential degradative effects on specific miRNAs might be applicable to selectively downregulate oncogenic Conflict of interest miRNA in different malignancies and thus might be amenable for developing a novel potential anticancer The authors declare no conflict of interest.

Oncogene Human polynucleotide phosphorylase and RNA degradation SK Das et al 1741 Acknowledgements National Foundation for Cancer Research (PBF) and NIH grant R01 CA134721 (DS). DS is the Harrison Endowed This study was supported in part by NIH grants R01 Scholar in Cancer Research. PBF holds the Thelma Newmeyer CA097318, R01 CA127641, CA134721, and P01 CA104177, Corman Chair in Cancer Research and is a SWCRF the Samuel Waxman Cancer Research Foundation and the Investigator.

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