uORF-mediated Translational Control of Cardiac Transcription Factor Expression

Short peptide-encoding sequences in the 5' untranslated region of messenger ribonucleic acids (mRNA), called upstream open reading frames (uORFs), are widespread in ~50% of human mRNAs. Translating these uORF sequences reduces the protein output of an mRNA main open reading frame (mORF). Our bioinformatic analysis of human and mouse ribosome profiling databases uncovered a group of cardiac mRNA transcripts containing translated uORFs, such as transcription factors, including GATA4. Biochemical analysis suggests that stabilizing the double-stranded RNA (dsRNA) structure downstream of the start codons of these peptide-encoding sequences enhances their translation, thereby inhibiting the translation of mORFs. This translational inhibitory mechanism is mitigated by DEAD-box RNA helicase DDX3X that unwinds dsRNA and inactivates uORF. Genetic depletion of GATA4 uORF activity using CRISPR-Cas9 mediated genomic editing of the start codon in human embryonic stem cells (ESC) provides evidence of uORF-mediated regulation of mORF translation and cardiomyocyte (CM) hypertrophy. In addition, an established CRISPR-Cas9-derived uORF start codon mutant knock-in mouse model shows spontaneous cardiac hypertrophy and will be used to characterize CM hypertrophy at baseline and under stress conditions. Based on our discovered molecular mechanism of DDX3X-regulated, dsRNA-dependent, uORF-mediated translational inhibition of mORF, we have developed two types of antisense oligonucleotides (ASOs) that can either enhance or reduce uORF translation by strengthening or disrupting dsRNA structures. The uORF-enhancing ASO locks the dsRNA structure and activates translation of the uORF, thereby reducing GATA4 mORF protein expression in human CMs. Treatment of mouse cardiomyopathy models with uORF-enhancing ASO reduces GATA4 protein expression, antagonizes cardiac hypertrophy, and restores cardiac function. Based on our findings, we hypothesize that cardiac transcription factor mRNA uORF-dsRNA element acts as a switch for translational control of mORF, regulating cardiac hypertrophy, and can be targeted by ASOs to modulate mORF protein translation and cardiac hypertrophy. We will focus on 3 Specific Aims. Aim 1. Elucidate mRNA structural elements and their interplay with the GATA4 uORF for regulating mORF translation. Aim 2. Determine the biological role of the GATA4 uORF in genetic knock-in mouse models and primary CM cell culture systems. Aim 3. Develop proof-of-concept translation-manipulating ASOs targeting the GATA4 uORF for short-term anti- hypertrophy intervention. These studies will provide novel insights into translational control mechanisms in cardiac biology. This project will promote novel therapeutic approaches (targeting uORF-dsRNA elements) to regulate cardiac hypertrophy. Our mechanism-based design of translation-manipulating ASOs can serve as a proof-of-concept model to apply to different pathogenic mRNA targets.