overexpression by mature miRNA transfection}{mutation}{siRNA anti-miRNA
overexpression by mature miRNA transfection
overexpression by miRNA precursor transfection
overexpression by miRNA retinoic acid induction
overexpression by miRNA precursor transfection}{downregulation by anti-miRNA oligonucleotide
mutation}{overexpression by miRNA precursor transfection
overexpression by mature miRNA transfection}{mutation
overexpression by miRNA-masking antisense oligodeoxynucleotides
downregulation by anti-miRNA oligonucleotide}{mutation}{overexpression by mature miRNA transfection
overexpression by mature miRNA mimics transfection}{mutation}{downregulation by anti-miRNA oligonucleotide
overexpression by mature miRNA transfection}{mutation}{downregulation by anti-miRNA oligonucleotide
downregulation by 2'-O-Me antisense miRNA oligonucleotide
overexpression by mature miRNA mimics transfection
overexpression by miRNA precursor transfection}{underexpression by anti-miRNA oligonucleotides
overexpression by miRNA mimics tranfection
downregulation by LNA antisense miRNA oligonucleotide

{Western blot}{Western blot}{Western blot}{activity assay}{activity assay}{activity assay}
{Western blot}{phenotypic analysis of target gene}

{[The mutation of the target sites  significantly enhanced the target gene's expression.]}
{As an additional control experiment, we used a mutated target mRNA sequence for MAFB lacking five of the complementary bases. As expected, the mutations completely abolished the interaction between miR- 130a and its target 3_UTRs (Fig. 2B)}
{As predicted, the 372/373 complementary sequences mediated strong inhibition of luciferase expression in Tera1 cells. Significantly, a potent inhibition of luciferase activity was also mediated by the 30UTR of LATS2 in either MCF-7 ectopically expressing miR-372 or in Tera1 cells but not by a construct mutated at both miR-372-predicted target sites. These results indicate that LATS2 is indeed a direct target of miR-372&3.}
{Relative luciferase activity was determined by inserting part of the 3'-UTR target sequence from Delta, recognized by dmiR-1, into the 3'-UTR of luciferase (Delta 3'-UTR-luc) in the presence or absence of dmiR-1 in Drosophila S2 cells. The basal level of Delta 3'-UTR-luc or Delta 3'-UTR-mut-luc activity in the presence of Ubi-Gal4 but not UAS-miR-1 was defined as 100%. Overexpression of dmiR-1 inhibited activity of the luciferase reporter withWTDelta 3'UTR, but not with the dmiR-binding-site mutated Delta 3'UTR.}
{Cotransfection of miR-133 with the luciferase reporter gene linked to the wild-type 3' UTR of Rhoa, Cdc42 or Whsc2 resulted in a significant (P o 0.01) decrease in luciferase activity (Fig. 4 and Supplementary Fig. 6). In contrast, cotransfection of a control miRNA (not complementary to the 3' UTRs of these three genes) with the wildtype 3' UTR constructs did not result in a decrease in luciferase activity; similarly, cotransfection of miR-133 with constructs containing mutated or deleted 3' UTR sequences also did not result in a decrease in luciferase activity (Fig. 4 and Supplementary Fig. 6).}
{To investigate the role of 3' UTR seed matches in RISC association and regulation for direct targets, we created a series of seed site deletions in luciferase reporters bearing the UTRs of Ctdsp1 and Vamp3. When the Vamp3 reporter was tested in cortical neurons in the presence of endogenous miR-124a, a loss of repression was observed in constructs lacking miR-124a seed complementary sites (Fig. 2A).}
{To confirm that the underexpression of Yan expression is mediated by miR-7 binding to yan mRNA, we generated a reporter gene with GFP coding sequence fused to the yan 30UTR. Expression of the GFP reporter was downregulated in photoreceptor cells, whereas a GFP reporter with the four miR-7 binding sites mutated was not downregulated (Figures 3H and 3I).}
{In a more stringent test, we generated an E(spl)m5 sensor carrying 2-nt mutations in each of its three GY-boxes. These targeted changes also abolished the ability of miR-7 to negatively regulate E(spl)m5 (Fig. 3L).}
