An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

Elizabeth O'Day,Minh T.N Le,Shunsuke Imai,Shen Mynn Tan,Rory Kirchner,Haribabu Arthanari,Oliver Hofmann,Gerhard Wagner,Judy Lieberman

doi:10.1074/jbc.m115.665521

Elizabeth O'Day, Minh T.N Le + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m115.665521

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Abstract

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.

Highlights

Lin28 is an evolutionary conserved RNA-binding protein that regulates miRNAs and mRNAs; Lin28 overexpression is linked to poor prognosis in cancer
Lin28-regulated Gene Sequences Are Enriched for G-quartet Motifs—We examined the sequences of the 12 let-7 loops and of all 15 Lin28 targets with an experimentally defined Lin28-responsive elements (LREs) (Table 1)
To determine whether Lin28 RNA targets might contain G4s, we first utilized the Quadruplex G-Rich Sequence (QGRS) Mapper, which predicts G4s in nucleic acids by identifying potential G-quartet sequences that match the following motif: GxNy1GxNy2GxNy3Gx, where x is the number of G-repeats and y1,y2,and y3 are the lengths of the gaps [43]

Summary

Background

Lin is an evolutionary conserved RNA-binding protein that regulates miRNAs and mRNAs; Lin overexpression is linked to poor prognosis in cancer. Because guanine rich nucleic acids can form stable planar structures called G-quartets (G4s) by G:G:G:G hydrogen bonding, we hypothesized that Lin might recognize structured RNAs that contain G4s. To address this hypothesis, we used bioinformatics, NMR spectroscopy, a fluorescent dye that binds to G4s, and gel shift assays to analyze the structural features of the pre-let-7s and their loops, which contain the 3Ј-GGAG motif; the LREs of DSG2, HSPA5, and SUN1, which contain the putative Lin consensus sequences AAGNNG, AAGNG(N), and (N)UGUG(N), respectively; and the LRE of OCT4, which contains the proposed “A bulge.”. We demonstrate that Lin may function to unwind G4s and that G4-stabilizing agents represent a novel class of Lin inhibitors

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