Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Proc Natl Acad Sci U S A ; 120(41): e2312126120, 2023 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-37792516

RESUMEN

The dynamic balance between tRNA supply and codon usage demand is a fundamental principle in the cellular translation economy. However, the regulation and functional consequences of this balance remain unclear. Here, we use PARIS2 interactome capture, structure modeling, conservation analysis, RNA-protein interaction analysis, and modification mapping to reveal the targets of hundreds of snoRNAs, many of which were previously considered orphans. We identify a snoRNA-tRNA interaction network that is required for global tRNA modifications, including 2'-O-methylation and others. Loss of Fibrillarin, the snoRNA-guided 2'-O-methyltransferase, induces global upregulation of tRNA fragments, a large group of regulatory RNAs. In particular, the snoRNAs D97/D133 guide the 2'-O-methylation of multiple tRNAs, especially for the amino acid methionine (Met), a protein-intrinsic antioxidant. Loss of D97/D133 snoRNAs in human HEK293 cells reduced target tRNA levels and induced codon adaptation of the transcriptome and translatome. Both single and double knockouts of D97 and D133 in HEK293 cells suppress Met-enriched proliferation-related gene expression programs, including, translation, splicing, and mitochondrial energy metabolism, and promote Met-depleted programs related to development, differentiation, and morphogenesis. In a mouse embryonic stem cell model of development, knockdown and knockout of D97/D133 promote differentiation to mesoderm and endoderm fates, such as cardiomyocytes, without compromising pluripotency, consistent with the enhanced development-related gene expression programs in human cells. This work solves a decades-old mystery about orphan snoRNAs and reveals a function of snoRNAs in controlling the codon-biased dichotomous cellular states of proliferation and development.


Asunto(s)
Uso de Codones , ARN Nucleolar Pequeño , Humanos , Animales , Ratones , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Uso de Codones/genética , Células HEK293 , ARN de Transferencia/genética , Codón
2.
Genome Res ; 32(5): 968-985, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35332099

RESUMEN

The recent development and application of methods based on the general principle of "crosslinking and proximity ligation" (crosslink-ligation) are revolutionizing RNA structure studies in living cells. However, extracting structure information from such data presents unique challenges. Here, we introduce a set of computational tools for the systematic analysis of data from a wide variety of crosslink-ligation methods, specifically focusing on read mapping, alignment classification, and clustering. We design a new strategy to map short reads with irregular gaps at high sensitivity and specificity. Analysis of previously published data reveals distinct properties and bias caused by the crosslinking reactions. We perform rigorous and exhaustive classification of alignments and discover eight types of arrangements that provide distinct information on RNA structures and interactions. To deconvolve the dense and intertwined gapped alignments, we develop a network/graph-based tool Crosslinked RNA Secondary Structure Analysis using Network Techniques (CRSSANT), which enables clustering of gapped alignments and discovery of new alternative and dynamic conformations. We discover that multiple crosslinking and ligation events can occur on the same RNA, generating multisegment alignments to report complex high-level RNA structures and multi-RNA interactions. We find that alignments with overlapped segments are produced from potential homodimers and develop a new method for their de novo identification. Analysis of overlapping alignments revealed potential new homodimers in cellular noncoding RNAs and RNA virus genomes in the Picornaviridae family. Together, this suite of computational tools enables rapid and efficient analysis of RNA structure and interaction data in living cells.


Asunto(s)
ARN no Traducido , ARN , Algoritmos , Análisis por Conglomerados , ARN/química , ARN/genética , ARN no Traducido/química , Análisis de Secuencia de ARN/métodos , Programas Informáticos
3.
J Immunol ; 205(3): 637-647, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32591403

RESUMEN

Atypical TCRδ found in sharks, amphibians, birds, and monotremes and TCRµ found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VHδ/Vµ) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCRα/δ gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 Jα, nine Jδ, eight Dδ, two Cα, and five Cδ gene segments in the TCRα/δ loci of the Chinese alligator, representing the most complicated TCRα/δ gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VHδ genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VHδ genes could be related to bird VHδ genes, VHδ/Vµ genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCRδ/TCRµ genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.