{Moreover, when we deleted either a w160 bp segment of the 3'UTR containing LCSs 1-4, or a w870 bp segment containing LCSs 5-8, no significant underexpression was observed in L4 animals, and only partial underexpression occurred in the adult stage (Figure 3C; Supplemental Figure S2), The consistent with the notion that these sequences are reGene quired for optimal target gene repression.}
{To investigate whether the 3' UTR-mediated underexpression of die-1 expression depends on the two mir-273-complementary sites, we mutated each of the sites in the sensor gfp construct. Each of the mutant sensor constructs failed to be downregulated in ASER (Fig. 3a).}
{To further establish the link between repression and the presence of seed matches within the 30 UTR, wild-type and mutant 30 UTR segments from eight genes downregulated by miR-1 (Fig. 4a) and two genes downregulated by miR-124 (Fig. 4b) were placed into an established luciferase reporter system3.When cotransfected with the cognate miRNA, six of the ten wild-type reporters exhibited significant (P , 0.001) repression relative to the corresponding constructs with mutant seed matches. Cotransfection of the noncognate miRNA typically had no effect.}
{Using both wild-type and mutant constructs of human ZEB1 and ZEB2 3_UTRs carrying mutations in all miR-200b/c/429 targeting sites, we now demonstrate that ectopic miR-200c suppresses by directly affecting the predicted miR-200c targeting sites (Fig. 3C)}
{Mutation of the miR-200b sites in ZEB1 and SIP1 prevented underexpression of the reporters by miR-200b (Fig. 3d), verifying that the effect of the microRNA is due to direct interaction with the binding sites in the 3'UTRs. The mutations also reduced the repression by endogenous microRNAs in MDCK cells (Fig. 3c).}{Mutation of the miR-200b sites in ZEB1 and SIP1 prevented underexpression of the reporters by miR-200b (Fig. 3d), verifying that the effect of the microRNA is due to direct interaction with the binding sites in the 3'UTRs. The mutations also reduced the repression by endogenous microRNAs in MDCK cells (Fig. 3c).}
{Deletion of the first three bases in three putative miR-106b/miR-93 binding sites, complementary to miRNA seed regions, abrogates this effect (MUT).}
{Moreover, miR-106b and miR-93 mimics inhibited the expression of a reporter vector containing p2130UTR, while mutation of the predicted miRNA-binding site abrogated this effect (Figure 4E).}
{Our results showed that miR-224 specifically interacted with the 3'UTR of API-5 to inhibit reporter activity since no inhibition of reporter activity was observed when the miR-224 binding sites in API-5 3'UTR was mutated (Fig. 3D).}
{Luciferase reporter assays using the 39 UTR of Mef2c demonstrated a miR-223-specific regulation of reporter gene expression (Fig. 3a, b). This underexpression was specific to the predicted miR-223 target sites, as mutation of the 39 UTR seed match sequences relieved the inhibitory activity of miR-223 (Fig. 3a, b).}
{Mutation of a single nucleotide in the predicted miR-26a binding site (Ezh2mut-Luc) abolished the suppression of the luciferase activity by miR-26a, indicating a specific suppressive effect of miR-26a on Ezh2 (Fig. 4d). These data demonstrate that Ezh2 is indeed a direct target of miR-26a and imply the involvement of this miRNA in suppression of Ezh2 during myogenesis.}
{Both HoxB1a and HoxB3a wildtype sensor constructs are strongly repressed by the microRNA (figure 2C), while the seed point mutant construct proves insensitive to repression by miR-10.}
{To test whether miR-iab4AS is functional and can directly target abd-A and Ubx, we constructed Luciferase reporters carrying the corresponding wild-type 3_ UTRs and control 3_ UTRs in which each seed site was disrupted by point substitutions. mir-iab-4AS potently repressed reporter activity for abd-A and Ubx (Fig. 2B).}
{In reporter assays, mutation of the miR-335 seed sequence in the SOX4 UTR as well as miR-335 inhibition with an antagomir significantly increased reporter expression (Fig. 4d). Restoration of miR-335 expression in LM2 cells reduced endogenous SOX4 messenger RNA expression, whereas miR-335 inhibition increased SOX4 expression in MDA-MB-231 cells (Fig. 4e).}
