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Regulation of Huntingtin Gene Expression by miRNA-137, -214, -148a, and Their Respective isomiRs

Kozlowska, Emilia, Krzyzosiak, Wlodzimierz, Koscianska, Edyta·2013·International Journal of Molecular Sciences
DOI 10.3390/ijms140816999PMC PMC3759948
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Abstract

With the advent of deep sequencing technology, a variety of miRNA length and sequence variants, termed isomiRNAs (isomiRs), have been discovered. However, the functional roles of these commonly detected isomiRs remain unknown. In this paper, we demonstrated that miRNAs regulate the expression of the HTT gene, whose mutation leads to Huntington’s disease (HD), a hereditary degenerative disorder. Specifically, we validated the interactions of canonical miRNAs, miR-137, miR-214, and miR-148a, with the HTT 3'UTR using a luciferase assay. Moreover, we applied synthetic miRNA mimics to examine whether a slight shifting of miRNA seed regions might alter the regulation of the HTT transcript. We also examined miR-137, miR-214, and miR-148a isomiRs and showed the activity of these isoforms on reporter constructs bearing appropriate sequences from the HTT 3'UTR. Hence, we demonstrated that certain 5'-end variants of miRNAs might be functional for the regulation of the same targets as canonical miRNAs.

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Figure 1 Graphical presentation of selected miRNA target site distribution in the 3′ untranslated region (3′UTR) of the huntingtin transcript. To predict miRNAs that potentially target the HTT 3′UTR, the TargetScanHuman algorithm (Release 6.2) [46] was used. (A) Regions of interaction for the miRNAs selected for experimental validation; (B) miRNA base pairing with an appropriate target sequence is schematically presented.
Figure 1 Graphical presentation of selected miRNA target site distribution in the 3′ untranslated region (3′UTR) of the huntingtin transcript. To predict miRNAs that potentially target the HTT 3′UTR, the TargetScanHuman algorithm (Release 6.2) [46] was used. (A) Regions of interaction for the miRNAs selected for experimental validation; (B) miRNA base pairing with an appropriate target sequence is schematically presented.CC BY · PubMed Central
Figure 2 Endogenous expression and overexpression of miR-137, miR-214, and miR-148a in HEK293T cells. Northern blot detection of miRs 137, 214, and 148a in non-treated HEK293T cells and cells transfected with miRNA-coding plasmids (System Biosciences, Open Biosystems). M denotes the size marker, end-labeled 17, 19, 21, 23, and 25-nt oligoribonucleotides. En and Ex indicate the miRNA levels, endogenous and expressed from appropriate vectors, respectively. Hybridization to U6 RNA provides a loading control.
Figure 2 Endogenous expression and overexpression of miR-137, miR-214, and miR-148a in HEK293T cells. Northern blot detection of miRs 137, 214, and 148a in non-treated HEK293T cells and cells transfected with miRNA-coding plasmids (System Biosciences, Open Biosystems). M denotes the size marker, end-labeled 17, 19, 21, 23, and 25-nt oligoribonucleotides. En and Ex indicate the miRNA levels, endogenous and expressed from appropriate vectors, respectively. Hybridization to U6 RNA provides a loading control.CC BY · PubMed Central
Figure 3 Regulation of the huntingtin (HTT) expression by canonical miRNAs. (A) Relative repression of the luciferase expression. Reporter constructs carrying a single binding site for miR-137, miR-214, and miR-148a were tested. For each luciferase. experiment, the miRNA activity on four constructs was measured in parallel: an empty pmirGlO vector (Control), a wild-type potential binding site for the appropriate miRNA (WT), a mutated binding site (MUT), and a site with full complementarity (PM). The firefly luciferase activity was normalized against Renilla luciferase activity. An average result from at least three independent experiments is shown (details in the text); (B) Relative HTT mRNA levels. Real-time PCR performed 48 h after transfection of HEK293T cells with miR-137, miR-214, and miR-148a. The bar graphs show the quantification of the HTT mRNA levels normalized to actin mRNAs based on data collected from three independent experiments; (C) Relative HTT protein levels. Western blot analysis of the cellular levels of HTT protein 72 h after transfection of HEK293T cells with miR-137, miR-214, and miR-148a. The bar graphs show the quantification of the protein levels detected in three western blot experiments. A representative blot is shown. The asterisks indicate statistical significance; a single asterisk at p-value < 0.05 and a double asterisk at p-value < 0.01.
