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Biological basis for restriction of microRNA targets to the 3' untranslated region in mammalian mRNAs.

Gu S, Jin L, Zhang F, Sarnow P, Kay MA - Nat. Struct. Mol. Biol. (2009)

Bottom Line: To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence.The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown.Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

View Article: PubMed Central - PubMed

Affiliation: The Center for Clinical Science Research, Room 2105, 269 Campus Drive, Stanford, California 94305-5164, USA.

ABSTRACT
MicroRNAs (miRNAs) interact with target sites located in the 3' untranslated regions (3' UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

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Insertion of rare-codons upstream of the extended miRNA ORF rescues miRNA-mediated knockdown(A) The maps of the reporter constructs used in this study. Plasmids containing tandem mir-30 target sequence in either 3'UTR (a1) or ORF (a2) are the same as those described in Figure 1. A cluster of rare codons (represented as a dark box) were inserted either upstream (a3) or downstream (a4) of mir-30 target sequences. In another construct, the upstream rare codons (a3) were replaced with optimal-codon sequences that code for the same peptide sequence. The arrows and grey box represent the position of the miRNA target sequences.(B) HEK293 cells and (C), (D) NIH3T3 cells were transfected with the reporter constructs illustrated in (A). Dual-luciferase assays were performed 36hrs post-transfection. FF-luciferase activities were normalized with RL-luciferase, and the percentage of relative enzyme activity compared to the negative control (treated with sh-scramble) was plotted. Error bars represent standard deviation from three independent experiments, each performed in triplicate.(E) Protein levels of reporter genes were analyzed by Western blot in transfected 3T3 cells.(F) NIH3T3 cells were transfected with constructs as indicated in the figure. Insertion of rare-codon cluster (dark box) upstream of mir-30 targets sites in the 3'UTR did not substantially change the miRNA-induced repression.(G) RNA levels of reporter genes were analyzed by Ribonuclease Protection Assay. The loading sequence of line 1 to 11 is same as noted in (E).
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Figure 3: Insertion of rare-codons upstream of the extended miRNA ORF rescues miRNA-mediated knockdown(A) The maps of the reporter constructs used in this study. Plasmids containing tandem mir-30 target sequence in either 3'UTR (a1) or ORF (a2) are the same as those described in Figure 1. A cluster of rare codons (represented as a dark box) were inserted either upstream (a3) or downstream (a4) of mir-30 target sequences. In another construct, the upstream rare codons (a3) were replaced with optimal-codon sequences that code for the same peptide sequence. The arrows and grey box represent the position of the miRNA target sequences.(B) HEK293 cells and (C), (D) NIH3T3 cells were transfected with the reporter constructs illustrated in (A). Dual-luciferase assays were performed 36hrs post-transfection. FF-luciferase activities were normalized with RL-luciferase, and the percentage of relative enzyme activity compared to the negative control (treated with sh-scramble) was plotted. Error bars represent standard deviation from three independent experiments, each performed in triplicate.(E) Protein levels of reporter genes were analyzed by Western blot in transfected 3T3 cells.(F) NIH3T3 cells were transfected with constructs as indicated in the figure. Insertion of rare-codon cluster (dark box) upstream of mir-30 targets sites in the 3'UTR did not substantially change the miRNA-induced repression.(G) RNA levels of reporter genes were analyzed by Ribonuclease Protection Assay. The loading sequence of line 1 to 11 is same as noted in (E).

Mentions: Since our results suggested that active translation of mRNAs precludes miRNA-induced knockdown, we predicted that ribosome hindrance would interfere with the ability of miRNA and its associated machinery to attach to its target site. To test this, we introduced a cluster (9 amino acids) of rare codons upstream of miRNA target sites located in extended luciferase ORF (Figure 3A), an approach used to cause ribosome pausing in eukaryotes26,27. Since we could not measure ribosome translocation directly, we constructed a number of different control sequences for direct comparisons. We inserted the same 9 amino acids in the identical location using an optimized set of codons, or placed the rare codons downstream of the miRNA target. When the rare codons were upstream of the target, miRNA-induced silencing from sh-mir-30 was restored to a level close to what was observed (rescue >80% and 70% in 293 and NIH 3T3 cells, respectively), while replacing the rare with optimal codons or placing the rare codons downstream of the miRNA target was unable to rescue miRNA-induced silencing (Figures 3B-E). This confirmed that the additional nucleotides or the extra amino acids were not responsible for the differential activity of the miRNA target. To eliminate the possibility that adding the 27 nucleotides altered the local RNA folding structure -- and, hence, the accessibility and efficacy of miRNA target sites -- we inserted these sequences upstream of mir-30 target sites, which remain in the 3'UTR in the FF-luciferase reporter construct. MiRNA-mediated repression was not changed (Figure 3F). RNA analyses (Figure 3G) confirmed that the rare/optimal codon clusters had no substantial effect on the steady-state mRNA levels.


