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. 2008 May;14(5):872-7.
doi: 10.1261/rna.972008. Epub 2008 Mar 26.

miR-148 targets human DNMT3b protein coding region

Anja M Duursma  1 , Martijn Kedde, Mariette Schrier, Carlos le Sage, Reuven Agami
Affiliations

miR-148 targets human DNMT3b protein coding region

Anja M Duursma et al. RNA. 2008 May.

Abstract

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

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Figures

FIGURE 1.
FIGURE 1.
Schematic representation of putative miR-148 sites in the Dnmt3b1 coding region. (A) miR-148a and miR-148b are highly complementary to nucleotides 2382–2412 of human Dnmt3b. This potential miR-148 target site is conserved in rhesus, mouse, rat, dog, horse, and armadillo. This site is referred to as target site #1. (B) Schematic representation of two possible miR-148 target sites in the Dnmt3b coding region.
FIGURE 2.
FIGURE 2.
miR-148 regulates exogenous DNMT3b protein expression through interaction with its protein coding region. (A) RPA was used to detect miR-148 levels in HeLa cells that were transfected with miR-148, a control miRNA (Ctrl) or in a stable polyclonal pool of miR-148a expressing cells. The protected fragments are indicated. Lane P shows the probes for cyclophilin and miR-148a. In lane Y, yeast RNA was used as control. (B) qRT-PCR to detect miR-148a expression was performed using the indicated cell lines. Error bars represent standard deviation. (C) MCF-7 cells were cotransfected with either miR-Vec-148a or a miR-Vec control, together with GFP-Dnmt3b and H2B-GFP. Whole-cell extracts were subjected to immunoblot analysis to detect GFP. H2B-GFP was used to demonstrate equal transfection efficiencies. Asterisk marks a nonspecific band and shows equal loading. (D,E) As described in C. HeLa cells were transfected with the indicated constructs.
FIGURE 3.
FIGURE 3.
miR-148 reduces endogenous Dnmt3b1 mRNA and protein level. (A) qRT-PCR of either miR-Vec-148 or control (Ctrl.) transfected HeLa cells. Specific primers were used to detect Dnmt3b1 and Dnmt3b3 as shown schematically. Error bars represent standard deviation, n = 4 and (***) P < 0.001. (B) 2102EP cells were electroporated with the indicated constructs and subjected to immunoblot analysis for DNMT3b and TUBULIN.
FIGURE 4.
FIGURE 4.
Suppression of endogenous miR-148 increases DNMT3b1 expression. (A) miR-148a level was detected by RPA as described in Figure 2A. (B) 2102EP cells were electroporated with either control (Ctrl.) or miR-148kd#2 constructs and subjected to immunoblot analysis with Dnmt3b and Rel A antibodies.
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