Genome-wide association studies (GWAS) have identified numerous RA associated genetic variants, most of which localize to non-coding regulatory regions (eg, enhancers), but their functional annotation remains a barrier to translating GWAS findings into mechanistic insights. This study hypothesized that these enhancer SNPs reside in the accessible chromatin domains of RA's target tissue (synovium) and modulate target gene expression by altering transcription factor (TF) binding affinity. Thus, leveraging prospectively collected RA synovial tissues, it aimed to identify key regulatory TFs and their roles in RA pathogenesis. The study integrated assay for transposase-accessible chromatin sequencing (ATAC-seq) data from synovial tissues of 7 RA patients with GWAS-derived RA-associated enhancer SNPs to prioritize TFs that bind these enhancers, with functional validation performed using primary fibroblast-like synoviocytes (FLSs) from RA synovial tissues and MH7A cell lines. Results showed that 1006 GWAS SNPs localized to enhancer regions and were significantly enriched in the open chromatin domains of RA synovium. Transcription factor enrichment analysis identified RUNX3 as the top candidate TF, which regulates FLS migration and invasion. Mechanistically, the G allele of rs1930785 binds RUNX3 in an allele-specific manner, upregulating the expression of its target gene TRAF1, which in turn exerts a protective effect on FLS homeostasis. The study confirms RUNX3 as a key TF regulating the activity of RA-associated enhancers, highlights the value of patient-derived synovial tissue data and the dual validation system, and lays a foundation for mechanistic research and targeted therapy for RA. Rheumatoid arthritis is a chronic inflammatory joint disease associated with numerous genetic variations whose functions remain poorly understood. Using joint tissue from patients, we investigated how these genetic differences affect cellular activity in affected joints. We identified a key regulatory protein called RUNX3, and found that the G allele of genetic variant rs1930785 enhances its binding to DNA and increases expression of the TRAF1 gene, helping to maintain normal joint cell function. These findings improve our understanding of RA genetics and may support the development of future targeted therapies.

Genome-wide association studies (GWAS) have identified numerous rheumatoid arthritis (RA) associated genetic variants, most of which localize to non-coding regulatory regions (eg, enhancers), but their functional annotation remains a barrier to translating GWAS findings into mechanistic insights. This study hypothesized that these enhancer single nucleotide polymorphisms (SNPs) reside in the accessible chromatin domains of RA's target tissue (synovium) and modulate target gene expression by altering transcription factor (TF) binding affinity. Thus, leveraging prospectively collected RA synovial tissues, it aimed to identify key regulatory TFs and their roles in RA pathogenesis. The study integrated assay for transposase-accessible chromatin sequencing (ATAC-seq) data from synovial tissues of 7 RA patients with GWAS-derived RA-associated enhancer SNPs to prioritize TFs that bind these enhancers, with functional validation performed using primary fibroblast-like synoviocytes (FLSs) from RA synovial tissues and MH7A cell lines. Results showed that 1006 GWAS SNPs localized to enhancer regions and were significantly enriched in the open chromatin domains of RA synovium; TF enrichment analysis identified RUNX3 as the top candidate TF, which regulates FLS migration and invasion. Mechanistically, the G allele of rs1930785 binds RUNX3 in an allele-specific manner, upregulating the expression of its target gene TRAF1, which in turn exerts a protective effect on FLS homeostasis. The study confirms RUNX3 as a key TF regulating the activity of RA-associated enhancers, highlights the value of patient-derived synovial tissue data and the dual validation system, and lays a foundation for mechanistic research and targeted therapy for RA.

