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Cancer immunotherapy has witnessed rapid advancement in recent years, with a particular focus on neoantigens as promising targets for personalized treatments. The convergence of immunogenomics, bioinformatics, and artificial intelligence (AI) has propelled the development of innovative neoantigen discovery tools and pipelines. These tools have revolutionized our ability to identify tumor-specific antigens, providing the foundation for precision cancer immunotherapy. AI-driven algorithms can process extensive amounts of data, identify patterns, and make predictions that were once challenging to achieve. However, the integration of AI comes with its own set of challenges, leaving space for further research. With particular focus on the computational approaches, in this article we have explored the current landscape of neoantigen prediction, the fundamental concepts behind, the challenges and their potential solutions providing a comprehensive overview of this rapidly evolving field.
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Antígenos de Neoplasias , Inteligencia Artificial , Inmunoterapia , Neoplasias , Medicina de Precisión , Humanos , Neoplasias/terapia , Neoplasias/inmunología , Medicina de Precisión/métodos , Antígenos de Neoplasias/inmunología , Inmunoterapia/métodos , Biología Computacional/métodos , AnimalesRESUMEN
The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.
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Bacteriófagos , Virus ARN , Virosis , Virus , Humanos , Biología Computacional , Virus/genéticaRESUMEN
Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.
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Acetilación , Cromatina , Lisina , Metilación , Procesamiento Proteico-Postraduccional , Sitio de Iniciación de la Transcripción , Animales , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Péptidos/química , Péptidos/metabolismo , Histona Desacetilasas/metabolismoRESUMEN
Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that concentrate and promote efficient biogenesis of snRNA-protein complexes involved in splicing (snRNPs). Depletion of the CB scaffolding protein coilin disperses snRNPs, making CBs a model system for studying the structure and function of MLOs. Although it is assumed that CBs form through condensation, the biomolecular interactions responsible remain elusive. Here, we discover the unexpected capacity of coilin's N-terminal domain (NTD) to form extensive fibrils in the cytoplasm and discrete nuclear puncta in vivo. Single amino acid mutational analysis reveals distinct molecular interactions between coilin NTD proteins to form fibrils and additional NTD interactions with the nuclear Nopp140 protein to form puncta. We provide evidence that Nopp140 has condensation capacity and is required for CB assembly. From these observations, we propose a model in which coilin NTD-NTD mediated assemblies make multivalent contacts with Nopp140 to achieve biomolecular condensation in the nucleus.
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Cuerpos Enrollados , Ribonucleoproteínas Nucleares Pequeñas , Aminoácidos/metabolismo , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Células HeLa , Humanos , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Factor de Empalme U2AF , Gránulos de Estrés , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Síndromes Mielodisplásicos/genética , Sitios de Empalme de ARN , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismo , Gránulos de Estrés/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Understanding the targeting and spreading patterns of long non-coding RNAs (lncRNAs) on chromatin requires a technique that can detect both high-intensity binding sites and reveal genome-wide changes in spreading patterns with high precision and confidence. Here we determine lncRNA localization using biotinylated locked nucleic acid (LNA)-containing oligonucleotides with toehold architecture capable of hybridizing to target RNA through strand-exchange reaction. During hybridization, a protecting strand competitively displaces contaminating species, leading to highly specific RNA capture of individual RNAs. Analysis of Drosophila roX2 lncRNA using this approach revealed that heat shock, unlike the unfolded protein response, leads to reduced spreading of roX2 on the X chromosome, but surprisingly also to relocalization to sites on autosomes. Our results demonstrate that this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of lncRNAs on chromatin.
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Cromatina/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Oligonucleótidos/química , ARN Largo no Codificante/química , Proteínas de Unión al ARN/genética , Cromosoma X/química , Animales , Emparejamiento Base , Sitios de Unión , Biotinilación , Cromatina/metabolismo , Mapeo Cromosómico , Cromosomas de Insectos/química , Cromosomas de Insectos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Respuesta al Choque Térmico , Nanotecnología/métodos , Hibridación de Ácido Nucleico , Oligonucleótidos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Cromosoma X/metabolismoRESUMEN
The diverse roles of cellular RNAs can be studied by purifying RNAs of interest together with the biomolecules they bind. Biotinylated antisense oligonucleotides that hybridize specifically to the RNA of interest provide a general approach to develop affinity reagents for these experiments. Such oligonucleotides can be used to enrich endogenous RNAs from cross-linked chromatin extracts to study the genomic binding sites of RNAs. These hybridization capture protocols are evolving modular experiments that are compatible with a range of cross-linkers and conditions. This review discusses the principles of these hybridization capture experiments as well as considerations and controls necessary to interpret the resulting data without being misled by artifactual signals.
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Cromatina/metabolismo , Hibridación de Ácido Nucleico , ARN Largo no Codificante/metabolismo , Animales , Compensación de Dosificación (Genética) , HumanosRESUMEN
The growing appreciation of the importance of long noncoding RNAs (lncRNAs), together with the awareness that some of these RNAs are associated with chromatin, has inspired the development of methods to detect their sites of interaction on a genome-wide scale at high resolution. Hybridization capture methods combine antisense oligonucleotide hybridization with enrichment of RNA from cross-linked chromatin extracts. These techniques have provided insight into lncRNA localization and the interactions of lncRNAs with protein to better understand biological roles of lncRNAs. Here, we review the core principles of hybridization capture methods, focusing on the three most commonly used protocols: capture hybridization analysis of RNA targets (CHART), chromatin isolation by RNA purification (ChIRP) and RNA affinity purification (RAP). We highlight the general principles of these techniques and discuss how differences in experimental procedures present distinct challenges to help researchers using these protocols or, more generally, interpreting the results of hybridization capture experiments.
