A Single Let-7 MicroRNA Bypasses LIN28-Mediated Repression

Abstract

Let-7 microRNAs (miRNAs) are critical regulators of animal development, stem cell differentiation, glucose metabolism, and tumorigenesis. Mammalian genomes contain 12 let-7 isoforms that suppress expression of a common set of target mRNAs. LIN28 proteins selectively block let-7 biogenesis in undifferentiated cells and in cancer. The current model for coordinate let-7 repression involves the LIN28 cold-shock domain (CSD) binding the terminal loop and the two CCHC-type zinc fingers recognizing a GGAG sequence motif in precursor let-7 (pre-let-7) RNAs. Here, we perform a systematic analysis of all let-7 miRNAs and find that a single let-7 family member, human let-7a-3 (and its murine ortholog let-7c-2), escapes LIN28-mediated regulation. Mechanistically, we find that the pre-let-7c-2 loop precludes LIN28A binding and regulation. These findings refine the current model of let-7 regulation by LIN28 proteins and have important implications for understanding the LIN28/let-7 axis in development and disease.

Figure 1. Systematic analysis of let-7 regulation by LIN28 proteins

(A) Western blot analyses showing expression of FLAG-LIN28A and FLAG-LIN28B upon doxycyline treatment. (B, C) q.RT-PCR analyses of let-7 expression in iLIN28A (B) and iLIN28B Hela cells (C). Results are the average of two biological replicate experiments represented as percentage of repression relative to mock treated cells. Error bars represent SD. (D) Electrophoretic mobility shift assay (EMSA) showing relative LIN28A binding to individual pre-let-7 RNAs. A twofold dilution series was used for the LIN28A titrations. Human precursors were used in these assays unless stated otherwise. See also Figure S1.

Figure 2. Human let-7a-3 and mouse let-7c-2 bypass LIN28A-mediated repression

(A) q.RT-PCR analyses of let-7g, let-7a and let-7c expression in Hela cells upon transfection of empty vectors (mock), let-7 pri-miRNA only or let-7 pri-miRNA and FLAG-LIN28A as indicated. (B) Schematic representation of the let-7c-2 locus. Arrowheads indicate guide RNA (gRNA) target sites used for CRISPR/Cas9 DNA editing. (C) PCR for genotyping analysis of let-7c-2^+/+ and let-7c-2^−/− TC1 mESC clones. (D) Sequencing analyses showing the deletion in the let-7c-2 locus. The upper track and the lower track correspond to the sequencing results obtained from the let-7c-2^+/+ clone #1 and the let-7c-2^−/− clone #1 respectively. (E) q.RT-PCR analyses for let-7c expression in let-7c-2^+/+ and let-7c-2^−/− clones. (F) q.RT-PCR analyses for let-7g and let-7c expression in KH2-iLIN28A mESCs treated with or without doxycycline (dox). (G) RT-qPCR analyses for let-7g and let-7c expression in TC1 mESCs transfected with control or Lin28a siRNA. Upper right panels represent Western blot results validating LIN28A knockdown. (A), (E-G) Results are the average of two independent experiments. Error bars represent SD.

Figure 3. The preE loop of pre-let-7c-2 precludes LIN28A binding and regulation

(A) Representation of different wild type and mutant preE sequences of let-7 constructs used in EMSA and transfection experiments. Mutations are underlined, preE stem is in blue and GGAG motif is in red. (B) EMSA showing specific binding of LIN28A to individual let-7 wild type and mutant pre-miRNA depicted in (A). (C) q.RT-PCR analyses of let-7a, let-7c and let-7g expression in Hela cells upon transfection of empty vectors (mock), let-7 pri-miRNA only or let-7 pri-miRNA and FLAG-LIN28A as indicated. Results are the average of two independent experiments. Error bars represent SD.