{The luciferase mRNA was stable in both deletion constructs (as demonstrated by quantitative RT-PCR of luciferase mRNA in MCF-7 cells transfected with either wild-type CD44-3'UTR luciferase reporter or deletion reporter; see Supplementary Information, Fig. S4). In transient transfection assays, deletion of both sites completely abrogated the inhibition of the luciferase signal by miR-373 (Fig. 4c). Together, these results suggest that miR-373 and miR-520c promote cell migration and invasion, at least in part, by limiting CD44 expression directly.}
{Thus, the effect of cellular endogenous miR-26a on translation of the luciferase mRNA was dependent on the presence of the miR-26a cognate binding site within the 3'-UTR, because expression of the luciferase reporter containing the mutant miR-26a binding site within the 3'-UTR was unaffected by the presence of endogenous cellular miR-26a.}
{This effect depends on the seed region hexamer complements in the p21 mRNA 3_ UTR, as mutations in these sequences rendered the reporter irresponsive to micro-RNA transfection (Fig. 4C).}
{However, no repression of Renilla luciferase translation was seen for the T allele construct when miR-433 was added (Figure 2D). After complete deletion of the miR-433 binding site (21 bp), we observed significantly higher Renilla luciferase translation compared to the constructs containing eitherCor Talleles (Figure 2E).}
{We also assayed truncated sensors consisting of 3_ UTR segments bounded by the miR-315 sites in each target and found that they recapitulated the quantitative repression by miR-315 mediated by their full-length counterparts. Finally, we introduced two point mutations into each of the miR-315 seed matches of the Axin and Notum sensors. These specific mutations abolished the response of these sensors to miR-315 (Fig. 4 B and C). Therefore, miR-315 directly represses Axin and Notum by the identified seed-match target sites.}
{N/A}{N/A}{The cells were also transfected to express miR-14. miR-14 expression reduced activity of the luciferase reporter containing the EcR 3_ UTR by 40%, but showed a reduced ability to regulate a comparable construct in which the seed regions of the three conserved miR-14 target sites had been mutated (Fig. 1B).}
{Four lines of evidence indicate that sqt, lft1, and lft2 are in vivo targets of miR-430. (i) Reporter mRNAs consisting of the green fluorescence protein (GFP) coding region and fulllength sqt, lft1, or lft2 3'UTRs were repressed in the wild type but not in MZdicer mutants, which lack all mature miRNAs including miR-430. Derepression of reporter genes was most pronounced for lft2 and least marked for lft1, suggesting that lft2 is more strongly repressed by miR-430 than sqt and lft1 (Figs. 1A and 2D and figs. S2 and S5).}
{Mutation of the 3 nucleotides within the seed sequences of both putative target sites in the 3'UTR abolished this underexpression (Fig. 7C).}
{The construct with the single let-7 b.s reduced the luciferase activity to 66.8 % of the control, while for the construct with the whole 3'UTR the reduction was on average up to 45.6 %. In the case of the c-MYC MUT_3'UTR sensor the luciferase activity reached 51.2 % of the control, suggesting that the mutation disturbing the potential b.s for let-7c restored the activity of luciferase when compared to the WT_3'UTR sensor, however, not efficiently compared with the construct bearing the let-7 binding site only (WT_b.s).}
{Coexpression of the luciferase reporters in S2 cells with miR-8 reduced luciferase mRNA levels to _60% (Figure 4C). A comparable reduction of luciferase enzyme activity was observed in response to miR-8 (Figure 4D), and this effect was largely eliminated when the miR-8 sites were mutated (Figure 4D).}{To address this, we generated transgenic flies expressing a luciferase reporter gene linked to the atrophin 30UTR or a version of this UTR in which the miR-8 sites were mutated. Q-PCR analysis showed that the level of luciferase mRNA increased in the miR-8 mutants for the construct with the intact miR-8 sites, but not when these sites were mutated (Figure 4B).}{Coexpression of the luciferase reporters in S2 cells with miR-8 reduced luciferase mRNA levels to _60% (Figure 4C). A comparable reduction of luciferase enzyme activity was observed in response to miR-8 (Figure 4D), and this effect was largely eliminated when the miR-8 sites were mutated (Figure 4D).}
