Targeted Inhibition of Oncogenic MiR-21 Maturation with Designed RNA-binding Proteins

Overview

Journal Nat Chem Biol

Specialties Biochemistry
Biology
Chemistry

Date 2016 Jul 19

PMID 27428511

Citations 25

Authors

Yu Chen

Fan Yang

Lorena Zubovic

Tom Pavelitz

Wen Yang

Katherine Godin

Matthew Walker

Suxin Zheng

Paolo Macchi

Gabriele Varani

Affiliations

Soon will be listed here.

Abstract

The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.

Citing Articles

Stimulus-activated ribonuclease targeting chimeras for tumor microenvironment activated cancer therapy.

Zhang Y, Zhu J, Qiu L, Lv Z, Zhao Z, Ren X Nat Commun. 2025; 16(1):1288.

PMID: 39900602 PMC: 11790973. DOI: 10.1038/s41467-025-56691-3.

Two distinct binding modes provide the RNA-binding protein RbFox with extraordinary sequence specificity.

Ye X, Yang W, Yi S, Zhao Y, Varani G, Jankowsky E Nat Commun. 2023; 14(1):701.

PMID: 36759600 PMC: 9911399. DOI: 10.1038/s41467-023-36394-3.

Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

Shortridge M, Chaubey B, Zhang H, Pavelitz T, Vidadala V, Tang C ACS Chem Biol. 2023; 18(2):237-250.

PMID: 36727622 PMC: 10593481. DOI: 10.1021/acschembio.2c00502.

Inverse In Vitro Selection Enables Comprehensive Analysis of Cross-Chiral L-Aptamer Interactions.

Piwko A, Han X, Kabza A, Dey S, Sczepanski J Chembiochem. 2022; 23(24):e202200520.

PMID: 36282114 PMC: 9798143. DOI: 10.1002/cbic.202200520.

A Slow Dynamic RNA Switch Regulates Processing of microRNA-21.

Shortridge M, Olsen G, Yang W, Walker M, Varani G J Mol Biol. 2022; 434(16):167694.

PMID: 35752213 PMC: 10593484. DOI: 10.1016/j.jmb.2022.167694.

References

Krol J, Loedige I, Filipowicz W . The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 2010; 11(9):597-610. DOI: 10.1038/nrg2843. View

MacRae I, Zhou K, Li F, Repic A, Brooks A, Cande W . Structural basis for double-stranded RNA processing by Dicer. Science. 2006; 311(5758):195-8. DOI: 10.1126/science.1121638. View

Auweter S, Oberstrass F, Allain F . Sequence-specific binding of single-stranded RNA: is there a code for recognition?. Nucleic Acids Res. 2006; 34(17):4943-59. PMC: 1635273. DOI: 10.1093/nar/gkl620. View

Chen Y, Zubovic L, Yang F, Godin K, Pavelitz T, Castellanos J . Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer. Nucleic Acids Res. 2016; 44(9):4381-95. PMC: 4872098. DOI: 10.1093/nar/gkw177. View

Trabucchi M, Briata P, Garcia-Mayoral M, Haase A, Filipowicz W, Ramos A . The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature. 2009; 459(7249):1010-4. PMC: 2768332. DOI: 10.1038/nature08025. View

Tenzer S, Moro A, Kuharev J, Francis A, Vidalino L, Provenzani A . Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach. J Proteome Res. 2013; 12(6):2869-84. DOI: 10.1021/pr400193j. View

Chen Y, Kortemme T, Robertson T, Baker D, Varani G . A new hydrogen-bonding potential for the design of protein-RNA interactions predicts specific contacts and discriminates decoys. Nucleic Acids Res. 2004; 32(17):5147-62. PMC: 521638. DOI: 10.1093/nar/gkh785. View

Das R, Laederach A, Pearlman S, Herschlag D, Altman R . SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA. 2005; 11(3):344-54. PMC: 1262685. DOI: 10.1261/rna.7214405. View

Schmittgen T, Jiang J, Liu Q, Yang L . A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res. 2004; 32(4):e43. PMC: 390315. DOI: 10.1093/nar/gnh040. View

10.

Lunse C, Michlewski G, Hopp C, Rentmeister A, Caceres J, Famulok M . An aptamer targeting the apical-loop domain modulates pri-miRNA processing. Angew Chem Int Ed Engl. 2010; 49(27):4674-7. DOI: 10.1002/anie.200906919. View

11.

Mackay J, Font J, Segal D . The prospects for designer single-stranded RNA-binding proteins. Nat Struct Mol Biol. 2011; 18(3):256-61. DOI: 10.1038/nsmb.2005. View

12.

Davis B, Hilyard A, Lagna G, Hata A . SMAD proteins control DROSHA-mediated microRNA maturation. Nature. 2008; 454(7200):56-61. PMC: 2653422. DOI: 10.1038/nature07086. View

13.

Auweter S, Fasan R, Reymond L, Underwood J, Black D, Pitsch S . Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO J. 2005; 25(1):163-73. PMC: 1356361. DOI: 10.1038/sj.emboj.7600918. View

14.

Nguyen T, Jo M, Choi Y, Park J, Kwon S, Hohng S . Functional Anatomy of the Human Microprocessor. Cell. 2015; 161(6):1374-87. DOI: 10.1016/j.cell.2015.05.010. View

15.

Minuchehr Z, Goliaei B . Propensity of amino acids in loop regions connecting beta-strands. Protein Pept Lett. 2005; 12(4):379-82. DOI: 10.2174/0929866053765617. View

16.

Cheong C, Hall T . Engineering RNA sequence specificity of Pumilio repeats. Proc Natl Acad Sci U S A. 2006; 103(37):13635-9. PMC: 1564246. DOI: 10.1073/pnas.0606294103. View

17.

Agrawal A, McLaughlin K, Jenkins J, Kielkopf C . Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA. Proc Natl Acad Sci U S A. 2014; 111(49):17420-5. PMC: 4267390. DOI: 10.1073/pnas.1412743111. View

18.

Wang Y, Wang Z, Hall T . Engineered proteins with Pumilio/fem-3 mRNA binding factor scaffold to manipulate RNA metabolism. FEBS J. 2013; 280(16):3755-67. PMC: 3768134. DOI: 10.1111/febs.12367. View

19.

Parisien M, Major F . The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature. 2008; 452(7183):51-5. DOI: 10.1038/nature06684. View

20.

Si M, Zhu S, Wu H, Lu Z, Wu F, Mo Y . miR-21-mediated tumor growth. Oncogene. 2006; 26(19):2799-803. DOI: 10.1038/sj.onc.1210083. View