Biological basis for restriction of microRNA targets to the 3' untranslated region in mammalian mRNAs.

Gu S, Jin L, Zhang F, Sarnow P, Kay MA - Nat. Struct. Mol. Biol. (2009)

Insertion of rare-codons upstream of the extended miRNA ORF rescues miRNA-mediated knockdown(A) The maps of the reporter constructs used in this study. Plasmids containing tandem mir-30 target sequence in either 3'UTR (a1) or ORF (a2) are the same as those described in Figure 1. A cluster of rare codons (represented as a dark box) were inserted either upstream (a3) or downstream (a4) of mir-30 target sequences. In another construct, the upstream rare codons (a3) were replaced with optimal-codon sequences that code for the same peptide sequence. The arrows and grey box represent the position of the miRNA target sequences.(B) HEK293 cells and (C), (D) NIH3T3 cells were transfected with the reporter constructs illustrated in (A). Dual-luciferase assays were performed 36hrs post-transfection. FF-luciferase activities were normalized with RL-luciferase, and the percentage of relative enzyme activity compared to the negative control (treated with sh-scramble) was plotted. Error bars represent standard deviation from three independent experiments, each performed in triplicate.(E) Protein levels of reporter genes were analyzed by Western blot in transfected 3T3 cells.(F) NIH3T3 cells were transfected with constructs as indicated in the figure. Insertion of rare-codon cluster (dark box) upstream of mir-30 targets sites in the 3'UTR did not substantially change the miRNA-induced repression.(G) RNA levels of reporter genes were analyzed by Ribonuclease Protection Assay. The loading sequence of line 1 to 11 is same as noted in (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713750&req=5

Figure 3: Insertion of rare-codons upstream of the extended miRNA ORF rescues miRNA-mediated knockdown(A) The maps of the reporter constructs used in this study. Plasmids containing tandem mir-30 target sequence in either 3'UTR (a1) or ORF (a2) are the same as those described in Figure 1. A cluster of rare codons (represented as a dark box) were inserted either upstream (a3) or downstream (a4) of mir-30 target sequences. In another construct, the upstream rare codons (a3) were replaced with optimal-codon sequences that code for the same peptide sequence. The arrows and grey box represent the position of the miRNA target sequences.(B) HEK293 cells and (C), (D) NIH3T3 cells were transfected with the reporter constructs illustrated in (A). Dual-luciferase assays were performed 36hrs post-transfection. FF-luciferase activities were normalized with RL-luciferase, and the percentage of relative enzyme activity compared to the negative control (treated with sh-scramble) was plotted. Error bars represent standard deviation from three independent experiments, each performed in triplicate.(E) Protein levels of reporter genes were analyzed by Western blot in transfected 3T3 cells.(F) NIH3T3 cells were transfected with constructs as indicated in the figure. Insertion of rare-codon cluster (dark box) upstream of mir-30 targets sites in the 3'UTR did not substantially change the miRNA-induced repression.(G) RNA levels of reporter genes were analyzed by Ribonuclease Protection Assay. The loading sequence of line 1 to 11 is same as noted in (E).
Mentions: Since our results suggested that active translation of mRNAs precludes miRNA-induced knockdown, we predicted that ribosome hindrance would interfere with the ability of miRNA and its associated machinery to attach to its target site. To test this, we introduced a cluster (9 amino acids) of rare codons upstream of miRNA target sites located in extended luciferase ORF (Figure 3A), an approach used to cause ribosome pausing in eukaryotes26,27. Since we could not measure ribosome translocation directly, we constructed a number of different control sequences for direct comparisons. We inserted the same 9 amino acids in the identical location using an optimized set of codons, or placed the rare codons downstream of the miRNA target. When the rare codons were upstream of the target, miRNA-induced silencing from sh-mir-30 was restored to a level close to what was observed (rescue >80% and 70% in 293 and NIH 3T3 cells, respectively), while replacing the rare with optimal codons or placing the rare codons downstream of the miRNA target was unable to rescue miRNA-induced silencing (Figures 3B-E). This confirmed that the additional nucleotides or the extra amino acids were not responsible for the differential activity of the miRNA target. To eliminate the possibility that adding the 27 nucleotides altered the local RNA folding structure -- and, hence, the accessibility and efficacy of miRNA target sites -- we inserted these sequences upstream of mir-30 target sites, which remain in the 3'UTR in the FF-luciferase reporter construct. MiRNA-mediated repression was not changed (Figure 3F). RNA analyses (Figure 3G) confirmed that the rare/optimal codon clusters had no substantial effect on the steady-state mRNA levels.

Bottom Line: To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence.The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown.Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

View Article: PubMed Central - PubMed

Affiliation: The Center for Clinical Science Research, Room 2105, 269 Campus Drive, Stanford, California 94305-5164, USA.

ABSTRACT
MicroRNAs (miRNAs) interact with target sites located in the 3' untranslated regions (3' UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

Show MeSH