Abstract P2055: Secondary Structure And Antisense Oligonucleotides Regulate Cardiac Messenger RNA Translation And Modulates Cardiomyocyte Hypertrophy

Abstract

Translation of upstream open reading frames (uORFs) typically abrogates translation for the main ORF (mORF) and regulates protein expression. uORFs are present in nearly 50% of messenger RNA (mRNA) transcripts, but their impact and regulation on biological processes are understudied. Mining of ribosome profiling data from next-generation sequencing analysis of human and mouse hearts reveals that most translated uORFs reside within mRNAs of transcription factors, which play essential roles in cardiomyocyte (CM) development and growth. We show that the uORF start codon synergizes with an immediate downstream double-stranded RNA (dsRNA) element to activate uORF and inhibit mORF translation. Biochemical and genetic evidence support that this mechanism is utilized by mRNAs encoding multiple transcription factors such as GATA4 (GATA binding protein 4), encoding a master transcription regulator of CM differentiation and pathological hypertrophy. Intriguingly, a trans-acting regulatory factor, DEAD-box RNA helicase DDX3X, is involved in unwinding the dsRNA structure and promoting mORF translation. We develop two types of antisense oligonucleotides (ASOs) that mimic the unwinding or stabilizing of the dsRNA secondary structure, thereby suppressing or enhancing the translation of this uORF, respectively. Genetic or chemical inactivation of this GATA4 uORF using CRISPR-Cas9 or ASOs in human embryonic stem cells lead to enhanced differentiation into CMs and cellular hypertrophy. At the organismal level, short-term treatment of isoproterenol and surgery-induced cardiac hypertrophy mouse models with uORF-enhancing ASOs reduces GATA4 mRNA translation and antagonizes cardiac hypertrophy and remodeling. As a summary, this uORF-dsRNA element is a new tunable regulator of cardiac transcription factor mRNA translation and can be targeted to alter cellular and organismal phenotypes. Moreover, mechanism-based translation-manipulating ASOs can serve as promising biotechnological tools to regulate gene expression in vitro and in vivo and can be adapted to create novel RNA-based therapeutics.