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.
Digital Object Identifier (DOI)
National Institutes of Health, Bethesda, MD (grants R01 NS061933, R01 GM089784 and K08 NS050110); Alzheimer Association (NIRG-89917). Funding for open access charge: National Institutes of Health (grants R21AG036875, R01NS061933).
Nelson, Peter T.; Wang, Wang-Xia; Mao, Guogen; Wilfred, Bernard R.; Xie, Kevin; Jennings, Mary H.; Gao, Zhen; and Wang, Xiaowei, "Specific Sequence Determinants of miR-15/107 MicroRNA Gene Group Targets" (2011). Pathology and Laboratory Medicine Faculty Publications. 8.
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Nelson_PT_et_al_Suppl_Fig_1.ppt (255 kB)
Supplemental Figure 1
Nelson_PT_et_al_Supplemental_Table_1.xlsx (2779 kB)
Supplemental Table 1
Nelson_PT_et_al_Suppl_Tables_2_and_3_REVISED_FINAL.ppt (407 kB)
Supplemental Tables 2 and 3
Nelson_PT_et_al_Supplemental_Figures_REVISED_FINAL.ppt (379 kB)
Supplemental_Figure_Legends.doc (35 kB)
Supplemental Table Legend