{To demonstrate that the effect of miR-150 on the luciferase gene is dependent on the presence of the miR-150 binding sites, we generated a panel of reporter constructs containing the c-Myb 30UTR with the miR-150 binding sites mutated individually or in combination (Figure 1B). Although the mutation of site 1 did not influence the inhibitory effect of miR-150 on reporter gene expression, mutation of sites 2 and 3 individually reduced the inhibitory effect to 2.5- to 3-fold, and mutation of both sites completely abolished the inhibitory effect, suggesting that these two sites function cooperatively in mediating c-Myb inhibition (Figure 1E).}
{To confirm that the 3-UTR of IMP-1 is directly targeted by let-7, wemutated its LCS2 and LCS5 seed matches in two fragments, one carrying LCS2 and LCS3 and one carrying LCS5, respectively (Fig. 2C). Mutation of either of these two LCS significantly inhibited luciferase repression by let-7. Taken together, these data show that, similar to HMGA2, IMP-1 protein expression is under control of endogenous let-7 through targeting LCS in its 3-UTR.}
{To investigate the direct interaction between the miRs and p27 mRNA and the relative functionalities of the putative binding sites, we separately cloned the four "seeds" downstream to the luciferase open reading frame. Interestingly,only the presence of wild-type "seed-1" caused a 70% inhibition of the luciferase activity, whereas visible but not significant effects were observed for the other three sites (Fig. 6C;data not shown). Mutations within the seed-1 sequence totally abolished this repression, indicating the specificity of the action (Fig. 6C).}
{Nucleotide substitutions disrupting the mir-29c-binding site(s) were introduced in the 3_ UTRs of collagen 1A1, 3A1, and 4A2 cloned downstream of the firefly luciferase gene (Fig. 3A).}
{The 3_ UTRs of all of these 10 candidate target genes (Collagen 1A1, 1A2, 3A1, 4A1, 4A2,15A1, FUSIP1iso1, laminin _1, SPARC, and TDG) elicited significantly decreased luciferase activities (P values from 3 _ 10_3 to 1.2 _ 10_7) in mir-29c transfected cells (Fig. 2).}
{Transfection of let-7 decreased the expression of only full-length HMGA2 mRNA, but not the truncated HMGA2 mRNA without the 3_UTR.}
{We observed a 3- to 4-fold decrease in FF luciferase activity of LCS-containing mRNAs relative to those with reversed,mutated LCSs, whether under the control of the strong or weak promoter (Fig. 1C).}
{On the contrary, when we used, as a reporter construct, a plasmid harboring the 3_ UTR of p27 mRNA where two core binding sites for miR-221 and miR-222 were inactivated by site-directed mutagenesis, we observed only a very slight effect on luciferase activity, compared with LNCaP cells that did not express any microRNA.}
{}{Cotransfections of HEK293 cells with pGL3 S1-5 and concentrations of miR-200b ranging, from 2.5 nM to 30 nM resulted in a dose-dependent repression of the reporter (Fig. 3A). Repression was specific to miR-200b as a mutated version of miR-200b containing two point mutations in the seed region failed to repress, as did a structurally unrelated miRNA, miR-10a (Fig. 3A).}
{We found that the mutated p27-30UTR (DM) was completely refractory to the miR-221- and miR-222-suppressive effect (Figure 3C). As each single mutant retained at least some sensitivity to miRNAs 221&222 (data not shown), this result indicates that both predicted sites are required for miR-221&222 effects.}
{Notably, inhibition of endogenous miRNA by overexpressing the miR-24 inhibitor led to up-regulation of DHFR protein in WT but not in mutant cells (Fig. 1C).}{miR-24 mimics and inhibitors were then transfected in WT and mutant DHFR 3_ UTRexpressing DG44 cells. Overexpression of the miR-24 mimics in WT cells led to down-regulation of DHFR protein (Fig. 1C). In cells containing the 829C3T SNP, overexpression of the miR-24 mimic had no effect on the expression level of DHFR protein, suggesting that miR-24-mediated regulation of DHFR was lost in mutant DHFR-expressing cells.}
