The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR

Abstract

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3'UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3'UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3'UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.

Figure 1.

LCSs are necessary and sufficient for proper down-regulation in vivo. (A) Schematic of in vivo LCS constructs. All share a common col-10 promoter fused to the Escherichia coli lacZ reporter gene. Each construct contains various LCSs from the Caenorhabditis elegans lin-41 3′UTR inserted into the unc-54 3′UTR. pMV18 contains Caenorhabditis briggsae lin-41 DNA. (+++) 0%-12% of animals with hypodermal expression at the adult stage); (++) 13%-32%); (+) 33%-60%); (-) >60%. (B) In vivo lacZ expression analysis. We analyzed multiple lines for all constructs and averaged the data for each construct. Error bars indicate standard deviation between lines/trials where indicated. See Supplemental materials for number of lines/animals scored. Numbers below graph refer to ratio of hypodermal lacZ expression between adult vs. L₁-L₃ animals.

Figure 1.

LCSs are necessary and sufficient for proper down-regulation in vivo. (A) Schematic of in vivo LCS constructs. All share a common col-10 promoter fused to the Escherichia coli lacZ reporter gene. Each construct contains various LCSs from the Caenorhabditis elegans lin-41 3′UTR inserted into the unc-54 3′UTR. pMV18 contains Caenorhabditis briggsae lin-41 DNA. (+++) 0%-12% of animals with hypodermal expression at the adult stage); (++) 13%-32%); (+) 33%-60%); (-) >60%. (B) In vivo lacZ expression analysis. We analyzed multiple lines for all constructs and averaged the data for each construct. Error bars indicate standard deviation between lines/trials where indicated. See Supplemental materials for number of lines/animals scored. Numbers below graph refer to ratio of hypodermal lacZ expression between adult vs. L₁-L₃ animals.

Figure 2.

Effect of point mutations in LCS 1 and LCS 2. (A) The sequence of the wild-type LCS 1 and LCS 2, the predicted base pairing with the let-7 RNA, and mutations compensatory to let-7(n2853) in the LCSs of the lin-41 3′UTR. Arrows indicate base nucleotide substitutions (C to U) made in LCS 1, LCS 2, or both, and predicted base pairing with the let-7(n2853) RNA. (B-D) See Supplemental materials for number of lines/animals scored. All data were analyzed as in Fig. 1B. Numbers below graph refer to ratio of hypodermal expression between adult vs. L₁-L₃ animals. (B) Down-regulation of col-10-lacZ-LCS 1, LCS 2, LCS 3, and LCS 5(pMV1) is abrogated in let-7(n2853) animals. (C) Effect of LCS 1^* and LCS 2^* mutations in a wild-type background. (D) A let-7(n2853) compensatory mutation in LCS 2 and LCS 1 partially restores down-regulation in a let-7(n2853) background. Lines that showed the most severe lack of down-regulation at the adult stage in a wild-type background were assayed.

Figure 2.

Effect of point mutations in LCS 1 and LCS 2. (A) The sequence of the wild-type LCS 1 and LCS 2, the predicted base pairing with the let-7 RNA, and mutations compensatory to let-7(n2853) in the LCSs of the lin-41 3′UTR. Arrows indicate base nucleotide substitutions (C to U) made in LCS 1, LCS 2, or both, and predicted base pairing with the let-7(n2853) RNA. (B-D) See Supplemental materials for number of lines/animals scored. All data were analyzed as in Fig. 1B. Numbers below graph refer to ratio of hypodermal expression between adult vs. L₁-L₃ animals. (B) Down-regulation of col-10-lacZ-LCS 1, LCS 2, LCS 3, and LCS 5(pMV1) is abrogated in let-7(n2853) animals. (C) Effect of LCS 1^* and LCS 2^* mutations in a wild-type background. (D) A let-7(n2853) compensatory mutation in LCS 2 and LCS 1 partially restores down-regulation in a let-7(n2853) background. Lines that showed the most severe lack of down-regulation at the adult stage in a wild-type background were assayed.

Figure 2.

Effect of point mutations in LCS 1 and LCS 2. (A) The sequence of the wild-type LCS 1 and LCS 2, the predicted base pairing with the let-7 RNA, and mutations compensatory to let-7(n2853) in the LCSs of the lin-41 3′UTR. Arrows indicate base nucleotide substitutions (C to U) made in LCS 1, LCS 2, or both, and predicted base pairing with the let-7(n2853) RNA. (B-D) See Supplemental materials for number of lines/animals scored. All data were analyzed as in Fig. 1B. Numbers below graph refer to ratio of hypodermal expression between adult vs. L₁-L₃ animals. (B) Down-regulation of col-10-lacZ-LCS 1, LCS 2, LCS 3, and LCS 5(pMV1) is abrogated in let-7(n2853) animals. (C) Effect of LCS 1^* and LCS 2^* mutations in a wild-type background. (D) A let-7(n2853) compensatory mutation in LCS 2 and LCS 1 partially restores down-regulation in a let-7(n2853) background. Lines that showed the most severe lack of down-regulation at the adult stage in a wild-type background were assayed.

Figure 3.

let-7 can bind to sequences from the lin-41 3′UTR in vitro. (A) Schematic of in vitro LCS constructs used to generate RNAs. (B) Wild-type let-7 RNA, but not let-7(n2853) mutant RNA can bind the full-length lin-41 3′UTR RNA. Lanes as indicated; retarded products indicated by two large arrows (lane 1), and two small arrows (lane 4). (C) LCS 1 and LCS 2 are sufficient to bind let-7 RNA in vitro. Lanes as indicated. The let-7 free probe has run off the gel and is not shown.

Figure 3.

let-7 can bind to sequences from the lin-41 3′UTR in vitro. (A) Schematic of in vitro LCS constructs used to generate RNAs. (B) Wild-type let-7 RNA, but not let-7(n2853) mutant RNA can bind the full-length lin-41 3′UTR RNA. Lanes as indicated; retarded products indicated by two large arrows (lane 1), and two small arrows (lane 4). (C) LCS 1 and LCS 2 are sufficient to bind let-7 RNA in vitro. Lanes as indicated. The let-7 free probe has run off the gel and is not shown.