Loop nucleotides control primary and mature miRNA function in target recognition and repression

Abstract

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5’ end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs’ sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.

Acknowledgements

We thank the members of Chen lab for the comments and suggestions.

Funding

This work was supported by grants from National Institute of Health (HL081612 and 1DP1 OD00643501) and the W. M. Keck foundation to C.-Z. C.

Figures and Tables

Figure 1 Identification of pri/pre-miRNA loop mutations that potentiate cel-let-7 activity and partially rescue defective mature let-7 biogenesis of cel-let-7 miRNAs in human cells. (A) Schematic diagrams depict the pre-miRNA sequences and structures of the wild-type cel-let-7 gene and loop mutants. (B) Seed-dependent repression of target reporter by the cel-let-7 and loop mutants. Average results of at least six independent trials ± S.D. are shown. Statistical significance was determined by an unpaired two-tailed Student's t test. (C) Expression of pre- and mature miRNA made from wild-type and loop mutant cel-let-7 genes was determined by quantitative northern blot. Representative results of four northern blot analyses from independent transfections are shown. Blots were also probed for U6 small nuclear RNA as a loading control. (D & E) LP4 mutations in cel-let-7 result in increases in pre- (n=2) and mature let-7 (n=3 to 6) with correct 5′ ends. The 5′ ends of (D) mature let-7 and (E) pre-let-7 made from the wild-type cel-let-7 and cel-let-7_LP4 genes were determined by primer extension analyses.

Figure 2 Loop mutations that inhibit biogenesis of full-length mature let-7. (A) Schematic diagrams depicting the pre-miRNA sequences and structures of cel-let-7_LP4 and loop mutants. (B) A northern blot analysis representative of three experiments shows pre-miRNA and mature miRNA expression from cel-let-7-LP4 and loop mutants. (C) The 5′ ends of mature let-7 miRNAs made from the type cel-let-7 and pri-let-7 loop mutants were determined by primer extension analyses (n=6). (D) The 5′ ends of pre-let-7 miRNAs made from the type cel-let-7 and pri-let-7 loop mutants were determined by primer extension analyses (n=2).

Figure 3 The activities of cel-let-7_LP4 and loop mutants in target repression correlate with the formation of specific pri-let-7 RNAs and target complexes. (A) Repression of luciferase reporter expression by cel-let-7_LP4 and loop mutants. Representative results of at least six independent trials (± S.D.) are shown (*, p<0.0001 except as indicated). (B) Formation of pri-let-7 RNAs and target complexes predicts the activity of cel-let-7 and loop mutants in target repression. Lin-41_LCS reporter mRNAs were tagged with S1 aptamers and used to pull-down associated pri-let-7 RNA. S1-tagged reporter mRNAs with the Lin41-LCS_sm UTR or without the Lin41-LCS UTR were used as negative controls. The ratios of wild-type to mutant pri-let-7 RNA and corresponding S1-tagged target RNAs with or without the wild-type lin-41-LCS UTR or with a seed mutant lin-41-LCS UTR in the pull-down RNA samples were determined by using qPCR analyses (n=4, ±S.D., *, p< 0.001). (C & D) Standard curve-based qPCR was carried out to determine the average copy numbers of (C) pri-let-7 and (D) mature let-7 per target in the pull-down samples (n=4, ±S.D.). (E) The effects of cel-let-7-wt and cel-let-7-LP4 expression on the levels of target mRNA were determined by qPCR analyses (n=4, ±S.D., *, p< 0.001).

Figure 4 Repression activity by cel-let-7 and loop mutants may be controlled by both SD1-sensitive and SD1-insensitive components. (A) Schematic diagrams showing the predicted base parings between let-7 and modified lin-41_LCS. Altered nucleotides are indicated in blue, missing as underlined. (B & C) Repression of mutant lin-41_LCS reporters that abolish SD1 pairing by (B) wild-type cel-let-7 and cel-let-7-LP4 and (C) double loop mutant cel-let-7-LP4 genes. (D) siRNA duplexes of 20 base pairs (truncated let-7) and 22 base pairs (full-length mature let-7) were transfected into cells expressing mutant lin-41_LCS reporters that abolish SD1 pairing and reporter activity was measured. Representative results of at least six independent trials (± S.D.) are shown in panels B, C, and D (*, p< 0.01).