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A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Goldoni D, Yarham JM, McGahon MK, O'Connor A, Guduric-Fuchs J, Edgar K, McDonald DM, Simpson DA, Collins A - Biochem. J. (2012)

Bottom Line: The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis.The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells.This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University of Belfast, Institute of Clinical Science, Block A, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, UK.

ABSTRACT
Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

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Construct design and predicted target site(A) Structure of pmChKir2.1UTR after linearization with PasI. Location of features: pUC origin of replication, bp 182–825; CMV promoter (human CMV immediate early promoter), bp 908–1486; mCherry (human codon optimized), bp 1510–2229; Kir2.1 3′UTR, bp 2244–6161; SV40 early promoter (and enhancer sequences), bp 6274–6542; SV40 early polyA signals, bp 6441–6576; Kanr/Neor (Kanamycin/neomycin resistance gene), bp 6625–7419; HSV TK polyA (herpes simplex virus thymidine kinase polyadenylation signals), bp 7655–7673; and the primers (5′ ends of complementary), forward bp 2128 and reverse bp 6171. (B) Predicted base pairing between miR-212 and the 3′UTR of human Kir2.1 (KCNJ2). Upper sequence, bp 2677–2683 of the KCNJ2 3′UTR (5′→3′); lower sequence, miR-212 (3′→5′).
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Figure 1: Construct design and predicted target site(A) Structure of pmChKir2.1UTR after linearization with PasI. Location of features: pUC origin of replication, bp 182–825; CMV promoter (human CMV immediate early promoter), bp 908–1486; mCherry (human codon optimized), bp 1510–2229; Kir2.1 3′UTR, bp 2244–6161; SV40 early promoter (and enhancer sequences), bp 6274–6542; SV40 early polyA signals, bp 6441–6576; Kanr/Neor (Kanamycin/neomycin resistance gene), bp 6625–7419; HSV TK polyA (herpes simplex virus thymidine kinase polyadenylation signals), bp 7655–7673; and the primers (5′ ends of complementary), forward bp 2128 and reverse bp 6171. (B) Predicted base pairing between miR-212 and the 3′UTR of human Kir2.1 (KCNJ2). Upper sequence, bp 2677–2683 of the KCNJ2 3′UTR (5′→3′); lower sequence, miR-212 (3′→5′).

Mentions: The pmR-mCherry plasmid (Clontech) was modified by removal of 774 bp downstream of the BamHI site in the multiple cloning site following the mCherry stop codon. First, a fragment incorporating bases 2168–2670 of pmR-mCherry was amplified using Phusion® Hot Start II High-Fidelity DNA Polymerase (Fermentas) with the forward primer, 5′-ATATATGGATCCTATGTATCCGCTCATGAGACAATAACCCTG-3′ and the reverse primer, 5′-CCCAAGCGGCCGGAGAAC-3′, and restricted with BamHI and EagI (Fermentas). This was then ligated into the gel-purified 3468 bp fragment generated by restriction of pmR-mCherry with BamHI and EagI. The resulting plasmid was restricted with XhoI (Fermentas) and BamHI and ligated to the 3.9 kb human Kir2.1 3′UTR sequence (GenBank® accession number NM_000891) amplified from human genomic DNA (Bioline) by a two-stage nested PCR using Phusion® Hot Start II High-Fidelity DNA Polymerase and the forward primer (outer), 5′-TGCCCTCACAAGCAAAGAGG-3′; the reverse primer (outer), 5′-CTGCACCTGTCTGTTGAGGC-3′; the forward primer (inner), 5′-AGGTCTCTCGAGCCCTGACATAGACCTTCACAAC-3′; and the reverse primer (inner) 5′-GACAGAGGATCCCATTTCTATGGCTCTATGC-3′, restricted with XhoI and BamHI. The final product (pmChKir2.1UTR) has the CMV (cytomegalovirus) promoter driving transcription of mCherry with the human Kir2.1 3′UTR (Figure 1A). The second stage of the nested PCR was performed with the forward primer, 5′-ATGCCTACTAGTCCCTGACATAGACCTTCACAAC-3′ and the reverse primer, 5′-GACAGAGTCGACCATTTCTATGGCTCTATGC-3′. The product was restricted with SpeI and SalI (Fermentas) and ligated into the NheI (Fermentas) and SalI sites of pmirGLO (Promega), downstream of the firefly luciferase coding sequence, to give pmirGLOKir2.1UTR.


