Analyzing the functions of human polynucleotide phosphorylase (hPNPaseold-35)

RNA degradation plays a fundamental role in maintaining cellular homeostasis, along with being a part of normal regulatory mechanisms, 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. hPNPaseold-35 has progressed a long way from being just a 3’-5’ exoribonuclease to a functionally relevant molecule implicated in a multitude of diverse and important biological effects. hPNPaseold-35 plays central roles in diverse physiological processes including growth inhibition, senescence, mtRNA import, mitochondrial homeostasis, and RNA degradation, all while primarily being localized in the mitochondrial IMS (inter membrane space). hPNPaseold-35 also holds immense promise as a therapeutic agent due to its ability to degrade specific miRNA (miR-221) and mRNA (c-myc) species, and this property can be exploited in treating malignancies that are characterized by upregulation of harmful miRNA or mRNA molecules. But apart from these two targets, little is known about any other targets hPNPaseold-35 may degrade. Thus, the primary objective of this dissertation research was to identify targets other than c-myc or miR-221 that hPNPaseold-35 could directly degrade to discover newer and biologically relevant therapeutic targets for the treatment of hPNPaseold-35 –associated disease states. In order to do this we performed extensive microarray analyses following hPNPaseold-35 overexpression and depletion in mammalian cell lines, and were able to identify transcripts that could be potentially regulated by hPNPaseold-35 directly or indirectly. Apart from this we also analyzed the 3’UTR of c-myc in order to identify any specific sequence or secondary structural elements necessary for hPNPaseold-35 mediated degradation. Lastly, we identified certain residues in hPNPaseold-35 that have been under positive natural selection through evolution

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

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

Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (<i>hPNPase^old-35</i>) Using Melanoma as a Model

<div><p>Human Polynucleotide Phosphorylase (<i>hPNPase^old-35</i> or <i>PNPT1</i>) 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 <i>c-myc</i> and miR-221. To further elucidate the functional implications of <i>hPNPase^old-35</i> in cellular physiology, we knocked-down and overexpressed <i>hPNPase^old-35</i> in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of <i>hPNPase^old-35</i> resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of <i>hPNPase^old-35</i> caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our <i>hPNPase^old-35</i> knockdown and overexpression datasets allowed us to identify 77 potential “<i>direct”</i> and 61 potential “<i>indirect”</i> targets of <i>hPNPase^old-35</i> which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to <i>hPNPase^old-35</i> 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.</p></div

Functional analysis of genes dysregulated as a result of <i>hPNPase^old-35</i> overexpression.

<p>(A) The biological functions and states associated with genes differentially expressed when <i>hPNPase^old-35</i> is overexpressed in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with genes dysregulated (proportions shown in graphs) after <i>hPNPase^old-35</i> overexpression in melanoma cells, as identified by IPA Toxicogenomic Analysis.</p

Generation of a melanoma cell culture model for hPNPase^old-35 expression.

<p>(A) Phase contrast LM (top) and GFP fluorescent micrographs (bottom) of HO-1 melanoma cell lines following transduction with GFP expressing scrambled shRNA (HO-1 Csh) and <i>hPNPase^old-35</i> shRNA1 (shown in clone 4; cl4) and 2 (shown is clone 9; cl9) expressing lentiviruses and selection with puromycin. qRT-PCR expression of <i>hPNPase^old-35</i> (<i>hPNPase^old-35</i> knockdown) normalized to control (shScramble). Mean values normalized to a GAPDH internal reference; error bars represent mean ± S.E. of three replicate experiments. Anti-hPNPase^old-35 and EF1α loading control immunoblots. (B) qRT-PCR expression of <i>hPNPase^old-35</i> in HO-1 cells infected with Ad.<i>hPNPase^old-35</i> normalized to cells infected with Ad.<i>Vec</i> for 36 h. Immunoblot showing hPNPase^old-35 overexpression compared to Ad.<i>Vec</i> post 36 hour of infection. Error bars represent mean ± S.E of three replicate experiments. <i>* P<0.02</i>, <i>*** P<0.001</i>.</p

Venn diagrams representing number of genes significantly altered when <i>hPNPase^old-35</i> is knocked down or overexpressed in human melanoma cells.

<p>Shown are total number of dysregulated genes (A), genes “<i>directly</i>” (B) and “<i>indirectly</i>” (C) regulated by <i>hPNPase^old-35</i>.</p

Functional analysis of <i>hPNPase^old-35</i>-putative “<i>indirectly</i>” regulated genes.

<p>(A) The biological functions and states associated with <i>hPNPase^old-35</i>-putative “<i>indirectly</i>” regulated genes in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with <i>hPNPase^old-35</i>-putative “<i>indirectly</i>” regulated genes, as identified by IPA Toxicogenomic Analysis.</p

Real time qRT-PCR validation of microarray findings.

<p>(A) qRT-PCR verification of <i>hPNPase^old-35</i>-putative “<i>directly</i>” regulated genes identified by microarray analyses in response to <i>hPNPase^old-35</i> (i) knockdown or (ii) overexpression in HO-1 melanoma cells. (B) qRT-PCR verification of <i>hPNPase^old-35</i>-putative “<i>indirectly</i>” regulated genes identified by microarray analyses in response to <i>hPNPase^old-35</i> (i) knockdown or (ii) overexpression in HO-1 melanoma cells. Error bars represent mean ± S.E. error of three replicate experiments.</p

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

<div><p>One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. <i>Mycobacterium tuberculosis</i> (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro <i>vs</i>. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of <i>rpoB>aroK>ppk>rpoC>ilvB</i>. <i>RpoB</i> was used as the cidality control. In-vitro and in-vivo studies feature <i>aroK</i> (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute infection phase for the high fidelity translation, the compound efficacy may also be evaluated in the low pH, in addition to the standard replication condition.</p></div