{Further confirmation that miR-16-1 regulates CCND1 by binding to the 3'UTR was accomplished by mutating both miR-16-1 binding sites in the 3'UTR. The mimic to miR-16-1 could not shutdown GFP-M (double mutant) reporter construct (Figure 4C).}
{By contrast, GFP abundance was unaltered in mir-1 mutants for reporter constructs containing the unc-38 30UTR or a mutagenized unc-29 30UTR in which nucleotides in the three predicted miR-1 binding sites were scrambled (Figures 2G and 2H).}
{Deletion of both miRE1 and miRE2 from the 3' UTR of the luciferase reporter nearly abolished repression by miR-125a and miR-125b, whereas deletion of either element alone reduced but did not eliminate repression (Fig. 2B).}
{To test whether the human lin-28 gene can be repressed by miR-125a and/or miR-125b, we constructed a reporter in which the 2.7-kb 3_ UTR of human lin-28 (Fig. 2A) was fused to a translational unit encoding firefly luciferase Luc-lin 28).Expression of this reporter in transfected 293T cells was reduced by more than 80% upon cotransfection with a human gene encoding either miR-125a or miR-125b but not with otherwise identical miRNA genes lacking the pre-miR-125a or pre-miR-125b segment (Fig. 2B).}
{In contrast, deletion of the upstream portion, which contains all three predicted MREs (‚ñ≥5), abolished silencing (Figure 2J and K).}
{In 293 cells, miR-203,but not GFP, is able to reduce luciferase activity in this construct by 30% (Figure 5C) indicating that this microRNA may directly target the ABL1 30UTR. This reduction is abolished by a 3'UTR that contains specific point mutations in the seed region of the miR-203 target sequence (Figure 5C), indicating that miR-203 can directly influence ABL1 protein levels through specific binding to its 3'UTR}
{Consequently, pmiR-221 and pmiR-222, but not pDNR-CMV or pmiR-145, inhibited luciferase activity (Figure 5B). In the mutated control groups,the inhibitory effect of pmiR-221 and miR-222 disappeared(Figure 5B). The results suggested that miR-221 and miR-222 were able to bind to p27(Kip1) and p57(Kip2) directly and inhibit their expression}
{The pGL3-CCNE1 and pGL3-CCNE1-mut vectors were then co-transfected with miR-15b mimics or control into U87 cells. A Renilla luciferase vector(pRL-TK) was used to normalize differences in transfection efficiency.Luciferase activity in U87 cells co-transfected with miR-15b mimics and pGL3-CCNE1 vector was decreased when compared with controls (Fig. 4C).}
{Co-transfection of pMIR124-GR3‚ÄôUTRWT with 50nM miR-124a significantly (p<0.01) repressed this luciferase activity (25.0 + 4.4% reduction vs pMIR124-GR3‚ÄôUTRWT, n=4; Figure 4A). Mutations in the predicted miR-124a seed region abolished this repression, thus strongly indicating that miR124 binds to the predicted target site in the rat GR mRNA}
{We found that the deletion of the (AACCAAA) repeat (del. repeat) was sufficient to inhibit the miR-9 mediated downregulation (Fig. 4B) of the 3'UTR.}
{Furthermore, miR-204 and miR-510 had no significant repressive effect on luciferase activity when overexpressed in cells transfected with luciferase-UTR constructs containing the respective mutated seed sequences (Fig. 2D).}
{co-transfection of pGL3-Pum2mut with anti-miR-134 does not result in an increase in the activity of the reporter (Figure 5C).}
{Overexpression of miR-499 caused a 32% decrease in luciferase activity of the Sox6 3' -UTR reporter gene but not the parent vector lacking the Sox6 3 -UTR.}
{the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (Fig. 4). As expected the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences.}
{we find that in mir-52 rescued embryos, marked by DLG-1mCHERRY (Figure 4B), and in otherwise wild-type embryos (Figure S2), a col-10gfpcdh-3 transgene under the control of the wild-type cdh-3 3‚Ä≤ UTR (WT, Figure 4C) is downregulated. Importantly, a similar transgene but with a 4-bp mutation in the seed region of the predicted miR-52 binding site (Figure S3, MUT) does not show this effect (Figure 4F). In contrast, both WT and MUT transgenes are expressed in mir-51 family mutant embryos (Figure 4, I and L). A second transgene with an unrelated 3‚Ä≤ UTR (myo-2mcherryunc-54) is not regulated in a miR-52-dependent manner (Figure 4, B, E, H, and K). Taken together, these data show that miR-52 directly regulates the CDH-3 3‚Ä≤ UTR in vivo.}
