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A functional assay for microRNA target identification and validation.

Gäken J, Mohamedali AM, Jiang J, Malik F, Stangl D, Smith AE, Chronis C, Kulasekararaj AG, Thomas NS, Farzaneh F, Tavassoli M, Mufti GJ - Nucleic Acids Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematological Medicine, King's College London, Rayne Institute, London SE5 9NU, UK. joop.gaken@kcl.ac.uk

ABSTRACT
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.

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Schematic representation of the functional assay for miRNA target discovery.
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gks145-F1: Schematic representation of the functional assay for miRNA target discovery.

Mentions: Cells that do not express the miRNA of interest are transfected with a library of genes cloned downstream of a TKzeo fusion gene that provides resistance to zeocin and sensitivity to GCV (see Figure 1 for a schematic representation of the target identification strategy). Zeocin selection results in the isolation of transduced cells (i.e. the target cell library). The subsequent introduction of the miRNA of interest into the target cell library results in the down-regulation of TKzeo in cells containing a cDNA with a target site for the introduced miRNA abrogating the zeocin resistance but by the same token gaining resistance to GCV due to reduced expression of TKzeo.Figure 1.


A functional assay for microRNA target identification and validation.

Gäken J, Mohamedali AM, Jiang J, Malik F, Stangl D, Smith AE, Chronis C, Kulasekararaj AG, Thomas NS, Farzaneh F, Tavassoli M, Mufti GJ - Nucleic Acids Res. (2012)

Schematic representation of the functional assay for miRNA target discovery.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378903&req=5

gks145-F1: Schematic representation of the functional assay for miRNA target discovery.
Mentions: Cells that do not express the miRNA of interest are transfected with a library of genes cloned downstream of a TKzeo fusion gene that provides resistance to zeocin and sensitivity to GCV (see Figure 1 for a schematic representation of the target identification strategy). Zeocin selection results in the isolation of transduced cells (i.e. the target cell library). The subsequent introduction of the miRNA of interest into the target cell library results in the down-regulation of TKzeo in cells containing a cDNA with a target site for the introduced miRNA abrogating the zeocin resistance but by the same token gaining resistance to GCV due to reduced expression of TKzeo.Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematological Medicine, King's College London, Rayne Institute, London SE5 9NU, UK. joop.gaken@kcl.ac.uk

ABSTRACT
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.

Show MeSH