Figure 3 Regulation of the huntingtin (HTT) expression by canonical miRNAs. (A) Relative repression of the luciferase expression. Reporter constructs carrying a single binding site for miR-137, miR-214, and miR-148a were tested. For each luciferase. experiment, the miRNA activity on four constructs was measured in parallel: an empty pmirGlO vector (Control), a wild-type potential binding site for the appropriate miRNA (WT), a mutated binding site (MUT), and a site with full complementarity (PM). The firefly luciferase activity was normalized against Renilla luciferase activity. An average result from at least three independent experiments is shown (details in the text); (B) Relative HTT mRNA levels. Real-time PCR performed 48 h after transfection of HEK293T cells with miR-137, miR-214, and miR-148a. The bar graphs show the quantification of the HTT mRNA levels normalized to actin mRNAs based on data collected from three independent experiments; (C) Relative HTT protein levels. Western blot analysis of the cellular levels of HTT protein 72 h after transfection of HEK293T cells with miR-137, miR-214, and miR-148a. The bar graphs show the quantification of the protein levels detected in three western blot experiments. A representative blot is shown. The asterisks indicate statistical significance; a single asterisk at p-value < 0.05 and a double asterisk at p-value < 0.01.CC BY · PubMed Central
Figure 4 Graphical presentation of selected isomiR variants and their potential to target different genes. (A) Nucleotide sequences of miR-137, miR-214, miR-148a, and their isoforms. miRNA sequences are marked in red, and isomiR sequences are shown in blue, green, and violet for −1-, +1-, and +2-nt seed shifting, respectively. The miRNA seed sequences are labeled with black rectangles. Information on the miRNA lengths, as well as their potential for targeting the HTT gene and isomiR expression levels, is also provided. (*) Ability to interact with the HTT 3′UTR, as predicted by the TargetScanHuman algorithm (Release 6.2) for miRNAs and the TargetScan Custom (Release 5.2) for isomiRs [46], (**) isomiR read number according to the YM500 database [53]; (B) Venn diagrams showing the predicted miRNA targets for selected isomiRs. Potential targets for the 5′-end variants of miR-137, miR-214, and miR-148a were predicted using the TargetScan Custom algorithm (Release 5.2) and are shown as overlaps in the Venn diagrams. Targets for the canonical miRNAs are compared with the targets for the miRNAs with the shifted seed regions and are depicted in the same colors as in panel A. The numbers inside the circles denote the numbers of potential targets predicted for the appropriate miRNA variants.
Figure 4 Graphical presentation of selected isomiR variants and their potential to target different genes. (A) Nucleotide sequences of miR-137, miR-214, miR-148a, and their isoforms. miRNA sequences are marked in red, and isomiR sequences are shown in blue, green, and violet for −1-, +1-, and +2-nt seed shifting, respectively. The miRNA seed sequences are labeled with black rectangles. Information on the miRNA lengths, as well as their potential for targeting the HTT gene and isomiR expression levels, is also provided. (*) Ability to interact with the HTT 3′UTR, as predicted by the TargetScanHuman algorithm (Release 6.2) for miRNAs and the TargetScan Custom (Release 5.2) for isomiRs [46], (**) isomiR read number according to the YM500 database [53]; (B) Venn diagrams showing the predicted miRNA targets for selected isomiRs. Potential targets for the 5′-end variants of miR-137, miR-214, and miR-148a were predicted using the TargetScan Custom algorithm (Release 5.2) and are shown as overlaps in the Venn diagrams. Targets for the canonical miRNAs are compared with the targets for the miRNAs with the shifted seed regions and are depicted in the same colors as in panel A. The numbers inside the circles denote the numbers of potential targets predicted for the appropriate miRNA variants.CC BY · PubMed Central
