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 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.

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 discoveries: dsRNA within GATA4 uORF activates uORF translation and inhibits mORF translation. ASOs that target the dsRNA can either inhibit or enhance GATA4 mORF translation. ASOs can be used to impede hypertrophy in human cardiomyocytes and mouse hearts.uORF- and mORF-targeting ASOs can be used to control translation of multiple mRNAs.

RNA binding protein PRRC2B mediates translation of specific mRNAs 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.

Antiproliferative activity of a series of 5­(1H­1,2,3­triazolyl) methyl­ and 5­acetamidomethyl­oxazolidinone derivatives.

In the face of increasing resistance to the existing antibiotics, oxazolidinones (exemplified by linezolid) have been developed as promising antibacterial agents, but may have other useful actions. In the present study, a series of 5­(1H­1,2,3­triazoly) l­methyl­, 5­acetamidomethyl­morpholino and N­substituted­piperazino oxazolidinone derivatives were investigated to determine whether they are active against eukaryotic cells. An MTT assay, validated by cell counting, was used to assess the effect of nine oxazolidinone derivatives (concentrations 100 nM­10 µM) on the proliferation of MCF7 human breast cancer cells. The three most active compounds were then tested on MDA231 breast cancer cells. Cytotoxicity of the selected derivatives was determined by assessing the extent of apoptosis by flow cytometry. The antimetastatic potential of these compounds was assessed on MDA231 cells using wound healing and agarose invasion assays. The 5­triazolylmethyl piperazino­oxazolidinone derivatives containing 4­N­(2­chlorocinnamoyl), 4­N­(4­nitrobenzoyl) and 4­N­methylsulfonyl moieties exhibited the most potent cytostatic activity against cancer, inhibiting proliferation by up to 70%, in the same order as their reported antibacterial activity against Staphylococcus aureus, but at higher concentrations. Unexpectedly, several derivatives stimulated proliferation at 100 nM, well below their antibacterial minimum inhibitory concentrations. Certain compounds also retarded the motility and invasion of MDA231 cells. Three of the tested derivatives had no effect on the eukaryotic cell lines, demonstrating their preferential activity against bacteria. Two compounds actually stimulated eukaryotic cell proliferation. The remaining three exhibited potent cytostatic activity against and cancer cells, displaying differences in response at low and high concentrations, which may suggest multiple targets on eukaryotic cells. These latter compounds may be useful as anticancer agents.