A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Goldoni D, Yarham JM, McGahon MK, O'Connor A, Guduric-Fuchs J, Edgar K, McDonald DM, Simpson DA, Collins A - Biochem. J. (2012)

Construct design and predicted target site(A) Structure of pmChKir2.1UTR after linearization with PasI. Location of features: pUC origin of replication, bp 182–825; CMV promoter (human CMV immediate early promoter), bp 908–1486; mCherry (human codon optimized), bp 1510–2229; Kir2.1 3′UTR, bp 2244–6161; SV40 early promoter (and enhancer sequences), bp 6274–6542; SV40 early polyA signals, bp 6441–6576; Kanr/Neor (Kanamycin/neomycin resistance gene), bp 6625–7419; HSV TK polyA (herpes simplex virus thymidine kinase polyadenylation signals), bp 7655–7673; and the primers (5′ ends of complementary), forward bp 2128 and reverse bp 6171. (B) Predicted base pairing between miR-212 and the 3′UTR of human Kir2.1 (KCNJ2). Upper sequence, bp 2677–2683 of the KCNJ2 3′UTR (5′→3′); lower sequence, miR-212 (3′→5′).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475433&req=5

Figure 1: Construct design and predicted target site(A) Structure of pmChKir2.1UTR after linearization with PasI. Location of features: pUC origin of replication, bp 182–825; CMV promoter (human CMV immediate early promoter), bp 908–1486; mCherry (human codon optimized), bp 1510–2229; Kir2.1 3′UTR, bp 2244–6161; SV40 early promoter (and enhancer sequences), bp 6274–6542; SV40 early polyA signals, bp 6441–6576; Kanr/Neor (Kanamycin/neomycin resistance gene), bp 6625–7419; HSV TK polyA (herpes simplex virus thymidine kinase polyadenylation signals), bp 7655–7673; and the primers (5′ ends of complementary), forward bp 2128 and reverse bp 6171. (B) Predicted base pairing between miR-212 and the 3′UTR of human Kir2.1 (KCNJ2). Upper sequence, bp 2677–2683 of the KCNJ2 3′UTR (5′→3′); lower sequence, miR-212 (3′→5′).
Mentions: The pmR-mCherry plasmid (Clontech) was modified by removal of 774 bp downstream of the BamHI site in the multiple cloning site following the mCherry stop codon. First, a fragment incorporating bases 2168–2670 of pmR-mCherry was amplified using Phusion® Hot Start II High-Fidelity DNA Polymerase (Fermentas) with the forward primer, 5′-ATATATGGATCCTATGTATCCGCTCATGAGACAATAACCCTG-3′ and the reverse primer, 5′-CCCAAGCGGCCGGAGAAC-3′, and restricted with BamHI and EagI (Fermentas). This was then ligated into the gel-purified 3468 bp fragment generated by restriction of pmR-mCherry with BamHI and EagI. The resulting plasmid was restricted with XhoI (Fermentas) and BamHI and ligated to the 3.9 kb human Kir2.1 3′UTR sequence (GenBank® accession number NM_000891) amplified from human genomic DNA (Bioline) by a two-stage nested PCR using Phusion® Hot Start II High-Fidelity DNA Polymerase and the forward primer (outer), 5′-TGCCCTCACAAGCAAAGAGG-3′; the reverse primer (outer), 5′-CTGCACCTGTCTGTTGAGGC-3′; the forward primer (inner), 5′-AGGTCTCTCGAGCCCTGACATAGACCTTCACAAC-3′; and the reverse primer (inner) 5′-GACAGAGGATCCCATTTCTATGGCTCTATGC-3′, restricted with XhoI and BamHI. The final product (pmChKir2.1UTR) has the CMV (cytomegalovirus) promoter driving transcription of mCherry with the human Kir2.1 3′UTR (Figure 1A). The second stage of the nested PCR was performed with the forward primer, 5′-ATGCCTACTAGTCCCTGACATAGACCTTCACAAC-3′ and the reverse primer, 5′-GACAGAGTCGACCATTTCTATGGCTCTATGC-3′. The product was restricted with SpeI and SalI (Fermentas) and ligated into the NheI (Fermentas) and SalI sites of pmirGLO (Promega), downstream of the firefly luciferase coding sequence, to give pmirGLOKir2.1UTR.

Bottom Line: The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis.The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells.This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University of Belfast, Institute of Clinical Science, Block A, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, UK.

ABSTRACT
Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Show MeSH
Related in: MedlinePlus