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

Abstract

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.

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, where it serves as a guide to target and regulate specific mRNAs. By executing exonuclease activity hPNPase^old-35 specifically degrades mature miRNAs. However, their substrate recognition mechanism is unknown.

Figure 2. hPNPase^old-35 target miRNAs

HO-1 cells were either infected with Ad.vec or Ad.hPNPase^old-35 at a m.o.i. of 5000 vp/cells for three 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 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 hPNPase^old-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., PNAS, 2010)

Figure 3. hPNPase^old-35 preferentially degrades miR-221

(A) Comparative degradation between miR-221, miR-222 and miR-106b at a 2 h time point using in vitro degradation assays. P value was calculated using student’s t-test by comparing the specific 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 translated hPNPase^old-35 was confirmed by immunoprecipitation and Northern blotting.

Figure 4. Schematic representation of the diversified functions of hPNPase^old-35

Upon Type I IFNs binding with its corresponding receptor, hPNPase^old-35 transcription is promoted through activation of the JAK/STAT pathway. Once transcribed, hPNPase^old-35 is either imported into the mitochondria 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, e.g., induction of cell cycle arrest by targeting c-myc. In addition to maintaining mitochondrial homeostasis, hPNPase^old-35 actively participates to block the translation of oxidative stress-mediated damaged RNA by sequestering it. In addition, although not evident, hPNPase^old-35 might also be involved in removal of truncated mtRNA from mitochondria.