title: Specific sequence determinants of miR-15/107 microRNA gene group targets.
abstract: MicroRNAs (miRNAs) target mRNAs in human cells via complex mechanisms that are still incompletely understood. Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, 'RIP-Chip' experiments enable direct analyses of miRNA targets. RIP-Chip studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), and five control miRNAs. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. Computational analyses queried for determinants of miRNA:mRNA binding. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3' portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3'-untranslated region targeting, and stable AGO association versus mRNA knockdown. Future studies should take this important miRNA-to-miRNA variability into account.
authors: Department of Pathology and Division of Neuropathology, University of Kentucky Medical Center, Lexington, KY 40536, USA. pnels2@email.uky.edu
authors: Peter T
authors: PT
authors: Nelson
authors: Wang-Xia
authors: WX
authors: Wang
authors: Guogen
authors: G
authors: Mao
authors: Bernard R
authors: BR
authors: Wilfred
authors: Kevin
authors: K
authors: Xie
authors: Mary H
authors: MH
authors: Jennings
authors: Zhen
authors: Z
authors: Gao
authors: Xiaowei
authors: X
authors: Wang
mesh_terms: D060565:Argonaute Proteins / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D015536:Down-Regulation; D020869:Gene Expression Profiling; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000737:chemistry* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*
chemical_list: D060565:Argonaute Proteins; C549045:MIRN107 microRNA, human; C505361:MIRN15 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger