Khor, E. | bioRxiv Science

bioRxiv2023

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

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 identified a double-stranded RNA (dsRNA) structure residing within the GATA4 uORF that augments uORF translation and inhibits mORF translation. 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 general utility of uORF-dsRNA- or mORF- targeting ASO to regulate mORF translation for other mRNAs. Our work demonstrates a regulatory paradigm that controls translational efficiency and a useful strategy to alter protein expression and cellular phenotypes by targeting or generating dsRNA downstream of a uORF or mORF start codon. Bullet points for discoveriesO_LIdsRNA within GATA4 uORF activates uORF translation and inhibits mORF translation. C_LIO_LIASOs that target the dsRNA can either inhibit or enhance GATA4 mORF translation. C_LIO_LIASOs can be used to impede hypertrophy in human cardiomyocytes and mouse hearts. C_LIO_LIuORF- and mORF-targeting ASOs can be used to control translation of multiple mRNAs. C_LI

molecular biology↗

bioRxiv2022

RNA binding protein PRRC2B mediates translation of specific proteins and regulates cell cycle progression

Accumulating evidence suggests that posttranscriptional control of gene expression, including RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and sequencing (PAR-CLIP-seq), we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites near the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by polysome-associated RNA-seq, resulting in reduced G1/S phase transition and cell proliferation. Antisense oligonucleotides blocking PRRC2B interactions with CCND2 mRNA decreased its translation, thus inhibiting G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome analysis revealed RNA-independent interactions with eukaryotic translation initiation factors 3 (eIF3) and 4G2 (eIF4G2). The interaction with translation initiation factors is essential for PRRC2B function since the eIF3/eIF4G2-interacting defective mutant, unlike wild-type PRRC2B, failed to rescue the translation deficiency or cell proliferation inhibition caused by PRRC2B knockdown. Altogether, our findings reveal that PRRC2B is essential for efficiently translating specific proteins required for cell cycle progression and cell proliferation. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/520836v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@cc9385org.highwire.dtl.DTLVardef@e92f55org.highwire.dtl.DTLVardef@1228c7corg.highwire.dtl.DTLVardef@1352179_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