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Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Sikand K, Singh J, Ebron JS, Shukla GC - PLoS ONE (2012)

Bottom Line: The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked.Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data.Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT
Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

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Related in: MedlinePlus

Conservation of miR-644a target site.Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.
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pone-0047510-g003: Conservation of miR-644a target site.Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.

Mentions: Next, we asked if the observed reduction in GAPDH and β-actin mRNA levels is a consequence of miR-644a interacting with the 3′ UTRs of these mRNAs. A search for GAPDH- and β-actin-targeting miRNAs using TargetScan Human (release 6.1) revealed a potential binding site for miR-644a in GAPDH 3′ UTR and β-actin 3′ UTR suggesting that miR-644a could be directly regulating the expression of these genes by binding to the predicted target sites. We checked the evolutionary conservation of miR-644a target site in GAPDH 3′ UTR (Figure 3A) and β-actin 3′ UTR (Figure 3B) in seven mammalian genomes. The seed binding region of miR-644a target site (shown in bold, Figures 3A, B) was found to be highly conserved in both sets of 3′ UTRs. In order to validate the direct interaction of miR-644a with its cognate target site, we cloned GAPDH 3′ UTR containing the wild type (WT) or mutated (MUT) miR-644a target site in a firefly luciferase reporter vector (Figure 4A). Similar luciferase reporter constructs were made using a segment of β-actin 3′ UTR (Figure 5A). In the GAPDH MUT-3′ UTR construct, nucleotides 1183 to 1187 of the target site were mutated to their complementary nucleotides to disrupt any potential base-pairing interaction of miR-644a (Figure 4A). In the β-actin MUT-3′ UTR construct, nucleotides 1562 and 1563 of miR-644a binding site were mutated (Figure 5A). Each reporter construct (WT-3′ UTR or MUT-3′ UTR) was cotransfected with either miR-644a mimic or NC mimic in CHO-K1 cells and luciferase activity was measured 24 hours post-transfection. In experiments where miR-644a mimic was cotransfected with WT-3′ UTR luciferase reporter construct, we observed a marked repression of luciferase activity (Figures 4B and 5B). As expected, in experiments where miR-644a mimic was cotransfected with MUT-3′ UTR construct, a reversal of luciferase expression was observed (Figures 4B and 5B). Taken together, these data show that miR-644a represses GAPDH and β-actin expression by directly interacting with its target sequence in the respective 3′ UTRs.


Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Sikand K, Singh J, Ebron JS, Shukla GC - PLoS ONE (2012)

Conservation of miR-644a target site.Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472982&req=5

pone-0047510-g003: Conservation of miR-644a target site.Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.
Mentions: Next, we asked if the observed reduction in GAPDH and β-actin mRNA levels is a consequence of miR-644a interacting with the 3′ UTRs of these mRNAs. A search for GAPDH- and β-actin-targeting miRNAs using TargetScan Human (release 6.1) revealed a potential binding site for miR-644a in GAPDH 3′ UTR and β-actin 3′ UTR suggesting that miR-644a could be directly regulating the expression of these genes by binding to the predicted target sites. We checked the evolutionary conservation of miR-644a target site in GAPDH 3′ UTR (Figure 3A) and β-actin 3′ UTR (Figure 3B) in seven mammalian genomes. The seed binding region of miR-644a target site (shown in bold, Figures 3A, B) was found to be highly conserved in both sets of 3′ UTRs. In order to validate the direct interaction of miR-644a with its cognate target site, we cloned GAPDH 3′ UTR containing the wild type (WT) or mutated (MUT) miR-644a target site in a firefly luciferase reporter vector (Figure 4A). Similar luciferase reporter constructs were made using a segment of β-actin 3′ UTR (Figure 5A). In the GAPDH MUT-3′ UTR construct, nucleotides 1183 to 1187 of the target site were mutated to their complementary nucleotides to disrupt any potential base-pairing interaction of miR-644a (Figure 4A). In the β-actin MUT-3′ UTR construct, nucleotides 1562 and 1563 of miR-644a binding site were mutated (Figure 5A). Each reporter construct (WT-3′ UTR or MUT-3′ UTR) was cotransfected with either miR-644a mimic or NC mimic in CHO-K1 cells and luciferase activity was measured 24 hours post-transfection. In experiments where miR-644a mimic was cotransfected with WT-3′ UTR luciferase reporter construct, we observed a marked repression of luciferase activity (Figures 4B and 5B). As expected, in experiments where miR-644a mimic was cotransfected with MUT-3′ UTR construct, a reversal of luciferase expression was observed (Figures 4B and 5B). Taken together, these data show that miR-644a represses GAPDH and β-actin expression by directly interacting with its target sequence in the respective 3′ UTRs.

Bottom Line: The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked.Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data.Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT
Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

Show MeSH
Related in: MedlinePlus