Using luciferase gene as a reporter to demonstrate that miRNA derived from zebrafish miR-1 targeted the 3′ untranslated region (UTR) of the zebrafish hand2 gene and suppressed gene expression in zebrafish embryos. (A) To validate whether the hand2 gene was the target gene of ZF-miR-1, we engineered three constructs: cmlc2-Luciferase-hand2-3′UTR, cmlc2-EGFP and cmlc2-EGFP-miR-1. (B) Compared to the luciferase activity driven by injection of the combined cmlc2-Luciferase-hand2-3′UTR and cmlc2-EGFP, the relative luciferase activity was dramatically reduced to 10% when the zebrafish embryos were injected with the combined cmlc2-Luciferase-hand2-3′UTR and cmlc2-EGFP-miR-1. The error bar indicates standard deviation (n = 6).

Using fluorescent protein genes as reporters to demonstrate that miRNA derived from zebrafish miR-1 targeted the 3′UTR of the zebrafish hand2 gene and suppressed gene expression in the cell line. (A) To validate whether the hand2 gene was the target gene of ZF-miR-1, we engineered three constructs: pCMV-EGFP-hand2-3′UTR, pCMV-DsRed and pCMV-DsRed-miR-1. Then, these plasmids were transfected into COS-1 cells. (B) The expression of reporter genes was observed under fluorescence microscope at the wavelengths suitable for detection of green fluorescent protein (GFP) or red fluorescent protein (RFP).

Using luciferase activity assay to confirm the putative target genes for zebrafish miR-1 predicted from microarray analysis. (A) The 3′UTRs of these two cDNAs were separately engineered at the downstream of luciferase reporter gene (phRG-TK-3′UTR, as indicated) and transfected into COS-1 cells with pre-miR-1. Two other constructs—phRG-TK and CMV-pre-miR-1, served as control plasmids. (B) The luciferase activity driven by the 3′UTR from either suclg1 or parvalbumin 4 mRNA is presented in histograms. Y axis represents the relative value for Renilla reniformis luciferase to firefly luciferase after normalization with phRG-TK-transfected cells. Compared with the luciferase from the phRG-TK-transfected cells, the luciferase activity was repressed in the cells transfected with suclg1 3′UTR and pre-miR-1. The error bar indicates standard deviation (n = 6).

The mature microRNA (miRNA) was processed from the pre-miRNA labeled with digoxigenin (DIG). Pre-miRNAs of Cel-pre-lin4 (C-lin4), Cel-pre-let7 (C-let7), Cel-pre-let7M (C-let7M) from Caenorhabditis elegans and pre-miRNA of ZF-pre-let7 (ZF-let7) from zebrafish were labeled with DIG, and northern blot analysis was performed. The pre-miRNA was neither processed into mature miRNA in the absence of cell extracts from C. elegans (lanes 1, 4 and 7) and zebrafish (lane 10) nor in the absence of dicer (Dicer−), which was depleted by anti-dicer immunoprecipitation (lanes 3, 6, 9 and 12). However, the mature miRNA was normally processed in the presence of cell extracts (lanes 2, 5, 8 and 11), indicating that the DIG-tagged pre-miRNA does not affect the formation of mature miRNA.

Using the pull-down assay to determine the genes that are targeted by miRNA. Four miRNA-related genes were studied: two from Caenorhabditis elegans (let-7 and lin-4, Cel) and two from zebrafish (let-7 and miR-1, ZF). The target genes for Cel-pre-let7, Cel-pre-lin4 and ZF-pre-let7 are known, whereas the target gene(s) for ZF-pre-miR-1 is unknown. After pre-let7 (Cel-pre-let7, ZF-pre-let7), pre-lin4 (Cel-pre-lin4) and pre-miR-1 (ZF-pre-miR-1) were labeled with digoxigenin (DIG) and incubated with the cell extracts, RT–PCR was used to amplify the mRNA obtained from the pull-down assay. (A) The secondary structure and free energy predicted by the software program mfold (version 3.2) is shown. For the negative control, we designed a mutated sequence of pre-let7 of C. elegans (Cel-pre-let7M). The altered sequences of Cel-pre-let7M are indicated by boldface type. (B) After pull-down by DIG-Cel-pre-let7, we used RT–PCR to detect whether the known target genes, lin-41 and hb1-1, had been obtained. As expected, both lin-41 and hbl-1 were positive, but not EFT-2 (upper panel). Meanwhile, lin41, hbl-1 and EFT-2 were not positive when DIG-Cel-pre-let7M was used for pull-down and RT–PCR was used for detection (middle panel). Following a similar strategy, we detected the target genes lin-14 and lin-28 for Cel-pre-lin4 when DIG-Cel-pre-lin4 was used for pull-down (bottom panel). (C) After pull-down by DIG-ZF-pre-let7, the target gene zebrafish lin-41 was obtained, as expected, whereas neither β-actin nor myf5-intron1 (the first intron segment of the zebrafish myf5 gene) was positive, suggesting that there was no contamination of genomic DNA in the template (upper panel). Interestingly, when DIG-ZF-pre-miR-1 was used, a novel putative target gene, hand2, was obtained (bottom panel). M, molecular marker; RT, reverse transcriptase was added; RT–, reverse transcriptase was not added; P, positive control, template DNA was from C. elegans cDNA; and N, negative control, template DNA was not added.