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Cromatina/metabolismo , Hibridación de Ácido Nucleico/métodos , ARN/metabolismo , Animales , Reactivos de Enlaces Cruzados/metabolismo , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells.
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Cromatina/aislamiento & purificación , Genómica/métodos , Hibridación de Ácido Nucleico/métodos , ARN Largo no Codificante/aislamiento & purificación , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Fragmentación del ADN , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Oligonucleótidos/genética , ARN Largo no Codificante/genéticaRESUMEN
Initially identified as a marker of coiled bodies (now Cajal bodies or CBs), the protein coilin was discovered a quarter of century ago. Coilin is now known to scaffold the CB, but its structure and function are poorly understood. Nearly devoid of predicted structural motifs, coilin has numerous reported molecular interactions that must underlie its role in the formation and function of CBs. In this review, we summarize what we have learned in the past 25 years about coilin's structure, post-transcriptional modifications, and interactions with RNA and proteins. We show that genes with homology to human coilin are found in primitive metazoans and comment on differences among model organisms. Coilin's function in Cajal body formation and RNP metabolism will be discussed in the light of these developments.
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Cuerpos Enrollados/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/historiaRESUMEN
Coilin protein scaffolds Cajal bodies (CBs)-subnuclear compartments enriched in small nuclear RNAs (snRNAs)-and promotes efficient spliceosomal snRNP assembly. The molecular function of coilin, which is intrinsically disordered with no defined motifs, is poorly understood. We use UV crosslinking and immunoprecipitation (iCLIP) to determine whether mammalian coilin binds RNA in vivo and to identify targets. Robust detection of snRNA transcripts correlated with coilin ChIP-seq peaks on snRNA genes, indicating that coilin binding to nascent snRNAs is a site-specific CB nucleator. Surprisingly, several hundred small nucleolar RNAs (snoRNAs) were identified as coilin interactors, including numerous unannotated mouse and human snoRNAs. We show that all classes of snoRNAs concentrate in CBs. Moreover, snoRNAs lacking specific CB retention signals traffic through CBs en route to nucleoli, consistent with the role of CBs in small RNP assembly. Thus, coilin couples snRNA and snoRNA biogenesis, making CBs the cellular hub of small ncRNA metabolism.
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Cuerpos Enrollados/metabolismo , Proteínas Nucleares/metabolismo , ARN Pequeño no Traducido/metabolismo , Animales , Ciclo Celular , Nucléolo Celular/metabolismo , Células HeLa , Humanos , Ratones , Unión Proteica , Transporte de ARNRESUMEN
Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Mastocitos/citología , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de IgE/metabolismo , Tetraspanina 29/fisiología , Animales , Antígenos/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Quimiotaxis , Citoesqueleto/metabolismo , Glucuronidasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Fosforilación , Unión Proteica , Ratas , Ratas Wistar , Tirosina/químicaRESUMEN
The cell nucleus contains dozens of subcompartments that separate biochemical processes into confined spaces. Cajal bodies (CBs) were discovered more than 100 years ago, but only extensive research in the past decades revealed the surprising complexity of molecular and cellular functions taking place in these structures. Many protein and RNA species are modified and assembled within CBs, which have emerged as a meeting place and factory for ribonucleoprotein (RNP) particles involved in splicing, ribosome biogenesis and telomere maintenance. Recently, a distinct structure near histone gene clusters--the Histone locus body (HLB)--was discovered. Involved in histone mRNA 3'-end formation, HLBs can share several components with CBs. Whether the appearance of distinct HLBs is simply a matter of altered affinity between these structures or of an alternate mode of CB assembly is unknown. However, both structures share basic assembly properties, in which transcription plays a decisive role in initiation. After this seeding event, additional components associate in random order. This appears to be a widespread mechanism for body assembly. CB assembly encompasses an additional layer of complexity, whereby a set of pre-existing substructures can be integrated into mature CBs. We propose this as a multi-seeding model of CB assembly.
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Núcleo Celular/metabolismo , Cuerpos Enrollados/genética , Cuerpos Enrollados/metabolismo , Animales , Histonas/genética , Humanos , ARN/genética , ARN/metabolismo , Empalme del ARN , Ribonucleoproteínas Nucleares Pequeñas/biosíntesis , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/biosíntesis , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
Arginine dimethylation plays critical roles in the assembly of ribonucleoprotein complexes in pre-mRNA splicing and piRNA pathways. We report solution structures of SMN and SPF30 Tudor domains bound to symmetric and asymmetric dimethylated arginine (DMA) that is inherent in the RNP complexes. An aromatic cage in the Tudor domain mediates dimethylarginine recognition by electrostatic stabilization through cation-π interactions. Distinct from extended Tudor domains, dimethylarginine binding by the SMN and SPF30 Tudor domains is independent of proximal residues in the ligand. Yet, enhanced micromolar affinities are obtained by external cooperativity when multiple methylation marks are presented in arginine- and glycine-rich peptide ligands. A hydrogen bond network in the SMN Tudor domain, including Glu134 and a tyrosine hydroxyl of the aromatic cage, enhances cation-π interactions and is impaired by a mutation causing an E134K substitution associated with spinal muscular atrophy. Our structural analysis enables the design of an optimized binding pocket and the prediction of DMA binding properties of Tudor domains.