Asunto(s)
Caimanes y Cocodrilos , Evolución Molecular , Sitios Genéticos , Receptores de Antígenos de Linfocitos T alfa-beta , Proteínas de Reptiles , Caimanes y Cocodrilos/genética , Caimanes y Cocodrilos/inmunología , Animales , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas de Reptiles/genética , Proteínas de Reptiles/inmunología
4.
Immunology ; 163(4): 448-459, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33738807

RESUMEN

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.


Asunto(s)
Calostro/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Intestino Delgado/metabolismo , Placenta/metabolismo , Receptores Fc/metabolismo , Porcinos/inmunología , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Lactancia Materna , Sistemas CRISPR-Cas , Bovinos , Femenino , Técnicas de Inactivación de Genes , Antígenos de Histocompatibilidad Clase I/genética , Homeostasis , Humanos , Inmunidad Materno-Adquirida , Inmunoglobulina G/metabolismo , Embarazo , Conejos , Receptores Fc/genética , Ovinos
5.
Methods Enzymol ; 691: 253-281, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37914449

RESUMEN

RNA structures and interactions in living cells drive a variety of biological processes and play critical roles in physiology and disease states. However, studies of RNA structures and interactions have been challenging due to limitations in available technologies. Direct determination of structures in vitro has been only possible to a small number of RNAs with limited sizes and conformations. We recently introduced two chemical crosslink-ligation techniques that enabled studies of transcriptome-wide secondary and tertiary structures and their dynamics. In a dramatically improved version of the psoralen analysis of RNA interactions and structures (PARIS2) method, we detailed the synthesis and use of amotosalen, a highly soluble psoralen analogue, and enhanced enzymology for higher efficiency duplex capture. We also introduced spatial 2'-hydroxyl acylation reversible crosslinking (SHARC) with exonuclease (exo) trimming, a method which utilizes a novel crosslinker class that targets the 2'-OH to capture three-dimensional (3D) structures. Both are powerful orthogonal approaches for solving in vivo RNA structure and interactions, integrating crosslinking, exo trimming, proximity ligation, and high throughput sequencing. In this chapter, we present a detailed protocol for the methods and highlight steps that outperform existing crosslink-ligation approaches.


Asunto(s)
Furocumarinas , ARN , ARN/química , Transcriptoma
6.
Biotechnol Appl Biochem ; 59(6): 405-10, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23586948

RESUMEN

A luminescent strain was isolated and identified as Vibrio campbellii strain LZ5 by 16S rDNA analysis. It grew well in broad living conditions, and the relative fluorescence intensity was stable at pH ranging from 5 to 7.5, NaCl concentrations from 0% to 3%, KCl from 1.5% to 5%, and CaCl2 from 1% to 3%. In contrast, the relative fluorescence intensity was negatively correlated with both CdCl2 and HgCl2 concentrations from 0 to 1 mg/L. The luminescent fraction from the cell lysate was purified by ion-exchange chromatography, and identified as a luciferase by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Furthermore, the biofilm of V. campbellii LZ5 was formed constantly under different conditions, and was not affected by heavy metal ions. The data collectively reveal that strain LZ5 has the potential to be developed into a biosensor for real-time monitoring of heavy metals.


Asunto(s)
Técnicas Biosensibles/métodos , Ambiente , Contaminantes Ambientales/análisis , Luminiscencia , Metales Pesados/análisis , Vibrio/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Concentración de Iones de Hidrógeno , Luciferasas/metabolismo , Metales Pesados/química , Metales Pesados/toxicidad , Océanos y Mares , Vibrio/crecimiento & desarrollo , Vibrio/metabolismo , Vibrio/fisiología
7.
Nat Commun ; 13(1): 911, 2022 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-35177610

RESUMEN

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.