{Relative to the virgin luciferase reporter, annexation of the N-ras 3‚Ä≤-UTR resulted in an ~5-fold reduction in the luciferase activity (Fig. 5D). Deletion and point mutation analyses showed that the proximal miR-214 recognition site had a stronger role in attenuating the expression, whereas mutating both sequences severely curtailed the transcriptional repression activity of N-ras 3‚Ä≤-UTR (Fig. 5, C and D). }
{ The LNA-let-7 inhibitor (1, 5, or 25 pmol) or scrambled control (25 pmol) was co-transfected into the HEK239 cells together with the psiCHECK/MOR1UTR or the psiCHECK/MOR1UTRmut. The LNA-let-7 inhibitor increased the reporter gene activity in a dose-dependant manner. Co-transfection with 25 pmol LNA-let-7 inhibitor completely restored the luciferase repression by psiCHECK/MOR1UTR. In contrast, the LNA-let-7 inhibitor did not change the luciferase signal when co-transfected with the psiCHECK/MOR1UTRmut that lacks the let-7 binding sequence (Fig. 1C). }
{A significant decrease in luciferase activity was observed in the construct bearing an intact miR-21 binding site when compared with the control (Fig. 8B; P < 0.05). The decrease in luciferase activity was lost on mutation of the putative binding site for miR-21 by site-directed mutagenesis (Fig. 8C; P = not significant). }
{To verify whether the Ybx1 3‚Ä≤-UTR is a direct target of miR-216a, Luc reporter vectors containing the Ybx1 3‚Ä≤-UTR were constructed (Fig. 2E). miR-216a mimic significantly inhibited Luc activity of Ybx1 3‚Ä≤-UTR vector by almost 70% relative to a negative control mimic (NC Mimic) but not in the two control constructs, namely control without 3‚Ä≤-UTR (first pair of bars) or that with Ybx1 3‚Ä≤-UTR that has base substitutions in the miR-216a site (third pair of bars) (Fig. 2F). }
{To validate whether miR-29a directly recognizes the 3‚Ä≤UTR of PTEN mRNA or not, firstly, we cloned two sequences with the predicted target site of miR-29a (PTEN 3‚Ä≤UTR-1 and PTEN 3‚Ä≤UTR-2) or predicted target site deleted sequences (PTEN 3‚Ä≤UTR-1-del and PTEN 3‚Ä≤UTR-2-del) into downstream of the pGL3-control luciferase reporter gene vector, respectively. When the PTEN 3‚Ä≤UTR wild-type or deletion-type vector was cotransfected with miR-29a, the luciferase activity of the PTEN 3‚Ä≤UTR wild-type vector was significantly decreased compared with the deletion-type vector in a dose-dependent manner (Fig. 3B). Moreover, when both miR-29a and the specific miR-29a inhibitor were cotransfected with the PTEN 3‚Ä≤UTR wild-type vector, the luciferase activity of PTEN 3‚Ä≤UTR wild-type vector was rescued by the inhibitor of miR-29a in a dose dependent manner (Fig. 3C). }
{"Deletion of the miR-30e-5p site (Œî miR-30e-5p reporter) did not alter the inhibitory effect of miR-9 on luciferase reporter activity in co-transfected Cos cells (Fig. 4D, middle bars). However, deletion of the miR-9 seed site (Œî miR-9 reporter) abrogated the inhibitory effects of miR-9 on luciferase reporter activity in co-transfected Cos cells (Fig. 4D, right hand bars). In contrast, miR-30e-5p had no inhibitory effect on luciferase activity when co-transfected into Cos cells with any of the reporter constructs (Fig. 4E). These findings suggest that miR-9 exerts an inhibitory effect via direct interaction with its predicted seed site in the 3‚Ä≤UTR of PDGFR-Œ≤."}
{To validate whether miR-206 recognizes the 3‚Ä≤UTR of TIMP-3, two constructs containing each miR-206 seed sequence and the immediately surrounding sequences in TIMP-3 were cloned downstream of the luciferase open reading frame; pLUC-1090/97 and pLUC-1683/89 (Figure 9E and data not shown). The luciferase activity of each construct was evaluated following their cotransfection in HEK-293 cells either with miR-206 or a negative control miRNA. Luciferase activity from pLUC-1683/89 was markedly inhibited by miR-206 overexpression whereas this effect was prevented by the deletion of the seed complementary nucleotides in pLUC-M (Figure 9E); in contrast, miR-206 seed-pairing site at position 1090/97 was not functional in the assayed conditions (data not shown). Thus, the seed sequence 1683/89 represents the specific target of miR-206 and is responsible for miR-206-mediated TIMP-3 down-regulation.}
