MicroRNA-181a modulates gene expression of zinc finger family members by directly targeting their coding regions

Abstract

MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that specifically bind to the 3' untranslated region (3'UTR) of target genes in animals. However, some recent studies have demonstrated that miRNAs also target the coding regions of mammalian genes. Here, we show that miRNA-181a downregulates the expression of a large number of zinc finger genes (ZNFs). Bioinformatics analysis revealed that these ZNFs contain many miR-181a seed-matched sites within their coding sequences (CDS). In particular, miR-181a 8-mer-matched sequences were mostly localized to the regions coding for the ZNF C2H2 domain. A series of reporter assays confirmed that miR-181a inhibits the expression of ZNFs by directly targeting their CDS. These inhibitory effects might be due to the multiple target sites located within the ZNF genes. In conclusion, our findings indicate that some miRNA species may regulate gene family by targeting their coding regions, thus providing an important and novel perspective for decoding the complex mechanism of miRNA/mRNA interplay.

Figure 1.

Distribution of miR-181a seed-matched sites in the ZNFs downregulated by miR-181a. Top: the number of miR-181a seed-matched sites within the transcripts of these ZNFs (including the 5′UTR, ORF and 3′UTR). MiR-181a seed-matched sequences are classified as 8-mer (TGAATGTA), 7-mer-m8 (TGAATGT, 7m8) and 7-mer-A1 (GAATGTA, 7A1). Note: ZNF658 has 14 8-mer-matched sites that are not fully shown in the figure. Bottom: quantitation of mRNA levels of ZNFs in miR-181a-transfused HepG2 cells relative to control cells according to the mRNA profile. The values were converted to the Log2 ratio of miR-181a versus control.

Figure 2.

Inhibition of miR-181a increases the expression levels of the ZNF genes in Huh-7 cells. (A) The relative expression of mature miR-181a was determined by the ABI TaqMan miRNA assay in Huh-7 cells transfected with either miR-181a inhibitors or negative control. U6 snRNA served as an internal control. (B) The relative expression of the ZNF genes was determined by SYBR real-time PCR in Huh-7 cells transfected with either miR-181a inhibitors or negative control. The β-actin served as an internal control. The values were converted to the Log2 ratio of miR-181a inhibitors versus control. Results are representatives of three independent experiments. Error bars represent the SEM.

Figure 3.

The miR-181a seed-matched sites in the coding regions of ZNFs. (A) The number of miR-181a seed-matched sites within the coding regions of 709 ZNFs. MiR-181a seed-matched sequences are classified as 8-mer (TGAATGTA), 7-mer-m8 (TGAATGT, 7m8) and 7-mer-A1 (GAATGTA, 7A1). (B) MiR-181a 8-mer-matched sites in ZNFs were mostly located in the regions coding for the C2H2 domain. The sequence GAA encodes glutamic acid (E), and TGT encodes cysteine (C). Y is tyrosine, F is phenylalanine, H is histidine and X is any amino acid.

Figure 4.

MiR-181a directly targets ZNFs within the CDS as determined by HA-tagged construct and luciferase reporter assays. (A) Vectors encoding the wild-type CDS of ZNF83, ZNF37A, ZNF180 and RhoGDI tagged with HA were constructed and co-transfected with pri-miR-181a/151 vectors or mature miR-181a/151 mimics into HEK 293T cells. After 48 h, protein was extracted and probed with HA antibody. (B) MiR-181a inhibits luciferase activity when the ZNF CDS region is located in the luciferase gene 3′UTR. HEK 293T cells were seeded in 96-well plates and co-transfected with pWPT-GFP or pWPT-miR-181a, luciferase reporter (p-luc, p-luc-ZNF37A, p-luc-ZNF83 or p-luc-ZNF180) and ‘Renilla’ pRL-CMV internal control plasmid using Lipofectamine 2000. Luciferase activity was measured by the dual-luciferase reporter assay system after 48 h transfection. Results are representatives of three independent experiments. Error bars represent the SEM.

Figure 5.

The effects of miR-181a on the expression of ZNF37A CDS variants. (A) Schematic diagram of various ZNF37A CDS constructs tagged with HA. (B–E) Western-blot analysis of HA-tagged protein expression in HEK 293T cells transfected with (B) various ZNF37A CDS fragments; (C) either pWPT-GFP or pWPT-miR-181a and various fragments of the ZNF37A CDS; (D) either control or miR-181a mimics and mutated variants of the ZNF37A CDS; and (E) either control or miR-181a mutated mimics and mutated variants of the ZNF37A CDS. β-actin was used as an internal control. Quantification was performed with Image-Pro Plus software.