Figure 5 Correlation between the results of the luciferase experiments conducted with miR-137-coding plasmid and synthetic miR-137 mimics. (A) Relative repression of the luciferase expression. Reporter constructs carrying a single binding site for miR-137 were tested; miRNA activity on four constructs was measured in parallel (Control, WT, MUT, and PM), as described in Figure 3. Left—miRNA expression from the synthetic oligonucleotide (miR-137 mimic), right—miRNA overexpressed from the miR-137 vector. The firefly luciferase activity was normalized against Renilla luciferase activity. The standard errors are calculated from three independent experiments; (B) The relative repression of the luciferase expression resulted from the miRNA mimic activity. Four reporter constructs were tested (Control, WT, MUT, and PM) but with the addition of miR-137 mimic at different final concentrations, specifically 10, 30, and 50 nM, as denoted in the figure. The standard errors were calculated from one experiment performed in triplicate.
Figure 5 Correlation between the results of the luciferase experiments conducted with miR-137-coding plasmid and synthetic miR-137 mimics. (A) Relative repression of the luciferase expression. Reporter constructs carrying a single binding site for miR-137 were tested; miRNA activity on four constructs was measured in parallel (Control, WT, MUT, and PM), as described in Figure 3. Left—miRNA expression from the synthetic oligonucleotide (miR-137 mimic), right—miRNA overexpressed from the miR-137 vector. The firefly luciferase activity was normalized against Renilla luciferase activity. The standard errors are calculated from three independent experiments; (B) The relative repression of the luciferase expression resulted from the miRNA mimic activity. Four reporter constructs were tested (Control, WT, MUT, and PM) but with the addition of miR-137 mimic at different final concentrations, specifically 10, 30, and 50 nM, as denoted in the figure. The standard errors were calculated from one experiment performed in triplicate.CC BY · PubMed Central
Figure 6 Regulation of the huntingtin expression by isomiRs. Relative repression of the luciferase expression for miR-214, miR-137, miR-148a, and their isomiRs (+1, +2, or −1). Reporter constructs carrying single binding sites for the appropriate miRNAs were tested, namely miR-137 (A), miR-214 (B), and miR-148a (C), as depicted in the figure. For each luciferase experiment, the miRNA activity on four constructs (Control, WT, MUT and PM) was measured in parallel, as described in Figures 3 and 5. The firefly luciferase activity was normalized against Renilla luciferase activity. The standard errors were calculated from three independent experiments. The asterisks indicate statistical significance; a single asterisk at p-value < 0.05 and a double asterisk at p-value < 0.01.
Figure 6 Regulation of the huntingtin expression by isomiRs. Relative repression of the luciferase expression for miR-214, miR-137, miR-148a, and their isomiRs (+1, +2, or −1). Reporter constructs carrying single binding sites for the appropriate miRNAs were tested, namely miR-137 (A), miR-214 (B), and miR-148a (C), as depicted in the figure. For each luciferase experiment, the miRNA activity on four constructs (Control, WT, MUT and PM) was measured in parallel, as described in Figures 3 and 5. The firefly luciferase activity was normalized against Renilla luciferase activity. The standard errors were calculated from three independent experiments. The asterisks indicate statistical significance; a single asterisk at p-value < 0.05 and a double asterisk at p-value < 0.01.CC BY · PubMed Central

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Metadata

Authors
Kozlowska, E., Krzyzosiak, W., & Koscianska, E.
Affiliations
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland
ORCID
Koscianska, Edyta
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2013-08-19
Volume
14
Issue
8
Pages
16999-17016
ISSN
1422-0067
DOI
10.3390/ijms140816999
PMC
PMC3759948
Cited by
45
References
3
Language
en
Licence
creativecommons.org/licenses/by/3.0/ (vor)

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