Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis.
Dicer is a crucial RNase III enzyme in miRNA biogenesis pathway. Although numerous studies have been carried out to investigate the role of miRNAs and Dicer in the regulation of biological processes, few studies have examined proteomic alterations upon knockout of Dicer. We employed a Cre-loxP-based inducible knockout mouse system to investigate the proteome regulated by Dicer-dependent miRNAs. We utilized spiked liver lysates from metabolically labeled mice to quantify the subtle changes in the liver proteome upon deletion of Dicer. We identified 2137 proteins using high resolution tandem mass spectrometry analysis. The upregulated proteins included several enzymes involved in peroxisomal β-oxidation of fatty acids and a large majority of the upregulated proteins involved in lipid metabolism were known PPARα targets. MRM-based assays were carried out to confirm the upregulation of enzymes including peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, cytochrome P450 3A13, cytochrome P450 3A41 and myristoylated alanine-rich protein kinase C substrate. Further, miRNA-124 which is predicted to regulate expression of peroxisomal bifunctional enzyme was confirmed to be downregulated in the Dicer knockout mice. Our study demonstrates the strength of coupling knockout mouse models and quantitative proteomic strategies to investigate functions of individual proteins in vivo.


CONCLUSIONS
Dicer dependent miRNA biogenesis is the major pathway for generation of mature miRNAs. We developed SILAC mouse-based proteomics screen to identify protein targets of Dicer-dependent miRNAs in liver of Dicer knockout mice. We spiked liver lysates of induced and uninduced Dicer knockout mice with liver lysate of SILAC labeled mice for identification of dysregulated proteome. We quantitated 1217 proteins of which 257 were upregulated in induced Dicer knockout mice. We observed enrichment of PPAR-α targets and proteins involved in lipid metabolism among upregulated proteins. We further carried out MRM-based validation of peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, Cyp3A13, Cyp3A41 and myristoylated alanine-rich protein kinase C substrate. We further validated upregulation of peroxisomal bifunctional enzyme using Western blot analysis and downregulation of its predicted upstream miRNA, miR-124 using qRT-PCR. Our study demonstrates that upon ablation of Dicer, certain Dicer-dependent miRNAs are dysregulated which result in dysregulation of their target proteins such as proteins associated with lipid metabolism. Our study illustrates the use of SILAC strategy for quantitative proteomic investigations of animal model systems.
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India; Manipal University, Madhav Nagar, Manipal 576104, India.
Nandini A
NA
Sahasrabuddhe
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Tai-Chung
TC
Huang
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Sartaj
S
Ahmad
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Min-Sik
MS
Kim
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Yi
Y
Yang
Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Bidyut
B
Ghosh
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Steven D
SD
Leach
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Harsha
H
Gowda
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Babu L
BL
Somani
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India; Manipal University, Madhav Nagar, Manipal 576104, India; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Raghothama
R
Chaerkady
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: pandey@jhmi.edu.
Akhilesh
A
Pandey
D000818:Animals; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism / Q000502:physiology*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D047493:PPAR alpha / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D035683:MicroRNAs; C540079:Mirn124 microRNA, mouse; D047493:PPAR alpha; D020543:Proteome; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
Dicer; Peroxisomal β-oxidation; Quantitative proteomics; SILAC mouse