Dataset Information

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.

ABSTRACT: Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

SUBMITTER: Jovanovic M

PROVIDER: S-EPMC3444237 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.

Jovanovic Marko M Reiter Lukas L Picotti Paola P Lange Vinzenz V Bogan Erica E Hurschler Benjamin A BA Blenkiron Cherie C Lehrbach Nicolas J NJ Ding Xavier C XC Weiss Manuel M Schrimpf Sabine P SP Miska Eric A EA Grosshans Helge H Aebersold Ruedi R Hengartner Michael O MO

Nature methods 20100912 10

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and lucif ...[more]

PMID: 20835247

Similar Datasets

Project description:Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIS1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

Project description:Celastrol, a natural substance isolated from plant extracts used in traditional Chinese medicine, has been extensively investigated as a possible drug for treatment of cancer, autoimmune diseases, and protein misfolding disorders. Although studies focusing on celastrol's effects in specific cellular pathways have revealed a considerable number of targets in a diverse array of in vitro models there is an essential need for investigations that can provide a global view of its effects. To assess cellular effects of celastrol and to identify target proteins as biomarkers for monitoring treatment regimes, we performed large-scale quantitative proteomics in cultured human lymphoblastoid cells, a cell type that can be readily prepared from human blood samples. Celastrol substantially modified the proteome composition and 158 of the close to 1800 proteins with robust quantitation showed at least a 1.5 fold change in protein levels. Up-regulated proteins play key roles in cytoprotection with a prominent group involved in quality control and processing of proteins traversing the endoplasmic reticulum. Increased levels of proteins essential for the cellular protection against oxidative stress including heme oxygenase 1, several peroxiredoxins and thioredoxins as well as proteins involved in the control of iron homeostasis were also observed. Specific analysis of the mitochondrial proteome strongly indicated that the mitochondrial association of certain antioxidant defense and apoptosis-regulating proteins increased in cells exposed to celastrol. Analysis of selected mRNA transcripts showed that celastrol activated several different stress response pathways and dose response studies furthermore showed that continuous exposure to sub-micromolar concentrations of celastrol is associated with reduced cellular viability and proliferation. The extensive catalog of regulated proteins presented here identifies numerous cellular effects of celastrol and constitutes a valuable biomarker tool for the development and monitoration of disease treatment strategies.

Dataset Information

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.

Publications

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets