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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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GCV selection of library transfected cells with or without mir-130a. Cells (106 MCF7 cells) transfected with p3′TKzeo + library (A) or MCF7 cells transfected with p3′TKzeo + library + pBabepuro empty vector (B) or transfected with p3′TKzeo + library + pBabepuro-mir-130a (C) were plated in 10-cm plates and selected in 8 μM GCV. After 10 days plates were stained with Coomassie blue.
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gks145-F3: GCV selection of library transfected cells with or without mir-130a. Cells (106 MCF7 cells) transfected with p3′TKzeo + library (A) or MCF7 cells transfected with p3′TKzeo + library + pBabepuro empty vector (B) or transfected with p3′TKzeo + library + pBabepuro-mir-130a (C) were plated in 10-cm plates and selected in 8 μM GCV. After 10 days plates were stained with Coomassie blue.

Mentions: Zeocin-resistant MCF7 cells were transfected with pBabepuro-mir-130a and after 48 h selected in 1 μg/ml puromycin resulting in approximately 10 000 individual MCF7 clones. As control zeocin-resistant MCF7 cells were transfected with empty vector pBabepuro. After 96 h 8 μM GCV was added and cells were selected for 10 days in both puromycin and GCV which resulted in GCV-resistant clones in mir-130a transfected cells but not in control pBabepuro transfected cells (Figure 3). Genomic DNA was isolated from the GCV-resistant cells, PCR amplified, TOPO-TA cloned and 96 of the resulting bacterial clones were sequenced. Of these 96 clones, 70 contained a MAFB insert. In addition, we identified TPT1 (tumour protein translationally controlled 1) in 3 clones, PRR14 (proline rich 14) in 3, KIFAP3 (kinesin-associated protein 3) in 3, MITD1 (microtubule interacting and transport domain containing 1) in 2, CYP27A1 (cytochrome P450 family 27 subfamily A polypeptide 1) in 2. The remainder of the tested clones had no insert in the Topo TA vector (Table 1). To further validate the procedure an identical small scale experiment was performed to identify targets for mir-10a, sequencing of 24 bacterial clones identified 4 putative targets, STMN2 (Stathmin like 2) in 9 clones, KATNA1 (Katanin p60) in 4, and CRMP1 (Collapsin response mediator protein 1) in 3. A further three clones contained HoxA1 (Homeobox A1) a validated target for mir-10a (13) (Table 1). Expression of both mature mir-130a and mir-10a in the transfected cells was measured by QRT–PCR 48 h after transfection and after puromycin selection (Supplementary Figure S6).Figure 3.


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)

GCV selection of library transfected cells with or without mir-130a. Cells (106 MCF7 cells) transfected with p3′TKzeo + library (A) or MCF7 cells transfected with p3′TKzeo + library + pBabepuro empty vector (B) or transfected with p3′TKzeo + library + pBabepuro-mir-130a (C) were plated in 10-cm plates and selected in 8 μM GCV. After 10 days plates were stained with Coomassie blue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks145-F3: GCV selection of library transfected cells with or without mir-130a. Cells (106 MCF7 cells) transfected with p3′TKzeo + library (A) or MCF7 cells transfected with p3′TKzeo + library + pBabepuro empty vector (B) or transfected with p3′TKzeo + library + pBabepuro-mir-130a (C) were plated in 10-cm plates and selected in 8 μM GCV. After 10 days plates were stained with Coomassie blue.
Mentions: Zeocin-resistant MCF7 cells were transfected with pBabepuro-mir-130a and after 48 h selected in 1 μg/ml puromycin resulting in approximately 10 000 individual MCF7 clones. As control zeocin-resistant MCF7 cells were transfected with empty vector pBabepuro. After 96 h 8 μM GCV was added and cells were selected for 10 days in both puromycin and GCV which resulted in GCV-resistant clones in mir-130a transfected cells but not in control pBabepuro transfected cells (Figure 3). Genomic DNA was isolated from the GCV-resistant cells, PCR amplified, TOPO-TA cloned and 96 of the resulting bacterial clones were sequenced. Of these 96 clones, 70 contained a MAFB insert. In addition, we identified TPT1 (tumour protein translationally controlled 1) in 3 clones, PRR14 (proline rich 14) in 3, KIFAP3 (kinesin-associated protein 3) in 3, MITD1 (microtubule interacting and transport domain containing 1) in 2, CYP27A1 (cytochrome P450 family 27 subfamily A polypeptide 1) in 2. The remainder of the tested clones had no insert in the Topo TA vector (Table 1). To further validate the procedure an identical small scale experiment was performed to identify targets for mir-10a, sequencing of 24 bacterial clones identified 4 putative targets, STMN2 (Stathmin like 2) in 9 clones, KATNA1 (Katanin p60) in 4, and CRMP1 (Collapsin response mediator protein 1) in 3. A further three clones contained HoxA1 (Homeobox A1) a validated target for mir-10a (13) (Table 1). Expression of both mature mir-130a and mir-10a in the transfected cells was measured by QRT–PCR 48 h after transfection and after puromycin selection (Supplementary Figure S6).Figure 3.

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