Cartilage and synovium are essential tissues involved in joint-related diseases and traits, including osteoarthritis (OA), rheumatoid arthritis (RA), and human height. Although genome-wide association studies (GWAS) identify numerous risk loci for these traits, the molecular mechanisms underlying these associations, particularly those involving alternative splicing, remain poorly understood due to the lack of splicing-related genetic data in relevant tissues. To address this limitation, we generate a splicing quantitative trait loci (sQTL) resource for cartilage and synovium. We identify 2,796 independent cis-sQTLs and six trans-sGenes across the two tissues, including 179 tissue-specific cis-sQTLs. Fine-mapping analysis identifies 116 high-confidence functional sVariants predicted to affect splicing through splice site gain or loss, with approximately half located outside canonical splice site motifs. Integration of sQTL data with GWAS summary statistics reveals 12 osteoarthritis, 6 rheumatoid arthritis, and 183 height effector genes in joint tissues. Notably, seven of the 12 osteoarthritis effector genes show joint tissue-specific colocalization. Together with the finding that tissue-specific sGenes play crucial roles in tissue-related biological processes and diseases, our work highlights the significant impact of tissue-specific alternative splicing regulation on disease etiology and provides a valuable resource for further mechanistic validation.

BACKGROUND:Genome-wide association studies (GWAS) have identified more than one hundred risk loci for osteoarthritis (OA). Identifying the effector genes and deciphering the underlying regulatory mechanisms are of great importance but remains challenging due to limited availability of OA-related tissue data. This study aims to address this issue by generating a cartilage expression quantitative trait loci (eQTLs) and a functional fine-mapping resource. METHODS:We performed cis-eQTL analysis using genomics and cartilage transcriptomics data from 204 patients with OA (largest sample size to date). Cell type-interaction eQTL analysis (ci-eQTL) was conducted to explore the chondrocyte subtype dependency of eQTL effects. Co-localization analysis was used to nominate effector genes of OA GWAS risk loci. A deciphering pipeline was established to identify candidate causal variants in eQTL loci that regulate gene expression through the alteration of chromatin accessibility or disruption of transcription factors (TFs) binding to regulatory elements. FINDINGS:We identified 3352 independent eQTLs for 3109 genes, 120 eQTL-gene pairs showed chondrocyte subtype dependency. We identified 19 new OA risk genes. We identified 117 causal eQTLs exhibiting allele-specific open chromatin (ASoC) and 547 eQTLs involved in transcription factor binding disruption (TBD). Functional validation showed that the T allele of the OA risk variant rs11750646 enhances the AR binding affinity to an open chromatin region, thereby promoting the expression of the OA-related gene PIK3R1. INTERPRETATION:Our findings provide insights into the unique regulatory landscape of cartilage and elucidate potential mechanisms underlying OA pathogenesis. FUNDING:This work was supported by National Natural Science Foundation of China (32470639, 82372458, and 82170896); Science Fund for Distinguished Young Scholars of Shaanxi Province (2025JC-JCQN-054); Innovation Capability Support Program of Shaanxi Province (2022TD-44, 2024RS-CXTD-86); Key Research and Development Project of Shaanxi Province (2023-YBSF-180); China Postdoctoral Science Foundation (2024M752561); and the Fundamental Research Funds for the Central Universities.

Upstream open reading frames (uORFs) are short peptide-encoding sequences located in the 5' untranslated region (5' UTR) of mRNAs, enabling translational repression of main (m)ORFs. While uORFs are found in ~50% of mRNAs in humans, our understanding of their biological function remains limited. This study aims to elucidate the role of the uORF in the 5' UTR of the Gata4 (GATA binding protein 4) gene in cardiac biology by inactivating its start codon (ΔuORF) in the mouse genome. Our investigation reveals that mice with Gata4 uORF inactivation manifest spontaneous cardiac hypertrophy without apparent fibrosis as they age. Utilizing single-nucleus RNA sequencing (snRNA-seq), we uncovered significant transcriptional variations between wild-type (WT) and ΔORF mice. Notably, mRNAs associated with sarcomeres and contractile functions show heightened expression levels, reflecting the hypertrophic phenotype. Notably, at least nine upregulated genes are GATA4-bound targets in mouse ventricles. Functional assessments of isolated primary adult cardiomyocytes confirmed enhanced hypertrophy and contractility in ΔORF mice. Additionally, we employed single-nucleus transposase-accessible chromatin (snATAC)-seq to investigate changes in chromatin accessibility. Our results indicated increased accessibility within specific transcription-regulatory elements linked to elevated gene transcription. These putative cis-regulatory elements (pCREs) are significantly enriched in MEF2 (myocyte enhancer factor 2) binding motifs. In vitro luciferase reporter assays further supported the regulatory potential of three of these pCREs, highlighting their role in the transcriptional enhancement of three GATA4 target genes bound by MEF2 and GATA4. These findings illuminate the role of uORF in negatively regulating GATA4 protein expression and cardiomyocyte hypertrophy at the organismal level and provide a novel therapeutic target for cardiac pathogenesis.

Cardiac physiology and pathology have been extensively explored at the transcriptional level. Still, they are less understood at the translational level, including three major knowledge gaps: pathophysiological impact, molecular mechanisms, and therapeutic implications of translational control in cardiac biology and heart disease. This review aims to provide a summary of the most recent key findings in this emerging field of translational control in heart health and disease, covering the physiological functions, disease pathogenesis, biochemical mechanisms, and development of potential RNA-based, translation-manipulating drugs. Translation of mRNA to protein is the final step in the central dogma for protein synthesis. Translation machinery includes a family of essential “housekeeping” factors and enzymes required for mRNA translation. These translation factors ensure the accurate processing of mRNA to protein according to the genetic code and maintain the optimal quality and quantity of cellular proteins for normal cardiac function. Translation factors also regulate the efficiency, speed, and fidelity of protein production and play a role in cardiac pathological remodeling under stress conditions. This review first introduces the techniques and methods used to study the translational regulation of gene expression in the cardiac system. We then summarize discoveries of a variety of pathophysiological functions and molecular mechanisms of translational control in cardiac health and disease, focusing on two primary symptoms, cardiac hypertrophy and fibrosis. In these sessions, we discuss the translational regulation directed by specific regulatory factors in cardiac physiology and how their genetic mutations, expression dysregulation, or functional alterations contribute to the etiology of heart disease. Notably, translational control exhibits extensive crosstalk with other processes, including transcriptional regulation, mitochondrial metabolism, and sarcomere homeostasis. Furthermore, recent findings have revealed the role of translational regulation in cardiomyocyte proliferation and heart regeneration, providing new approaches for creating regenerative medicine. Because transcript-specific translational regulation of both pathological and protective proteins occurs in heart disease, target-selective translation inhibitors and enhancers can be developed. These inhibitors and enhancers offer valuable insights into novel therapeutic targets and the development of RNA-based drugs for heart disease treatment.

OBJECTIVES:This study aimed to address the lack of gene expression regulation data in synovial tissues and to identify genes associated with rheumatoid arthritis (RA) in the synovium, a primary target tissue for RA. METHODS:Gene expression prediction models were built for synovial tissue using matched genotype and gene expression data from 202 subjects. Using this model, we conducted a transcriptome-wide association study (TWAS), utilizing the largest rheumatoid arthritis (RA) genome-wide association study (GWAS) meta-analysis data (n = 276 020). Further analyses, including conditional and joint analysis, causal analysis, differential expression analysis and gene-set enrichment analysis, were conducted to deepen our understanding of genetic architecture and comorbidity aetiology of RA. RESULTS:Our analysis identified eight genes associated with rheumatoid arthritis (RA), including three novel genes: TPRA1 (PTWAS = 9.59 × 10-6), HIP1 (PTWAS = 1.47 × 10-5) and RP11-73E17.2 (PTWAS = 3.32 × 10-7). These genes differed from those identified in previous TWAS studies using alternative tissues and may play a crucial role in the target synovial tissue. We found four genes exhibited significant causal relationships with RA and were differentially expressed in RA patients. Furthermore, we explored potential drug repurposing opportunities for these genes. CONCLUSIONS:Our study is the first to model gene expression in synovial tissue, uncovering novel genetic determinants of rheumatoid arthritis (RA). This advancement not only deepens our understanding of RA's genetic architecture, but also offers promising avenues for targeted therapies and drug repurposing.

Head-to-body ratios (HBRs) are important anthropometric traits with direct relevance to human growth, development, and disease risk. However, the role of the proportions between head and body remains understudied, with the genetic basis of HBRs remaining largely unexplored. By applying deep learning models to 38,202 whole-body dual-energy X-ray absorptiometry images from the UK Biobank, we generated 10 distinct HBR phenotypes based on head (length/width) and various body dimensions. Our genome-wide association analyses identify 245 significant loci, with SNP-based heritability estimates ranging from 25% to 43%. Functional annotations show that genes prioritized for HBRs are enriched in chondrocytes in skeletal tissues and oligodendrocytes across multiple brain regions. Polygenic risk scores and mendelian randomization analyses further showed that HBRs are significantly associated with risks for cardiovascular, metabolic, musculoskeletal, and neuropsychiatric diseases, underscoring their potential value as health-related biomarkers. Evolutionary analyses show that HBR-associated variants are enriched in conserved genomic regions and human accelerated regions, particularly those influencing brain development. Overall, our study provides insights into the genetic architectures of HBRs, establishes their relevance to major human diseases, and offers evolutionary context for their biological significance.

Central obesity is associated with higher risk of developing a wide range of diseases independent of overall obesity. Genome-wide association studies (GWASs) have identified more than 300 susceptibility loci associated with central obesity. However, the functional understanding of these loci is limited by the fact that most loci are in non-coding regions. To address this issue, our study first prioritized 2,034 single-nucleotide polymorphisms (SNPs) based on fine-mapping and epigenomic annotation analysis. Subsequently, we employed self-transcribing active regulatory region sequencing (STARR-seq) to systematically evaluate the enhancer activity of these prioritized SNPs. The resulting data analysis identified 141 SNPs with allelic enhancer activity. Further analysis of allelic transcription factor (TF) binding prioritized 20 key TFs mediating the central-obesity-relevant genetic regulatory network. Finally, as an example, we illustrate the molecular mechanisms of how rs8079062 acts as an allele-specific enhancer to regulate the expression of its targeted RNF157. We also evaluated the role of RNF157 in the adipogenic differentiation process. In conclusion, our results provide an important resource for understanding the genetic regulatory mechanisms underlying central obesity.

Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of the CM-specific loss of Eprs1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. The transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression in multi-aminoacyl-tRNA synthetase complex components, fatty acids, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicates that multiple proline-rich proteins were reduced at the early stage, which might contribute to the cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcomes of loss-of-function of Eprs1 in vivo and provides valuable insights into the potential side effects on CMs, resulting from the EPRS1-targeting therapeutic approach.

The synovium is an important component of any synovial joint and is the major target tissue of inflammatory arthritis. However, the multi-omics landscape of synovium required for functional inference is absent from large-scale resources. Here we integrate genomics with transcriptomics and chromatin accessibility features of human synovium in up to 245 arthritic patients, to characterize the landscape of genetic regulation on gene expression and the regulatory mechanisms mediating arthritic diseases predisposition. We identify 4765 independent primary and 616 secondary cis -expression quantitative trait loci ( cis -eQTLs) in the synovium and find that the eQTLs with multiple independent signals have stronger effects and heritability than single independent eQTLs. Integration of genome-wide association studies (GWASs) and eQTLs identifies 84 arthritis related genes, revealing 38 novel genes which have not been reported by previous studies using eQTL data from the GTEx project or immune cells. We further develop a method called eQTac to identify variants that could affect gene expression by affecting chromatin accessibility and identify 1517 regions with potential regulatory function of chromatin accessibility. Altogether, our study provides a comprehensive synovium multi-omics resource for arthritic diseases and gains new insights into the regulation of gene expression.

