Asunto(s)
Anticuerpos/genética , Anticuerpos/inmunología , Afinidad de Anticuerpos , Linfocitos B/inmunología , Sistemas CRISPR-Cas , Citidina Desaminasa/genética , Edición Génica , Citidina Desaminasa/inmunología , Evolución Molecular Dirigida , Células HEK293 , Antígenos HLA/inmunología , Humanos , Hipermutación Somática de InmunoglobulinaRESUMEN
Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.
Asunto(s)
Antígenos CD/química , Antígenos CD/inmunología , Butirofilinas/química , Butirofilinas/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos/química , Antígenos/inmunología , Antígenos CD/metabolismo , Butirofilinas/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes Quiméricas/inmunología , FosforilaciónRESUMEN
In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T/inmunología , Antígenos/inmunología , Antígenos CD/química , Antígenos CD/genética , Butirofilinas , Células Cultivadas , Difosfonatos/inmunología , Humanos , Imidazoles/inmunología , Espacio Intracelular , Activación de Linfocitos/genética , Mutación/genética , Unión Proteica/genética , Ingeniería de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Ácido ZoledrónicoRESUMEN
Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.
Asunto(s)
Antígenos CD/metabolismo , Antígenos/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/metabolismo , Anticuerpos Bloqueadores , Anticuerpos Inmovilizados , Anticuerpos Monoclonales , Antígenos CD/química , Antígenos CD/genética , Butirofilinas , Células Cultivadas , Células Clonales , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Factores Inmunológicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño , Receptores de Antígenos de Linfocitos T/agonistas , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.
Asunto(s)
Bacteriófagos/genética , Proteínas de la Cápside/metabolismo , Exones , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Empalme Alternativo , Proteínas de la Cápside/genética , Línea Celular , Clonación Molecular , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Levivirus/genética , Proteínas de Unión a Poli(A)/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Antígeno Intracelular 1 de las Células TRESUMEN
In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.
Asunto(s)
Empalme Alternativo/genética , Exones/genética , Riñón/fisiología , Proteínas de Unión a Poli(A)/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Activación Transcripcional/genética , Sitios de Unión , Línea Celular , Humanos , Unión Proteica , Sitios de Empalme de ARN/genética , Antígeno Intracelular 1 de las Células TRESUMEN
Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vgamma9Vdelta2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vgamma9Vdelta2 T cell activation, we used stable beta(2)-microglobulin knockdown to generate tumor cells with a approximately 10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vgamma9Vdelta2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vgamma9Vdelta2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vgamma9Vdelta2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.
Asunto(s)
Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias/inmunología , Subgrupos de Linfocitos T/inmunología , Microglobulina beta-2/antagonistas & inhibidores , Anticuerpos/farmacología , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica/genética , Regulación hacia Abajo , Humanos , Activación de Linfocitos , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Receptores KIR , Receptores de Células Asesinas Naturales , Escape del Tumor , Microglobulina beta-2/genéticaRESUMEN
A considerable amount of smooth muscle phenotypic diversity is generated by tissue-specific and developmentally regulated splicing of alternative exons. The control mechanisms are unknown. We are using a myosin phosphatase targeting subunit-1 (MYPT1) alternative exon as a model to investigate this question. In the present study, we show that the RNA binding proteins TIA and PTB function as antagonistic enhancers and suppressors of splicing of the alternative exon, respectively. Each functions through a single U-rich element, containing two UCUU motifs, just downstream of the alternative exon 5' splice site. Tissue-specific down-regulation of TIA protein in the perinatal period allows PTB to bind to the U-rich element and suppress splicing of the alternative exon as the visceral smooth muscle acquires the fast-phasic smooth muscle contractile phenotype. This provides a novel role for PTB in the tissue-specific regulation of splicing of alternative exons during the generation of smooth muscle phenotypic diversity.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/genética , Exones/fisiología , Fosfatasa de Miosina de Cadena Ligera/genética , Fosfoproteínas Fosfatasas/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Pollos , Molleja de las Aves/metabolismo , Humanos , Contracción Muscular , Músculo Liso/fisiología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fenotipo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Unión a Poli(A) , Proteína Fosfatasa 1 , Ratas , Antígeno Intracelular 1 de las Células TRESUMEN
Alternative CD44 exons v8, v9, and v10 are spliced as a block in epithelial cells (for example SVK14 cells), but can be skipped as a block by other cells. Using a minigene approach, we show that downstream intronic UGG repeats participate in activation of v8 exon splicing in SVK14 cells. The repeats can activate splicing of a heterologous exon in SVK14 cells and act additively with a previously described v8 exon splicing enhancer in this context. An alternative v9 exon 5' splice site used by some cells to make an aberrant transcript is repressed by an immediately downstream (UGG)3 sequence in SVK14 cells. We conclude that UGG repeats both activate v8 exon splicing and repress use of the alternative v9 exon 5' splice site in SVK14 cells, thus participating in the coordination of correct epithelial cell splicing of the v8-10 block.
Asunto(s)
Empalme Alternativo/genética , Exones/genética , Receptores de Hialuranos/genética , Intrones/genética , Análisis de Secuencia de ADN/métodos , Repeticiones de Trinucleótidos/genética , Secuencia de Bases , ADN Recombinante/genética , Datos de Secuencia MolecularRESUMEN
We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.
Asunto(s)
Músculo Liso/fisiología , Fosfatasa de Miosina de Cadena Ligera/genética , Proteínas/metabolismo , Empalme del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Pollos , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Molleja de las Aves/citología , Molleja de las Aves/fisiología , Datos de Secuencia Molecular , Músculo Liso/citología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Proteínas/genética , Sitios de Empalme de ARN/fisiología , Proteínas de Unión al ARN/genética , Retículo Sarcoplasmático/metabolismoRESUMEN
The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.
Asunto(s)
Receptores de Hialuranos/química , Receptores de Hialuranos/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Secuencia de Bases , Núcleo Celular/metabolismo , Elementos de Facilitación Genéticos , Células Epiteliales/metabolismo , Exones , Prueba de Complementación Genética , Células HeLa , Humanos , Receptores de Hialuranos/biosíntesis , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Empalme Serina-Arginina , Transfección , Rayos UltravioletaRESUMEN
TIA-1 and TIAR are a pair of related RNA-binding proteins which have been implicated in apoptosis. We show that chicken DT40 cells with both tia-1 alleles and one tiar allele disrupted (tia-1(-/-)tiar(-/+) cells) are viable. However, their growth and survival in medium containing low serum levels is significantly reduced compared with DT40 cells. The remaining intact tiar allele in tia-1(-/-)tiar(-/+) cells can only be disrupted if TIA-1 expression is first restored to the cells by transfection of a TIA-1 expression vector. We conclude that DT40 cells require either TIA-1 or TIAR for viability. TIA-1 overexpression in tia-1(-/-)tiar(-/+) cells leads to a radical drop in TIAR levels, by inducing efficient splicing of two tiar alternative exons carrying in-frame stop codons. In wild-type DT40 cells, tiar transcripts including these exons can also be detected. These transcripts increase significantly in abundance in cycloheximide-treated cells, suggesting that splicing of the exons exposes mRNAs to nonsense-mediated mRNA decay. TIA-1 or TIAR depletion leads to a marked drop in splicing of the exons. The human tiar gene contains a corresponding pair of TIA-1-inducible alternative exons, and we show that there is very high sequence conservation between chickens and humans of the exon pair and parts of the flanking introns. The TIA-1/TIAR responsiveness of these alternative tiar exons is likely to be of physiological importance for controlling TIAR levels.