title: Identification of novel targets for miR-29a using miRNA proteomics.
abstract: MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3' Untranslated Regions (3' UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.
authors.affiliation: Functional Genomics Unit, Council of Scientific Industrial Research-Institute of Genomics and Integrative Biology, Delhi, India.
authors.forename: Rhishikesh
authors.initials: R
authors.lastname: Bargaje
authors.forename: Shivani
authors.initials: S
authors.lastname: Gupta
authors.forename: Ali
authors.initials: A
authors.lastname: Sarkeshik
authors.forename: Robin
authors.initials: R
authors.lastname: Park
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.forename: Maharnob
authors.initials: M
authors.lastname: Sarkar
authors.forename: Mahantappa
authors.initials: M
authors.lastname: Halimani
authors.forename: Soumya Sinha
authors.initials: SS
authors.lastname: Roy
authors.forename: John
authors.initials: J
authors.lastname: Yates
authors.forename: Beena
authors.initials: B
authors.lastname: Pillai
mesh_terms: D020413:3' Untranslated Regions; D025181:ATP Synthetase Complexes / Q000378:metabolism; D000465:Algorithms; D017209:Apoptosis; D018929:Cell Culture Techniques; D003198:Computer Simulation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis / Q000378:metabolism*; D008957:Models, Genetic; D040901:Proteomics / Q000379:methods*; D050995:Voltage-Dependent Anion Channel 1 / Q000378:metabolism; D050996:Voltage-Dependent Anion Channel 2 / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; C494762:VDAC1 protein, human; C494765:VDAC2 protein, human; D050996:Voltage-Dependent Anion Channel 2; D050995:Voltage-Dependent Anion Channel 1; D025181:ATP Synthetase Complexes