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Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy.

ABSTRACT: Translation of upstream open reading frames (uORFs) typically abrogates translation of main (m)ORFs. The molecular mechanism of uORF regulation in cells is not well understood. Here, we data-mined human and mouse heart ribosome profiling analyses and identified a double-stranded RNA (dsRNA) structure within the GATA4 uORF that cooperates with the start codon to augment uORF translation and inhibits mORF translation. A trans-acting RNA helicase DDX3X inhibits the GATA4 uORF-dsRNA activity and modulates the translational balance of uORF and mORF. Antisense oligonucleotides (ASOs) that disrupt this dsRNA structure promote mORF translation, while ASOs that base-pair immediately downstream (i.e., forming a bimolecular double-stranded region) of either the uORF or mORF start codon enhance uORF or mORF translation, respectively. Human cardiomyocytes and mice treated with a uORF-enhancing ASO showed reduced cardiac GATA4 protein levels and increased resistance to cardiomyocyte hypertrophy. We further show the broad utility of uORF-dsRNA- or mORF-targeting ASO to regulate mORF translation for other mRNAs. This work demonstrates that the uORF-dsRNA element regulates the translation of multiple mRNAs as a generalizable translational control mechanism. Moreover, we develop a valuable strategy to alter protein expression and cellular phenotypes by targeting or generating dsRNA downstream of a uORF or mORF start codon.

SUBMITTER: Hedaya OM

PROVIDER: S-EPMC10547706 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy.

Hedaya Omar M OM Venkata Subbaiah Kadiam C KC Jiang Feng F Xie Li Huitong LH Wu Jiangbin J Khor Eng-Soon ES Zhu Mingyi M Mathews David H DH Proschel Chris C Yao Peng P

Nature communications 20231003 1

Translation of upstream open reading frames (uORFs) typically abrogates translation of main (m)ORFs. The molecular mechanism of uORF regulation in cells is not well understood. Here, we data-mined human and mouse heart ribosome profiling analyses and identified a double-stranded RNA (dsRNA) structure within the GATA4 uORF that cooperates with the start codon to augment uORF translation and inhibits mORF translation. A trans-acting RNA helicase DDX3X inhibits the GATA4 uORF-dsRNA activity and mod ...[more]

PMID: 37789015

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Project description:BackgroundPathological cardiac hypertrophy is a major contributor of heart failure (HF), which seriously threatens human's health world widely. Deregulation of m6A RNA methylation, and m6A methyltransferases and de-methyltransferases have been demonstrated to act essential roles in cardiac hypertrophy and HF. Here, we studied the potential roles and its underlying mechanisms of m6A Reader YTHDF proteins in HF. In this study, we constructed HF mouse model by transverse aortic constriction surgery. Primary cardiomyocytes were isolated and stimulated with isoproterenol (ISO) or phenylephrine (PHE) to induce myocardial hypertrophy.ResultsThrough single-cell RNA-seq analysis, immunofluorescent staining, HE staining, Western blotting, and real time-PCR detections, we found that YTHDF2 mRNA and protein level, but not YTHDF1 or YTHDF3, was significantly increased during HF development. YTHDF2 overexpression could efficiently alleviate cardiac hypertrophy. Furthermore, through immunoprecipitation accompanied with mass spectrometry analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that ISO stimulation did not evidently affect YTHDF2-interacting proteins. However, ISO or PHE stimulation significantly increased YTHDF2 protein interacting with Myh7 (beta-myosin heavy chain) mRNA, an important cardiac hypertrophy marker, in an m6A-dependent manner. Knockdown of Myh7 or deletion of the YTH domain of YTHDF2 reversed the protective effects of YTHDF2 on cardiac hypertrophy. Finally, we found that ISO or PHE stimulation promoted YTHDF2 protein expression through enhancing Ythdf2 mRNA stability in an m6A-dependent manner in cardiomyocytes.ConclusionsOverall, our results indicate that the m6A Reader YTHDF2 suppresses cardiac hypertrophy via Myh7 mRNA decoy in an m6A-dependent manner. This study highlights the functional importance of YTHDF2-dependent cardiac m6A mRNA regulation during cardiac hypertrophy, and provides a novel mechanistic insight into the therapeutic mechanisms of YTHDF2.

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Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy.

Publications

Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy.

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