Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes

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(1)Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes Duursma, A.M.. Citation Duursma, A. M. (2008, May 14). Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes. Retrieved from https://hdl.handle.net/1887/12848 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/12848. Note: To cite this publication please use the final published version (if applicable)..

(2) Chapter 4. miR-148 targets human Dnmt3b protein coding region

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(21) MicroRNAs (miRNAs) are small non-coding 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.. Introduction

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(370) 1. J1 6. 29 3T. miR-148. Ctrl.. 1. 0,9. 1. miR-148. Ctrl.. ut#2. GFP-Dnmt3b1 GFP 0,5. H2B-GFP. ut#1. miR-148b. trl.. E. *. *. U 2O S 21 02 EP. H eL a. H eL a. HeLa. GFP-Dnmt3b1. GFP-Dnmt3b1. ET M C F7. 50. D. miR-148. Ctrl.. MCF-7. 100. m. mature miR-148a. C. 300. iR -1 48. iR-148a precursor. 350. BJ. Cyclophilin. 400. miR-148a. P Y. B Relative miR-148/ U6 expression. A. Ctrl. iR-148 Stable miR-148 trans.. Chapter 4. 0,4. 0,5. 0,4. coding region. A RPA was used to detect miR-148 level 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 non-specific band and shows equal loading. D and E As described in C. HeLa cells were transfected with the indicated constructs.. Furthermore, the translational fusion product was detected at the appropriate size by Immunoblotting analysis (data not shown). We observed a marked reduction of 60% of GFP-DNMT3b1 expression, but not of co-transfected H2B-GFP, in cells expressing miR-148 (Figure 2C). As expected from the sequence similarity between miR-148a and miR-148b, a similar reduction of GFPDNMT3b1 expression in cells expressing miR-148b was observed (Figure 2D). These results suggest that the presence of miR-148 represses the expression of DNMT3b1.. 60. To determine whether the observed reduction in DNMT3b1 expression by miR148 is dependent on the putative miR-148 target sites, we mutated these regions to significantly reduce recognition by the miRNAs. Importantly, mutating these sites did not change the frame of the fusion proteins, as was verified by sequencing and Immunoblotting analysis (data not shown). Transient co-transfection experiments revealed that mutating the highly homologous target site #1 almost completely abrogated miR-148-mediated regulation (Figure 2E)..

(371) miR-148 targets human Dnmt3b protein coding region. 120. ***. trl.. Dnmt3b1. Relative % of Dnmt3b/ bActin mRNA expression. Dnmt3b3. miR-148. Ctrl.. Dnmt3b1. Dnmt3bkd#2. B. Dnmt3bkd#1. A. 0,2. 0,1. 1,9. Dnmt3b1. 100. Tubulin. 80 60. 1. 0,3. 0,9. 40 20 0 Dnmt3b1. Dnmt3b3. 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.. However, a mutant of target site #2, which has much less complementarity to miR-148, still retained sensitivity to miR-148. This suggests that miR-148 targets Dnmt3b1 through a highly complementary sequence in the Dnmt3b1 coding region. The results above imply that the Dnmt3b3 splice variant, which lacks miR-148 target site #1, is resistant to miR-148 mediated regulation. To examine this issue, we primers for qRT-PCR that designed distinguish endogenous Dnmt3b3 from Dnmt3b1 and other variants that express the targeted exon such as Dnmt3b2 and Dnmt3b4 mRNA (Figure 3A). This analysis revealed that stable expression of miR148a in HeLa cells resulted in small but significant reduction (p<0.001) in Dnmt3b1 mRNA levels of 25%, while no reduction in Dnmt3b3 mRNA levels was observed (Figure 3A). Thus, the expression of miR-148a affects the mRNA stability of Dnmt3b1, but. not Dnmt3b3, leading to a relative increase in the abundance of Dnmt3b3 compared to Dnmt3b1 and all other Dnmt3b splice variants that express the targeted site. DNMT3b protein is highly expressed in undifferentiated embryonic stem cells, whereas its expression level is much reduced in somatic cells (Okano et al., 1998). To study the effect of miR-148a on endogenous DNMT3b protein expression we attempted to detect it in several tumor cell lines using immuno-blot analysis. In our hands, only 2102EP testicular germ cell tumor cells expressed endogenous DNMT3b in sufficient amount to allow detection by western blot (data not shown). Furthermore, these cells express moderate level of miR-148a (Figure 2B). To assess whether increasing miR-148 level would result in reduced endogenous DNMT3b1 protein level, we electroporated the miR-148 expression vector into 2102EP cells and subjected them to immunoblotting. 61.

(372) Chapter 4. analysis with a specific anti-Dnmt3b antibody. In line with the results above, over-expression of miR-148a reduced DNMT3b1 protein level up to 70 % compared to two different miRNA control constructs (Figure 3B). We used two short-hairpin RNA (shRNA) constructs targeting Dnmt3b and an expression vector for Dnmt3b1 to control for the specificity of the Dnmt3b antibody (Figure 3B). Lastly, we asked whether inhibition of miR-148a activity would elevate DNMT3b1 expression. We designed two shRNA constructs targeting the miR-148 precursor RNA. Electroporation of 21202EP cells with these constructs showed that one construct, miR-148kd#2, was functional in reducing endogenous miR-148 level as determined by RPA analysis (Figure 4A). Western blot analysis of cells electroporated with miR-148kd#2 indeed revealed increase in the DNMT3b1 protein level (Figure 4B). We therefore conclude that endogenous DNMT3b1 expression is controlled by miR148a in 2102EP cells. Our data reveal a novel function of miRNAmediated regulation. miRNA targeting of human coding regions in addition to 3’UTRs not only extends the range of potential target sites, but also allows for splice variant specific regulation. Significantly, different DNMT3b splice variants might play a role in regulating DNMT3b activity or target site preference.. Suppression. endogenous. 148. increases. expression.. DNMT3b1. A. miR-148a. level was detected by RPA as. described. 2A.. B. 2102EP. electroporated. B. miR-. in cells with. Figure. P Y. miR-148kd#2. of. Ctrl.. 4.. The mechanism by which miRNAs mediate repression is not completely understood. Whereas some evidence exists for interference with initiation of translation, others suggest that miRNAs prevent the ‘closed loop’ mRNA configuration induced by interaction of polyA binding proteins with initiation factors at. Ctrl. miR-148kd#1 miR-148kd#2. Figure. This could be an additional mechanism to the regulation of splice variant abundance by alternative splicing. Our results rule out the possibility that alternative splicing is induced by miR-148 targeting, since miR-148 reduced expression of exogenous DNMT3bGFP in a site-specific manner. It is interesting that only the high affinity miR-148 target site in Dnmt3b1 was affected by miR-148. In the future, it will be important to determine whether miRNA binding to CDS requires more complementarity with the target than interactions with 3’UTRs. Plant miRNAs interact with high sequence complementarity with their target CDS eliciting in most of the cases an RNA interference-mediated cleavage of the target mRNA (Llave et al., 2002; Rhoades et al., 2002). Although we observed reduction of Dnmt3b mRNA in the presence of miR-148, the reduction was less prominent than the observed reduction on protein level. This suggests that miR-148 induces both translational repression and mRNA degradation of Dnmt3b1. Further experiments should approach this point.. 1. 1,3. Cyclophilin. were. Dnmt3b1. either kd#2. control (Ctrl.) or miR-148. iR-148a precursor. constructs and subjected to immunoblot. analysis. Dnmt3b and Rel A antibodies.. ature miR-148a. 1. 62. Rel A. with. 1 0,3.

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