Asunto(s)
Modelos Moleculares , ARN/ultraestructura , Acilación , Reactivos de Enlaces Cruzados/química , Células HEK293 , Células HeLa , Humanos , Conformación de Ácido Nucleico , ARN/química , ARN/aislamiento & purificación , Pliegue del ARN , Elongación de la Transcripción Genética
8.
Nat Commun ; 12(1): 2344, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879794

RESUMEN

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.


Asunto(s)
Enfermedades Transmisibles/genética , Enfermedades Transmisibles/metabolismo , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Fotoquímica/métodos , ARN/química , ARN/metabolismo , Calcinosis/genética , Calcinosis/metabolismo , Quistes del Sistema Nervioso Central/genética , Quistes del Sistema Nervioso Central/metabolismo , Reactivos de Enlaces Cruzados , Enterovirus Humano D/genética , Furocumarinas , Genoma Viral , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Procesos Fotoquímicos , ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN Viral/química , ARN Viral/genética
9.
Vet Immunol Immunopathol ; 215: 109913, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31420069

RESUMEN

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos , Linfocitos B/inmunología , Células HEK293 , Humanos , Región Variable de Inmunoglobulina/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Transfección , Recombinación V(D)J
10.
Virology ; 531: 19-30, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30852268

RESUMEN

In this study, using a dual-functional, piggyBac transposon-based system, we developed a method to systematically decipher the host genes that may be associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection. A Marc145 cell library, which was randomly mutated by transfecting piggyBac plasmids, was challenged with PRRSV. The surviving cell clones were subjected to inverse PCR and high-throughput sequencing to map the integration sites of the transposon. Detailed annotation of the genes flanking the integration sites allowed us to generate a ranked list of candidate genes. Among the predicted genes with a high priority, four genes, CDK17, RNF168, BCL2L15, and TRIM33, were strongly correlated with PRRSV infection in both Marc145 cells and porcine primary alveolar macrophages. This study not only assists in identifying the genes essential for PRRSV infection but also confirms the possibility of using the piggyBac system to study other virus-host genetic interactions in a high-throughput manner.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Animales , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Plásmidos/genética , Plásmidos/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Replicación Viral
11.
Dev Comp Immunol ; 85: 31-43, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29574022

RESUMEN

All jawed vertebrates have four T cell receptor (TCR) chains that are expressed by thymus-derived lymphocytes and play a major role in animal immune defence. However, few studies have investigated the TCR chains of crocodilians compared with those of birds and mammals, despite their key evolutionary position linking amphibians, reptiles, birds and mammals. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization, evolution and expression of TRB and TRG loci in Alligator sinensis. According to the sequencing data, the Alligator sinensis TRB locus spans approximately 500 Kb of genomic DNA containing two D-J-C clusters and 43 V gene segments and is organized as Vß(39)-pJß1-pCß1-pDß1-Dß2- Jß2(12)-Cß2-Vß(4), whereas the TRG locus spans 115 Kb of DNA genomic sequence consisting of 18 V gene segments, nine J gene segments and one C gene segment and is organized in a classical translocon pattern as Vγ(18)-Jγ(9)-Cγ. Moreover, syntenic analysis of TRB and TRG chain loci suggested a high degree of conserved synteny in the genomic regions across mammals, birds and Alligator sinensis. By analysing the cloned TRB/TRG cDNA, we identified the usage pattern of V families in the expressed TRB and TRG. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed TRB and TRG sequences. Phylogenetic analysis revealed that TRB and TRG loci possess distinct evolutionary patterns. Most Alligator sinensis V subgroups have closely related orthologues in chicken and duck, and a small number of Alligator sinensis V subgroups have orthologues in mammals, which supports the hypothesis that crocodiles are the closest relatives of birds and mammals. Collectively, these data provide insights into TCR gene evolution in vertebrates and improve our understanding of the Alligator sinensis immune system.


Asunto(s)
Caimanes y Cocodrilos/genética , Genes Codificadores de los Receptores de Linfocitos T/genética , Animales , Aves/genética , Cromosomas Artificiales Bacterianos/genética , ADN Complementario , Evolución Molecular , Genoma/genética , Genómica/métodos , Mamíferos/genética , Filogenia , Sintenía/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA