Specific sequence determinants of miR-15/107 microRNA gene group targets. Homo sapiens

Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, ‘RIP-Chip’ experiments enable direct analyses of miRNA targets. 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. For detailed protocol and for full discussion of the results please see Nelson PT et al, Nucleic Acids Res. 2011 Oct;39(18):8163-72. Affymetrix microarrays which were performed 2 days following miRNA transfections: total lysate following miRNA-like transfections; and RNA from anti-Argonaute (2A8 monoclonal antibody) co-Ips Overall design: 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), another physiological miRNA (miR-320), miR-15b*, and three non-physiological additional miRNA-like molecules (miR-107-mut1, mir-107-mut2, and negative control miRNA from Ambion). For details on transfected miRNA sequences, see Nelson PT et al, Nucleic Acids Res. 2011 Oct;39(18):8163-72. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. See Wang WX et al, RNA (2010) 16:394-404 for detailed protocol of RIP-ChIP.