RESUMEN
Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.
Asunto(s)
Ciclo del Ácido Cítrico , Citosol , Malato Deshidrogenasa , Mitocondrias , Unión Proteica , Malato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Citosol/metabolismo , Citosol/enzimología , Humanos , Mitocondrias/metabolismo , Mitocondrias/genética , Mitocondrias/enzimología , ARN/metabolismo , ARN/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , NAD/metabolismo , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.
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Proteínas de Unión al ADN , Virus de la Influenza A , ARN Mensajero , ARN Viral , Proteínas de Unión al ARN , Replicación Viral , Humanos , Replicación Viral/genética , ARN Viral/metabolismo , ARN Viral/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Virus de la Influenza A/metabolismo , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Células HEK293 , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Unión Proteica , AnimalesRESUMEN
Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.
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Neoplasias Óseas , Osteosarcoma , Humanos , Proliferación Celular/genética , Línea Celular Tumoral , Osteosarcoma/metabolismo , Neoplasias Óseas/metabolismo , ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
ALK-positive NSCLC patients demonstrate initial responses to ALK tyrosine kinase inhibitor (TKI) treatments, but eventually develop resistance, causing rapid tumor relapse and poor survival rates. Growing evidence suggests that the combination of drug and immune therapies greatly improves patient survival; however, due to the low immunogenicity of the tumors, ALK-positive patients do not respond to currently available immunotherapies. Tumor-associated macrophages (TAMs) play a crucial role in facilitating lung cancer growth by suppressing tumoricidal immune activation and absorbing chemotherapeutics. However, they can also be programmed toward a pro-inflammatory tumor suppressive phenotype, which represents a highly active area of therapy development. Iron loading of TAMs can achieve such reprogramming correlating with an improved prognosis in lung cancer patients. We previously showed that superparamagnetic iron oxide nanoparticles containing core-cross-linked polymer micelles (SPION-CCPMs) target macrophages and stimulate pro-inflammatory activation. Here, we show that SPION-CCPMs stimulate TAMs to secrete reactive nitrogen species and cytokines that exert tumoricidal activity. We further show that SPION-CCPMs reshape the immunosuppressive Eml4-Alk lung tumor microenvironment (TME) toward a cytotoxic profile hallmarked by the recruitment of CD8+ T cells, suggesting a multifactorial benefit of SPION-CCPM application. When intratracheally instilled into lung cancer-bearing mice, SPION-CCPMs delay tumor growth and, after first line therapy with a TKI, halt the regrowth of relapsing tumors. These findings identify SPIONs-CCPMs as an adjuvant therapy, which remodels the TME, resulting in a delay in the appearance of resistant tumors.
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Crizotinib , Neoplasias Pulmonares , Nanopartículas Magnéticas de Óxido de Hierro , Microambiente Tumoral , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Microambiente Tumoral/efectos de los fármacos , Animales , Nanopartículas Magnéticas de Óxido de Hierro/química , Humanos , Ratones , Crizotinib/farmacología , Crizotinib/química , Antineoplásicos/farmacología , Antineoplásicos/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Línea Celular Tumoral , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/metabolismo , Proliferación Celular/efectos de los fármacos , FemeninoRESUMEN
Cardiac development and function are becoming increasingly well understood from different angles, including signalling, transcriptional and epigenetic mechanisms. By contrast, the importance of the post-transcriptional landscape of cardiac biology largely remains to be uncovered, building on the foundation of a few existing paradigms. The discovery during the past decade of hundreds of additional RNA-binding proteins in mammalian cells and organs, including the heart, is expected to accelerate progress and has raised intriguing possibilities for better understanding the intricacies of cardiac development, metabolism and adaptive alterations. In this Review, we discuss the progress and new concepts on RNA-binding proteins and RNA biology and appraise them in the context of common cardiovascular clinical conditions, from cell and organ-wide perspectives. We also discuss how a better understanding of cardiac RNA-binding proteins can fill crucial knowledge gaps in cardiology and might pave the way to developing better treatments to reduce cardiovascular morbidity and mortality.
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Enfermedades Cardiovasculares , Proteínas de Unión al ARN , Humanos , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/fisiopatología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , AnimalesRESUMEN
Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.
Asunto(s)
Proteínas de Unión al ARN , Programas Informáticos , Sitios de Unión , Inmunoprecipitación , Unión Proteica , ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-ß has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-ß in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-ß remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-ß driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-ß-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-ß at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-ß drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-ß stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-ß profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.
