A functional assay for microRNA target identification and validation.
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.
Department of Haematological Medicine, King's College London, Rayne Institute, London SE5 9NU, UK. joop.gaken@kcl.ac.uk
Joop
J
Gäken
Azim M
AM
Mohamedali
Jie
J
Jiang
Farooq
F
Malik
Doris
D
Stangl
Alexander E
AE
Smith
Constantinos
C
Chronis
Austin G
AG
Kulasekararaj
N Shaun B
NS
Thomas
Farzin
F
Farzaneh
Mahvash
M
Tavassoli
Ghulam J
GJ
Mufti
D020413:3' Untranslated Regions; D002460:Cell Line; D003001:Cloning, Molecular; D015536:Down-Regulation; D015774:Ganciclovir / Q000494:pharmacology; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D015723:Gene Library; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D020413:3' Untranslated Regions; C554019:MIRN130 microRNA, human; D035683:MicroRNAs; D015774:Ganciclovir