{To see if miR-196 could target the rarab 3‚Ä≤UTR, we used a firefly luciferase assay in cultured cells using Renilla luciferase as an internal standard. We attached the rarab 3‚Ä≤UTR with its three predicted miR-196 binding sites to luciferase coding sequence (Fig. 5E, F) and co-transfected this construct and miR-196 into human 293T cells. miR-196 led to reduced luciferase fluorescence from the rarab 3‚Ä≤UTR construct compared to the control (Fig. 5G), showing that miR-196 can act on the rarab 3‚Ä≤UTR to inhibit message stability and/or translation. To test this interaction in living embryos, we co-injected a construct containing the GFP coding region followed by the rarab 3‚Ä≤UTR (Fig. 5E) and then overexpressed or knocked down miR-196. Embryos injected with miR-196 had less GFP fluorescence than uninjected controls or controls injected with miR-196 mismatch duplex, but embryos experiencing miR-196 knockdown had more fluorescence than controls (Fig. 5H‚ÄìK, Supplementary Fig. S8). Deletion of the three predicted binding sites for miR-196 in the rarab 3‚Ä≤UTR led to no significant difference in GFP fluorescence compared to controls (Fig. 5L‚ÄìO). }
{"To further examine whether miR-9 and miR-181b targetNRP1 directly, the luciferase reporter containing the NRP1 30-UTR fragment with the miR-9 or miR-181b binding sites wastransfected into EA.hy926 cells. When miR-9 or miR-181b werecotransfected with the NRP1 reporter constructs, luciferaseactivity was significantly suppressed compared withcotransfection with positive and negative controls of pre-miRmiRNA precursors (Fig. 3A). However, when miR-9 or miR-181b were cotransfected with the deleted seed sequence of the30-UTR, the luciferase activity suppression was completelyabolished. Taken together, these results suggest that miR-9 andmiR-181b suppressed NRP1 through a specific 30-UTR bindingsite."}
{"Reporter constructs containing full-length SOX9 3 -UTR weremade in which the sites were deleted individually or in combination.Expression of the 266‚Äì288 mutant reporter (site 1 deletion)was 2-fold higher than that of the wild type; however,deletion of the 1386‚Äì1408 binding site (site 2) did not affectluciferase activity (Fig. 3B). Simultaneous deletion of both bindingsites had no further effect, suggesting that the region 266‚Äì288 (site 1) is the one required for the observed miRNA-mediatedrepression.Furthermore, miR-145 overexpressionreduced by 2-fold the expression of a luciferase reporter containingwild type SOX9 3 -UTR. This effect was completely lostby the deletion of the putative 266‚Äì288 miR-145 binding site,thus confirming site 1 as the relevant one for the observedmiRNA-mediated repression (Fig. 3C)."}
{MiR-469 (Fig. 4) significantly decreased luciferase activity in the plasmid containing full-length coding region (103/423 nt) along with 172 bp 3' non-coding region (423/594 nt) of Prm2 (103/594 nt) (psiCHECK-2/Prm2). Similar inhibition was noted in the construct of 3' deletion from nt 386 (103/386 nt) (psiCHECK-2/Prm2-a) when compared to the negative control group with scrambled RNA. However, miR-469 did not have any effect on the plasmid containing isolated fragment from 365-594 nt (psiCHECK-2/Prm2-b).}
{MRE was then proposed to be localized in the coding region rather at 3‚Äô-UTR. mRNA-fold program confirmed the formation of duplex structure of TP2 mRNA (nt 352-383) with perfect match at nt 376-383 to the 5‚Äô seed region of miR-469 (Fig 6B). Significant loss of luciferase activity was observed in the construct containing the MRE sequence alone (psiCHECK-2/TP2-MRE). Mutation of the proposed MRE site in the psiCHECK-2 vector containing either coding region of TP2 (psiCHECK-2/TP2-CD-X) or the isolated MRE element (psiCHECK-2/ TP2-MRE-X) completely recovered the luciferase activity in the presence of miR-469.}