Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

Abstract

Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.

The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes

“…Interestingly, global analyses have shown that the levels of many transcripts are reduced when PNPase expression is decreased in human melanoma cells (Sokhi et al 2013) and E. coli (Mohanty and Kushner 2003), indicating that the stabilizing effect of the enzyme might be more general and not restricted to regulatory RNAs. It was unclear from the available data whether the genetic effects of PNPase on sRNAs was through a direct protection or an indirect mechanism, e.g., by reducing the levels of other ribonucleases or cofactors including repressive RNAs that might normally contribute to the degradation of these sRNAs.…”

Analysis of Global Changes in Gene Expression Induced by Human Polynucleotide Phosphorylase (hPNPaseold‐35)

“…Although these findings are a major step forward in delineating the functional roles this enzyme plays in cellular physiology, there is still limited understanding at the molecular level regarding the specific RNA species the enzyme targets for degradation, given the role of hPNPase old-35 in RNA processing. To this end, our prior work showed that manipulation of hPNPase old-35 expression in melanoma cells, i.e., its depletion or overexpression, caused genome wide alterations in numerous genes and pathways, with some of the most significant changes associated with mitochondrial function, cholesterol biosynthesis, cell cycle and cellular growth and proliferation [Sokhi et al, 2013b]. Apart from the identification of global cellular patterns of gene expression changes resulting from manipulation of hPNPase old-35 , we were able to validate certain known cellular targets of hPNPase old-35 function (e.g., mitochondria) and identify novel potential targets of hPNPase old-35 , which could pave the way for future therapeutic opportunities pertaining to hPNPase old-35 related disease states.…”

Whole-exome sequencing identifies novel variants in PNPT1 causing oxidative phosphorylation defects and severe multisystem disease

“…The PNPase protein consists of five domains, including two RNase PH-like 3′-5′ exoribonucleases domains, two RNA-binding domains, KH and S1, and an α-helical domain. [12][13][14] The N terminus of this protein has a mitochondrial targeting sequence that is required for subcellular localization to mitochondria. 15 PNPase localizes predominantly to the mitochondrial intermembrane space, and assembles into a homotrimeric complex, 7,15 but has also been found to be located in the mitochondrial matrix because of its interaction with the matrix-localised RNA helicase hSUV3, and in the cytoplasm, where it is involved in the degradation of microRNA and mRNA, 16 as well as regulation of the importation of cytosolic RNAs into the mitochondrial matrix.…”

“…15 PNPase localizes predominantly to the mitochondrial intermembrane space, and assembles into a homotrimeric complex, 7,15 but has also been found to be located in the mitochondrial matrix because of its interaction with the matrix-localised RNA helicase hSUV3, and in the cytoplasm, where it is involved in the degradation of microRNA and mRNA, 16 as well as regulation of the importation of cytosolic RNAs into the mitochondrial matrix. 14,17 Moreover, PNPase has an in vivo role in mitochondrial morphology and respiration through unknown mechanisms, 18 maintenance of mitochondrial homeostasis and the ability to protect cells from oxidative stress. 19 The evolutionary comparison, together with the in silico analysis, predicts that the p.(Gln254Lys) and p.(Ala510Pro) variants in PNPT1 are most likely to be pathogenic, despite that the Gln254 and Ala510 residues are not located in any of the PNPase functional domains and prompted us to make further investigations.…”