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.

FAM210A regulates mitochondrial translation and maintains cardiac mitochondrial homeostasis.

AIMS: Mitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodelling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo. METHODS AND RESULTS: Tamoxifen-induced αMHCMCM-driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response, resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial-encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischaemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodelling and damage in ischaemia-induced heart failure. CONCLUSION: These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischaemic heart disease.

FAM210A Regulates Mitochondrial Translation and Maintains Cardiac Mitochondrial Homeostasis.

Aims: Mitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodeling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo . Methods and Results: Tamoxifen-induced αMHC MCM -driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response (ISR), resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodeling and damage in ischemia-induced heart failure. Conclusion: These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischemic heart disease. Translational Perspective: Mitochondrial homeostasis is critical for maintaining healthy cardiac function. Disruption of mitochondrial function causes severe cardiomyopathy and heart failure. In the present study, we show that FAM210A is a mitochondrial translation regulator required for maintaining cardiac mitochondrial homeostasis in vivo . Cardiomyocyte-specific FAM210A deficiency leads to mitochondrial dysfunction and spontaneous cardiomyopathy. Moreover, our results indicate that FAM210A is downregulated in human and mouse ischemic heart failure samples and overexpression of FAM210A protects hearts from myocardial infarction induced heart failure, suggesting that FAM210A mediated mitochondrial translation regulatory pathway can be a potential therapeutic target for ischemic heart disease.

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.

MicroRNA-574 regulates FAM210A expression and influences pathological cardiac remodeling.

Aberrant expression of mitochondrial proteins impairs cardiac function and causes heart disease. The mechanism of regulation of mitochondria encoded protein expression during cardiac disease, however, remains underexplored. Here, we show that multiple pathogenic cardiac stressors induce the expression of miR-574 guide and passenger strands (miR-574-5p/3p) in both humans and mice. miR-574 knockout mice exhibit severe cardiac disorder under different pathogenic cardiac stresses while miR-574-5p/3p mimics that are delivered systematically using nanoparticles reduce cardiac pathogenesis under disease insults. Transcriptomic analysis of miR-574-null hearts uncovers family with sequence similarity 210 member A (FAM210A) as a common target mRNA of miR-574-5p and miR-574-3p. The interactome capture analysis suggests that FAM210A interacts with mitochondrial translation elongation factor EF-Tu. Manipulating miR-574-5p/3p or FAM210A expression changes the protein expression of mitochondrial-encoded electron transport chain (ETC) genes but not nuclear-encoded mitochondrial ETC genes in both human AC16 cardiomyocyte cells and miR-574-null murine hearts. Together, we discovered that miR-574 regulates FAM210A expression and modulates mitochondrial-encoded protein expression, which may influence cardiac remodeling in heart failure.

Mammalian RNA switches: Molecular rheostats in gene regulation, disease, and medicine.

Alteration of RNA structure by environmental signals is a fundamental mechanism of gene regulation. For example, the riboswitch is a noncoding RNA regulatory element that binds a small molecule and causes a structural change in the RNA, thereby regulating transcription, splicing, or translation of an mRNA. The role of riboswitches in metabolite sensing and gene regulation in bacteria and other lower species was reported almost two decades ago, but riboswitches have not yet been discovered in mammals. An analog of the riboswitch, the protein-directed RNA switch (PDRS), has been identified as an important regulatory mechanism of gene expression in mammalian cells. RNA-binding proteins and microRNAs are two major executors of PDRS via their interaction with target transcripts in mammals. These protein-RNA interactions influence cellular functions by integrating environmental signals and intracellular pathways from disparate stimuli to modulate stability or translation of specific mRNAs. The discovery of a riboswitch in eukaryotes that is composed of a single class of thiamine pyrophosphate (TPP) suggests that additional ligand-sensing RNAs may be present to control eukaryotic or mammalian gene expression. In this review, we focus on protein-directed RNA switch mechanisms in mammals. We offer perspectives on the potential discovery of mammalian protein-directed and compound-dependent RNA switches that are related to human disease and medicine.

UPF1 helicase promotes TSN-mediated miRNA decay.

While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of ∼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.

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.

Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation.

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5µM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.