Upregulation of PARG in prostate cancer cells suppresses their malignant behavior and downregulates tumor-promoting genes.

Post-translational modification of nuclear proteins through the addition of poly(ADP-ribose) (pADPr) moieties is upregulated in many metastatic cancers, where the high levels of pADPr have often been associated with poor cancer prognosis. Although the inhibitors of poly(ADP-ribose) polymerases (PARPs) have been utilized as potent anti-cancer agents, their efficacy in clinical trials varied among patient groups and has often been unpredictable. Such outcome cannot be interpreted solely by the inability to keep PARP-driven DNA repair in check. The focus of studies on PARP-driven tumorigenesis have recently been shifted toward PARP-dependent regulation of transcription. Here we utilized the controlled overexpression of poly(ADP-ribose) glycohydrolase (PARG), a sole pADPr-degrading enzyme, to investigate pADPr-dependent gene regulation in prostate cancer PC-3 cells. We demonstrated that PARG upregulation reduces pADPr levels and inhibits the expression of genes in key tumor-promoted pathways, including TNFα/NF-kB, IL6/STAT3, MYC, and KRAS signaling, the genes involved in inflammation response, especially chemokines, and endothelial-mesenchymal transition. The observed effect of PARG on transcription was consistent across all tested prostate cancer cell lines and correlates with PARG-induced reduction of clonogenic potential of PC-3 cells in vitro and a significant growth inhibition of PC-3-derived tumors in nude mice in vivo.

PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment.

Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly (ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly (ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG's overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3 T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells' tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3 T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

Poly(ADP)-Ribosylation Inhibition: A Promising Approach for Clear Cell Renal Cell Carcinoma Therapy.

Poly(ADP-ribose) polymerase 1 (PARP-1) and glycohydrolase (PARG) enzymes regulate chromatin structure, transcription activation, and DNA repair by modulating poly(ADP-ribose) (pADPr) level. Interest in PARP-1 inhibitors has soared recently with the recognition of their antitumor efficacy. We have shown that the development of clear cell renal cell carcinoma (ccRCC) is associated with extreme accumulation of pADPr caused by the enhanced expression of PARP-1 and decreased PARG levels. The most severe misregulation of pADPr turnover is found in ccRCC specimens from metastatic lesions. Both, classical NAD-like and non-NAD-like PARP-1 inhibitors reduced viability and clonogenic potential of ccRCC cell lines and suppressed growth of ccRCC xenograft tumors. However, classical NAD-like PARP-1 inhibitors affected viability of normal kidney epithelial cells at high concentrations, while novel non-NAD-like PARP-1 inhibitors exhibited activity against malignant cells only. We have also utilized different approaches to reduce the pADPr level in ccRCC cells by stably overexpressing PARG and demonstrated the prominent antitumor effect of this "back-to-normal" intervention. We also generated ccRCC cell lines with stable overexpression of PARG under doxycycline induction. This genetic approach demonstrated significantly affected malignancy of ccRCC cells. Transcriptome analysis linked observed phenotype with changes in gene expression levels for lipid metabolism, interferon signaling, and angiogenesis pathways along with the changes in expression of key cancer-related genes.

The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3'-UTRs of yeast mitochondrial mRNAs.

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.

Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions.

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification.

The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity.