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Cancer continues to be a major global health challenge, accounting for 10 million deaths annually worldwide. Since the inception of genome-wide cancer sequencing studies 20 years ago, a core set of ~700 oncogenes and tumor suppressor genes has become the basis for cancer research. However, this research has been based largely on an understanding that the human genome encodes ~19 500 protein-coding genes. Complementing this genomic landscape, recent advances have described numerous microproteins which are now poised to redefine our understanding of oncogenic processes and open new avenues for therapeutic intervention. This review explores the emerging evidence for microprotein involvement in cancer mechanisms and discusses potential therapeutic applications, with an emphasis on highlighting recent advances in the field., Competing Interests: Declaration of interests J.R.P. has received research honoraria from Novartis Biosciences and is a paid consultant for ProFound Therapeutics. D.A.H. and S.v.H. declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study, we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling, including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover, we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart, including 551 genes, 504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall, our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease., (© 2024. The Author(s).)

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed., Competing Interests: Declaration of interests J.R.P. has received research honoraria from Novartis Biosciences and is a paid consultant for ProFound Therapeutics. J.G.A. is a paid consultant for Enara Bio and Moderna. J.L.A. is an advisor to Microneedle Solutions. T.F.M. is a consultant for and holds equity in Velia Therapeutics. J.S.W. is an advisor and holds equity in Velia Therapeutics. G.M. is co-founder and CSO of OHMX.bio. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer and PrognomIQ. N.T.I. hold equity in Velia Therapeutics and holds equity and serves as a scientific advisor to Tevard Biosciences. P.F. is a member of the scientific advisory board of Infinitopes. A.-R. C. is a member of the advisory board of ProFound Therapeutics.

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers., Competing Interests: Competing interests: ZS and JK are inventors on different patents for γδ TCR sequences, recognition mechanisms and isolation strategies. JK is scientific cofounder and shareholder of Gadeta (www.gadeta.nl). The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets., Competing Interests: Declaration of interests K.L.L. reports the following interests: equity in Travera; research funds from Bristol Myers Squibb, SEngine Precision Medicine, Multiple Myeloma Research Foundation, and Eli Lilly and Company; and being a consultant or on the scientific advisory board for Bristol Myers Squibb, Travera, and IntegraGen. P.B. receives grant funding from Novartis Institute of Biomedical Research, and has received grant funding from Deerfield Therapeutics, both for unrelated projects. P.B. has also served on a paid advisory board for qed Therapeutics, unrelated to this work. D.E.R. receives research funding from members of the Functional Genomics Consortium (AbbVie, BMS, Jannsen, Merck, and Vir) and is a director of Addgene, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein coding.", Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genes and translated open reading frames (ORFs) that emerged de novo from previously non-coding sequences provide species with opportunities for adaptation. When aberrantly activated, some human-specific de novo genes and ORFs have disease-promoting properties-for instance, driving tumour growth. Thousands of putative de novo coding sequences have been described in humans, but we still do not know what fraction of those ORFs has readily acquired a function. Here, we discuss the challenges and controversies surrounding the detection, mechanisms of origin, annotation, validation and characterization of de novo genes and ORFs. Through manual curation of literature and databases, we provide a thorough table with most de novo genes reported for humans to date. We re-evaluate each locus by tracing the enabling mutations and list proposed disease associations, protein characteristics and supporting evidence for translation and protein detection. This work will support future explorations of de novo genes and ORFs in humans., (© 2023. Springer Nature Limited.)

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Immunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL., Methods: Drug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids., Results: Drug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage., Conclusions: This study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors., (© 2022. The Author(s).)

Glucocorticoids such as dexamethasone (Dex) are widely used to treat both acute and chronic inflammatory conditions. They regulate immune responses by dampening cell-mediated immunity in a glucocorticoid receptor (GR)-dependent manner, by suppressing the expression of pro-inflammatory cytokines and chemokines and by stimulating the expression of anti-inflammatory mediators. Despite its evident clinical benefit, the mechanistic underpinnings of the gene regulatory networks transcriptionally controlled by GR in a context-specific manner remain mysterious. Next generation sequencing methods such mRNA sequencing (RNA-seq) and Ribosome profiling (ribo-seq) provide tools to investigate the transcriptional and post-transcriptional mechanisms that govern gene expression. Here, we integrate matched RNA-seq data with ribo-seq data from human acute monocytic leukemia (THP-1) cells treated with the TLR4 ligand lipopolysaccharide (LPS) and with Dex, to investigate the global transcriptional and translational regulation (translational efficiency, ΔTE) of Dex-responsive genes. We find that the expression of most of the Dex-responsive genes are regulated at both the transcriptional and the post-transcriptional level, with the transcriptional changes intensified on the translational level. Overrepresentation pathway analysis combined with STRING protein network analysis and manual functional exploration, identified these genes to encode immune effectors and immunomodulators that contribute to macrophage-mediated immunity and to the maintenance of macrophage-mediated immune homeostasis. Further research into the translational regulatory network underlying the GR anti-inflammatory response could pave the way for the development of novel immunomodulatory therapeutic regimens with fewer undesirable side effects., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function., Competing Interests: The authors have declared that no competing interest exist

The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases., (© 2022 Dahale et al.)

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8-all identified as multifunctional RBPs by our analysis-selectively influence target translation rates depending on 5' UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression., Competing Interests: The authors have declared that no competing interests exist.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9–generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Background: Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines., Results: We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates., Conclusions: We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin + cells, in a manner independent of the nearby TF FOXA2 . One-by-one disruption of each of LINC00261 's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles., Competing Interests: BG, Sv, VS, JS, FW, SB, SN, RW, IM, NH, MS No competing interests declared, (© 2020, Gaertner et al.)

Background: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored., Methods: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart., Results: We generated nucleotide-resolution translatome data during the transforming growth factor β1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor β1., Conclusions: We reveal widespread translational effects of transforming growth factor β1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fibrosis is a common pathology in cardiovascular disease. In the heart, fibrosis causes mechanical and electrical dysfunction and in the kidney, it predicts the onset of renal failure. Transforming growth factor β1 (TGFβ1) is the principal pro-fibrotic factor, but its inhibition is associated with side effects due to its pleiotropic roles. We hypothesized that downstream effectors of TGFβ1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicity. Here we show, using integrated imaging-genomics analyses of primary human fibroblasts, that upregulation of interleukin-11 (IL-11) is the dominant transcriptional response to TGFβ1 exposure and required for its pro-fibrotic effect. IL-11 and its receptor (IL11RA) are expressed specifically in fibroblasts, in which they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il-11 injection causes heart and kidney fibrosis and organ failure, whereas genetic deletion of Il11ra1 protects against disease. Therefore, inhibition of IL-11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These results reveal a central role of IL-11 in fibrosis and we propose that inhibition of IL-11 is a potential therapeutic strategy to treat fibrotic diseases.

Background: Germline chromothripsis causes complex genomic rearrangements that are likely to affect multiple genes and their regulatory contexts. The contribution of individual rearrangements and affected genes to the phenotypes of patients with complex germline genomic rearrangements is generally unknown., Methods: To dissect the impact of germline chromothripsis in a relevant developmental context, we performed trio-based RNA expression analysis on blood cells, induced pluripotent stem cells (iPSCs), and iPSC-derived neuronal cells from a patient with de novo germline chromothripsis and both healthy parents. In addition, Hi-C and 4C-seq experiments were performed to determine the effects of the genomic rearrangements on transcription regulation of genes in the proximity of the breakpoint junctions., Results: Sixty-seven genes are located within 1 Mb of the complex chromothripsis rearrangements involving 17 breakpoints on four chromosomes. We find that three of these genes (FOXP1, DPYD, and TWIST1) are both associated with developmental disorders and differentially expressed in the patient. Interestingly, the effect on TWIST1 expression was exclusively detectable in the patient's iPSC-derived neuronal cells, stressing the need for studying developmental disorders in the biologically relevant context. Chromosome conformation capture analyses show that TWIST1 lost genomic interactions with several enhancers due to the chromothripsis event, which likely led to deregulation of TWIST1 expression and contributed to the patient's craniosynostosis phenotype., Conclusions: We demonstrate that a combination of patient-derived iPSC differentiation and trio-based molecular profiling is a powerful approach to improve the interpretation of pathogenic complex genomic rearrangements. Here we have applied this approach to identify misexpression of TWIST1, FOXP1, and DPYD as key contributors to the complex congenital phenotype resulting from germline chromothripsis rearrangements.

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ∼1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease., Competing Interests: S.A.C. consults for Illumina.

Histone modifications are epigenetic marks that fundamentally impact the regulation of gene expression. Integrating histone modification information in the analysis of gene expression traits (eQTL mapping) has been shown to significantly enhance the prediction of eQTLs. In this chapter, we describe (1) how to perform quantitative trait locus (QTL) analysis using histone modification levels as traits and (2) how to integrate these data with information on RNA expression for the elucidation of the epigenetic control of transcript levels. We will provide a comprehensive introduction into the topic, describe in detail how ChIP-seq data are analyzed and elaborate on how to integrate ChIP-seq and RNA-seq data from a segregating disease animal model for the identification of the epigenetic control of RNA expression.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development., (© 2016 American Society of Plant Biologists. All rights reserved.)

The extent of translational control of gene expression in mammalian tissues remains largely unknown. Here we perform genome-wide RNA sequencing and ribosome profiling in heart and liver tissues to investigate strain-specific translational regulation in the spontaneously hypertensive rat (SHR/Ola). For the most part, transcriptional variation is equally apparent at the translational level and there is limited evidence of translational buffering. Remarkably, we observe hundreds of strain-specific differences in translation, almost doubling the number of differentially expressed genes. The integration of genetic, transcriptional and translational data sets reveals distinct signatures in 3'UTR variation, RNA-binding protein motifs and miRNA expression associated with translational regulation of gene expression. We show that a large number of genes associated with heart and liver traits in human genome-wide association studies are primarily translationally regulated. Capturing interindividual differences in the translated genome will lead to new insights into the genes and regulatory pathways underlying disease phenotypes.

Tumorigenesis is often associated with loss of tumor suppressor genes (such as TP53), genomic instability and telomere lengthening. Previously, we generated and characterized a rat p53 knockout model in which the homozygous rats predominantly develop hemangiosarcomas whereas the heterozygous rats mainly develop osteosarcomas. Using genome-wide analyses, we find that the tumors that arise in the heterozygous and homozygous Tp53C273X mutant animals are also different in their genomic instability profiles. While p53 was fully inactivated in both heterozygous and homozygous knockout rats, tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation as compared to the tumors from heterozygous animals. In addition, complex structural rearrangements such as chromothripsis and breakage-fusion-bridge cycles were never found in tumors from homozygous animals, while these were readily detectable in tumors from heterozygous animals. Finally, we measured telomere length and telomere lengthening pathway activity and found that tumors of homozygous animals have longer telomeres but do not show clear telomerase or alternative lengthening of telomeres (ALT) activity differences as compared to the tumors from heterozygous animals. Taken together, our results demonstrate that host p53 status in this rat p53 knockout model has a large effect on both tumor type and genomic instability characteristics, where full loss of functional p53 is not the main driver of large-scale structural variations. Our results also suggest that chromothripsis primarily occurs under p53 heterozygous rather than p53 null conditions.

Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding the complexity of the human brain and its functional diversity remain a major challenge. Distinct anatomical regions are involved in an array of processes, including organismal homeostasis, cognitive functions, and susceptibility to neurological pathologies, many of which define our species. Distal enhancers have emerged as key regulatory elements that acquire histone modifications in a cell- and species-specific manner, thus enforcing specific gene expression programs. Here, we survey the epigenomic landscape of promoters and cis-regulatory elements in 136 regions of the adult human brain. We identify a total of 83,553 promoter-distal H3K27ac-enriched regions showing global characteristics of brain enhancers. We use coregulation of enhancer elements across many distinct regions of the brain to uncover functionally distinct networks at high resolution and link these networks to specific neuroglial functions. Furthermore, we use these data to understand the relevance of noncoding genomic variations previously linked to Parkinson's disease incidence.