Asunto(s)
Células Endoteliales , Factor de Crecimiento Transformador beta , Ratones , Animales , Factor de Crecimiento Transformador beta/metabolismo , Células Endoteliales/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , ARNRESUMEN
RNA-protein interactions are central to cardiac function, but how activity of individual RNA-binding protein is regulated through signaling cascades in cardiomyocytes during heart failure development is largely unknown. The mechanistic target of rapamycin kinase is a central signaling hub that controls mRNA translation in cardiomyocytes; however, a direct link between mTOR signaling and RNA-binding proteins in the heart has not been established. Integrative transcriptome and translatome analysis revealed mTOR dependent translational upregulation of the RNA binding protein Ybx1 during early pathological remodeling independent of mRNA levels. Ybx1 is necessary for pathological cardiomyocyte growth by regulating protein synthesis. To identify the molecular mechanisms how Ybx1 regulates cellular growth and protein synthesis, we identified mRNAs bound to Ybx1. We discovered that eucaryotic elongation factor 2 (Eef2) mRNA is bound to Ybx1, and its translation is upregulated during cardiac hypertrophy dependent on Ybx1 expression. Eef2 itself is sufficient to drive pathological growth by increasing global protein translation. Finally, Ybx1 depletion in vivo preserved heart function during pathological cardiac hypertrophy. Thus, activation of mTORC1 links pathological signaling cascades to altered gene expression regulation by activation of Ybx1 which in turn promotes translation through increased expression of Eef2.
Asunto(s)
Insuficiencia Cardíaca , Serina-Treonina Quinasas TOR , Cardiomegalia/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Ratones , RatasRESUMEN
System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo enhanced RNA interactome capture (eRIC) to characterize the RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.
Asunto(s)
Proteoma , Proteínas de Unión al ARN , Animales , Ratones , Proteoma/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN , Mamíferos/genética , Células CultivadasRESUMEN
Small noncoding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small noncoding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small noncoding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nt in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognized interactions.
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Glucólisis , ARN Pequeño no Traducido , ARN de Transferencia , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Glucólisis/genética , Proteoma/genética , ARN/metabolismo , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismoRESUMEN
SUMMARY: Transcriptome-wide detection of binding sites of RNA-binding proteins is achieved using Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) and its derivative enhanced CLIP (eCLIP) sequencing methods. Here, we introduce htseq-clip, a python package developed for preprocessing, extracting and summarizing crosslink site counts from i/eCLIP experimental data. The package delivers crosslink site count matrices along with other metrics, which can be directly used for filtering and downstream analyses such as the identification of differential binding sites. AVAILABILITY AND IMPLEMENTATION: The Python package htseq-clip is available via pypi (python package index), bioconda and the Galaxy Tool Shed under the open source MIT License. The code is hosted at https://github.com/EMBL-Hentze-group/htseq-clip and documentation is available under https://htseq-clip.readthedocs.io/en/latest.
Asunto(s)
Programas Informáticos , Transcriptoma , Sitios de Unión , Proteínas de Unión al ARN/metabolismo , InmunoprecipitaciónRESUMEN
Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1's acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.
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Glucólisis , Fosfopiruvato Hidratasa , Animales , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Glucólisis/fisiología , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , ARN/metabolismoRESUMEN
Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1-1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.
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Arginina , Lisina , Autofagia/genética , ARN Bacteriano , Proteína Sequestosoma-1/química , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismoRESUMEN
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
Asunto(s)
Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Microscopía por Crioelectrón , Células HeLa , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Factores de Terminación de Péptidos/metabolismo , Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/efectos de los fármacos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.
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Hierro , Micelas , Animales , Inflamación/tratamiento farmacológico , Macrófagos , Ratones , PolímerosRESUMEN
RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins. Furthermore, we identify the cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo. We identify mRNAs bound to and regulated by Cpeb4 in cardiomyocytes. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two zinc finger transcription factors (Zeb1 and Zbtb20) are discovered. We show that Cpeb4 regulates the expression of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as a critical regulator of cardiomyocyte function by differential binding to specific mRNAs in response to pathological growth stimulation.
Asunto(s)
Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proliferación Celular , Humanos , RatonesRESUMEN
Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.
RESUMEN
RNA-binding proteins (RBPs) are critical effectors of gene expression, and as such their malfunction underlies the origin of many diseases. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. System-wide unbiased identification of RBPs has increased the number of recognized RBPs into the four-digit range and revealed new paradigms: from the prevalence of structurally disordered RNA-binding regions with roles in the formation of membraneless organelles to unsuspected and potentially pervasive connections between intermediary metabolism and RNA regulation. Together with an increasingly detailed understanding of molecular mechanisms of RBP function, these insights are facilitating the development of new therapies to treat malignancies. Here, we provide an overview of RBPs involved in human genetic disorders, both Mendelian and somatic, and discuss emerging aspects in the field with emphasis on molecular mechanisms of disease and therapeutic interventions.