Alternative splicing generates variant forms of proteins for a given gene and accounts for functional redundancy or diversification. A novel RNA-binding protein, Pro-rich Coiled-coil Containing Protein 2B (PRRC2B), has been reported by multiple laboratories to mediate uORF-dependent and independent regulation of translation initiation required for cell cycle progression and proliferation. We identified two alternative spliced isoforms in human and mouse hearts and HEK293T cells, full-length (FL) and exon 16-excluded isoform ΔE16. A congenital heart disease-associated human mutation-mimicry knock-in of the equivalent variant in the mouse genome leads to the depletion of the full-length Prrc2b mRNA but not the alternative spliced truncated form ΔE16, does not cause any apparent structural or functional disorders. In contrast, global genetic inactivation of the PRRC2B gene in the mouse genome, nullifying both mRNA isoforms, caused patent ductus arteriosus (PDA) and neonatal lethality in mice. Bulk and single nucleus transcriptome profiling analyses of embryonic mouse hearts demonstrated a significant overall downregulation of multiple smooth muscle-specific genes in Prrc2b mutant mice resulting from reduced smooth muscle cell number. Integrated analysis of proteomic changes in Prrc2b null mouse embryonic hearts and polysome-seq and RNA-seq multi-omics analysis in human HEK293T cells uncover conserved PRRC2B-regulated target mRNAs that encode essential factors required for cardiac and vascular development. Our findings reveal the connection between alternative splicing regulation of PRRC2B, PRRC2B-mediated translational control, and congenital cardiovascular development and disorder. This study may shed light on the significance of PRRC2B in human cardiovascular disease diagnosis and treatment.

Cartilage damage is a leading cause of osteoarthritis (OA) etiology, however, the underlying mechanism governing gene expression regulation in this progress is poorly understood. Here, we described a comprehensive profiling of transcriptional regulation of 235 primary human cartilage samples. We identified 3,352 independent significant expression quantitative trait loci (eQTLs) for 3,109 genes. We explored the candidate casual SNP and its underlying regulatory mechanism using our established functional fine-mapping pipeline by integrating the cartilage-specific ATAC-seq data. We identified 117 causal eQTLs that display allele-specific open chromatin (ASoC) and 547 transcription factor binding-disruption (TBD) eQTLs. We conducted cell type-interaction eQTL (ci-eQTL) analyses based on speculated chondrocyte subtype proportions and revealed the regulation relationship of 120 eQTL-gene pairs showed cell type dependency. Further, by integrating with genome-wide association studies (GWASs) data of OA, we nominated 43 candidate effector genes for OA risk loci. We verified that the T allele of the OA risk variant rs11750646 increased the AR binding affinity to an open chromatin region and promoted the expression of an OA-related gene PIK3R1. Altogether, our findings provide new insights into the unique regulatory landscape of cartilage and elucidate potential mechanisms underlying the OA pathogenesis. ### Competing Interest Statement The authors have declared no competing interest.

The human brain has been implicated in the pathogenesis of several complex diseases. Taking advantage of single-cell techniques, genome-wide association studies (GWAS) have taken it a step further and revealed brain cell-type-specific functions for disease loci. However, genetic causal associations inferred by Mendelian randomization (MR) studies usually include all instrumental variables from GWAS, which hampers the understanding of cell-specific causality. Here, we developed an analytical framework, Cell-Stratified MR (csMR), to investigate cell-stratified causality through colocalizing GWAS signals with single-cell eQTL from different brain cells. By applying to obesity-related traits, our results demonstrate the cell-type-specific effects of GWAS variants on gene expression, and indicate the benefits of csMR to identify cell-type-specific causal effect that is often hidden from bulk analyses. We also found csMR valuable to reveal distinct causal pathways between different obesity indicators. These findings suggest the value of our approach to prioritize target cells for extending genetic causation studies.

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.

Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA.

Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer -indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.

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.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has been widely used to facilitate efficient genome editing. Current popular sgRNA design tools only consider the sgRNA perfectly matched to the target site and provide the results without any on-target mismatch. We suppose taking on-target gRNA-DNA mismatches into consideration might provide better sgRNA with similar binding activity and reduced off-target sites. Here, we trained a seq2seq-attention model with feedback-loop architecture, to automatically generate sgRNAs with on-target mismatches. Dual-luciferase reporter experiment showed that multiple sgRNAs with three mismatches could achieve the 80% of the relative activity of the perfect matched sgRNA. Meanwhile, it could reduce the number of off-target sites using sgRNAs with on-target mismatches. Finally, we provided a freely accessible web server sgRNA design tool named ExsgRNA. Users could submit their target sequence to this server and get optimal sgRNAs with less off-targets and similar on-target activity compared with the perfect-matched sgRNA.

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.