Dataset Information

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

ABSTRACT: The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.

SUBMITTER: Molinaro RJ

PROVIDER: S-EPMC1751551 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

Molinaro Ross J RJ Jha Babal Kant BK Malathi Krishnamurthy K Varambally Sooryanarayana S Chinnaiyan Arul M AM Silverman Robert H RH

Nucleic acids research 20061201 22

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3 ...[more]

PMID: 17145707

Similar Datasets

Project description:Background2'-5' oligoadenylate synthetases (OAS) are interferon inducible enzymes that polymerizes ATP to 2'-5'-linked oligomers of adenylate (2-5As). As part of the innate immune response, these enzymes are activated by viral double stranded RNA or mRNAs with significant double stranded structure. The 2-5As in turn activate RNaseL that degrade single stranded RNAs. Three distinct forms of OAS exist in human cells (OAS1, 2 and 3) with each form having multiple spliced variants. The OAS enzymes and their spliced variants have different enzyme activities. OAS enzymes also play a significant role in regulating multiple cellular processes such as proliferation and apoptosis. Moreover, Single nucleotide polymorphisms that alter OAS activity are also associated with viral infection, diabetes and cancer. Thus detection of OAS enzyme activity with a simple spectrophotometric method in cells will be important in clinical research.ResultsHere we propose a modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase (OAS) enzyme activity in prostate cell lines as a model system. The OAS enzyme from prostate cancer cell lysates was purified using Polyinosinic: polycytidylic acid (poly I:C) bound activated sepharose beads. The activated OAS enzyme eluted from Sepharose beads showed expression of p46 isoform of OAS1, generally considered the most abundant OAS isoform in elutes from DU14 cell line but not in other prostate cell line. In this assay the phosphates generated by the OAS enzymatic reaction is coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (methylthioguanosine, a guanosine analogue; MESG) to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a catalyst purine nucleoside phosphorylase (phosphorylase) using a commercially available pyrophosphate kit. The absorbance of the purine base product is measured at 360 nm. The higher levels of phosphates detected in DU145 cell line indicates more activity of OAS in this prostate cancer cell line.ConclusionThe modified simple method detected OAS enzyme activity with sensitivity and specificity, which could help in detection of OAS enzymes avoiding the laborious and radioactive methods.

Project description:BackgroundOligoadenylate synthetases (OASs) are widely distributed in Metazoa including sponges, fish, reptiles, birds and mammals and show large variation, with one to twelve members in any given species. Upon double-stranded RNA (dsRNA) binding, avian and mammalian OASs generate the second messenger 2'-5'-linked oligoadenylate (2-5A), which activates ribonuclease L (RNaseL) and blocks viral replication. However, how Metazoa shape their OAS repertoires to keep evolutionary balance to virus infection is largely unknown. We performed comprehensive phylogenetic and functional analyses of OAS genes from evolutionarily lower to higher Metazoa to demonstrate how the OAS repertoires have developed anti-viral activity and diversified their functions.ResultsAncient Metazoa harbor OAS genes, but lack both upstream and downstream genes of the OAS-related pathways, indicating that ancient OASs are not interferon-induced genes involved in the innate immune system. Compared to OASs of ancient Metazoa (i.e. sponge), the corresponding ones of higher Metazoa present an increasing number of basic residues on the OAS/dsRNA interaction interface. Such an increase of basic residues might improve their binding affinity to dsRNA. Moreover, mutations of functional residues in the active pocket might lead to the fact that higher Metazoan OASs lose the ability to produce 3'-5'-linked oligoadenylate (3-5A) and turn into specific 2-5A synthetases. In addition, we found that multiple rounds of gene duplication and domain coupling events occurred in the OAS family and mutations at functionally critical sites were observed in most new OAS members.ConclusionsWe propose a model for the expansion of OAS members and provide comprehensive evidence of subsequent neo-functionalization and sub-functionalization. Our observations lay the foundation for interrogating the evolutionary transition of ancient OAS genes to host defense genes and provide important information for exploring the unknown function of the OAS gene family.

Dataset Information

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

Publications

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets