Claudia Kutter

Translation elongation relies on accurate codon-anticodon pairing. Here, we present tTEscanR, an R package designed to investigate this translational interface. By quantifying mRNA codon demand alongside tRNA anticodon availability, tTEscanR provides scalable estimates of translation rates directly from standard transcriptomic and chromatin accessibility count matrices, where genomic features are represented as rows and experimental conditions as columns. tTEscanR seamlessly integrates into existing bulk and single-cell pipelines and provides modular functions for quality control, filtering, normalization, statistical analysis, and visualization. A structured data object centralizes workflow outputs and associated metadata. A built-in multilevel visualization module generates customizable, publication-ready graphical outputs to facilitate data interpretation and reproducibility of the complex translational landscape. We demonstrate the utility of tTEscanR across cancer biology, aging, and neurodegeneration datasets, uncovering critical translational regulatory programs overlooked by conventional analyses. tTEscanR is available in an open-source repository as a standalone tool or workflow plug-in.

Over the past decade, thousands of putative human RNA-binding proteins (RBPs) have been identified, increasing the need for methods that define their RNA-binding capacities across diverse biological settings. Existing methods rely either on antibody-based in vivo capture (e.g. CLIP-seq), which depends on cross-linking efficiency and antibody availability, or on synthetic oligonucleotide-based assays (e.g. RNAcompete), which use artificial RNA substrates and cannot assess binding across the native transcriptome. To bridge this gap, we developed RNA affinity purification followed by sequencing (RAPseq), an in vitro method that profiles RBP-binding to native cellular RNA, enabling large-scale transcriptome-wide characterization of RNA–protein interactions without antibodies or synthetic probes. Using RAPseq, we characterized the RNA interactomes of 11 canonical RBPs and 26 non-canonical RBPs, and uncovered novel and specialized moonlighting RNA-binding activities. Applying RAPseq to vertebrate HUR proteins revealed recognition of a conserved RNA-binding motif but showed species-specific binding preferences. Profiling of five pathological IGF2BP family variants exhibited distinct gain- and loss-of-function binding patterns, with implications for cancer biology. Our combinatorial RBP-binding assay (co-RAPseq) uncovered cooperative RNA-binding by HUR and PTBP1, including de novo estimation of the optimal binding distance. Lastly, we introduce a modification-sensitive assay (mod-RAPseq) to distinguish between modification-dependent and -independent RNA-binding sites of YTHDF1 and YBX1. Overall, our simple, scalable, and versatile method enables exploration of complex RNA–protein interactions and the regulatory layers that shape post-transcriptional gene regulation.

Biological systems encompass intricate networks governed by RNA-protein interactions that play pivotal roles in cellular functions. RNA and proteins constituting 1.1% and 18% of the mammalian cell weight, respectively, orchestrate vital processes from genome organization to translation. To date, disentangling the functional fraction of the human genome has presented a major challenge, particularly for noncoding regions, yet recent discoveries have started to unveil a host of regulatory functions for noncoding RNAs (ncRNAs). While ncRNAs exist at different sizes, structures, degrees of evolutionary conservation and abundances within the cell, they partake in diverse roles either alone or in combination. However, certain ncRNA subtypes, including those that have been described or remain to be discovered, are poorly characterized given their heterogeneous nature. RNA activity is in most cases coordinated through interactions with RNA-binding proteins (RBPs). Extensive efforts are being made to accurately reconstruct RNA-RBP regulatory networks, which have provided unprecedented insight into cellular physiology and human disease. In this review, we provide a comprehensive view of RNAs and RBPs, focusing on how their interactions generate functional signals in living cells, particularly in the context of post-transcriptional regulatory processes and cancer.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1 . TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, includingPAPSS2,ATAD1,KLLN, andPTEN. We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9–modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9–modified cells, which could impact cancer research.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor’s vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Introduction: The occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. Rationale: To understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. Methods: To dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. Results: Only males developed liver steatosis. We found that selective activation of either ERα or ERβ reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1 . TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. Conclusions: Systemic activation of ERα or ERβ modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevance: We identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevance: The targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTS: steatosis in livers of high-fat diet (HFD) male mice was effectively reduced by selective activation of estrogen receptors (ERα and ERβ) with four different agonists. ER agonist treatments successfully reversed HFD-induced changes in gene regulation and expression, revealing new treatment targets involving previously unconnected molecular pathways. estrogen-sensitive enhancers regulated important genes, including TEAD1, emerging as pivotal NAFLD regulators significantly impacting metabolic processes. high TEAD1 gene expression in NAFLD patients correlated with disease severity, underscoring its clinical significance in disease progression. inhibiting TEAD with small molecules alleviated steatosis by suppressing lipogenic pathways, resembling some of the same beneficial effects as estrogen treatment.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3 ⁺ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.