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1.
Sensors (Basel) ; 23(12)2023 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-37420632

RESUMEN

We report on the development of scintillating bolometers based on lithium molybdate crystals that contain molybdenum that has depleted into the double-ß active isotope 100Mo (Li2100deplMoO4). We used two Li2100deplMoO4 cubic samples, each of which consisted of 45-millimeter sides and had a mass of 0.28 kg; these samples were produced following the purification and crystallization protocols developed for double-ß search experiments with 100Mo-enriched Li2MoO4 crystals. Bolometric Ge detectors were utilized to register the scintillation photons that were emitted by the Li2100deplMoO4 crystal scintillators. The measurements were performed in the CROSS cryogenic set-up at the Canfranc Underground Laboratory (Spain). We observed that the Li2100deplMoO4 scintillating bolometers were characterized by an excellent spectrometric performance (∼3-6 keV of FWHM at 0.24-2.6 MeV γs), moderate scintillation signal (∼0.3-0.6 keV/MeV scintillation-to-heat energy ratio, depending on the light collection conditions), and high radiopurity (228Th and 226Ra activities are below a few µBq/kg), which is comparable with the best reported results of low-temperature detectors that are based on Li2MoO4 using natural or 100Mo-enriched molybdenum content. The prospects of Li2100deplMoO4 bolometers for use in rare-event search experiments are briefly discussed.


Asunto(s)
Molibdeno , Radio (Elemento) , Isótopos , Conteo por Cintilación/métodos , Litio , Iones
2.
Genes Chromosomes Cancer ; 58(6): 341-356, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30474255

RESUMEN

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.


Asunto(s)
Cariotipo Anormal , Inestabilidad Genómica , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Progeria/genética , Telomerasa/genética , Homeostasis del Telómero , Línea Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Telomerasa/metabolismo
3.
Biogerontology ; 20(3): 337-358, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31041622

RESUMEN

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, premature ageing syndrome in children. HGPS is normally caused by a mutation in the LMNA gene, encoding nuclear lamin A. The classical mutation in HGPS leads to the production of a toxic truncated version of lamin A, progerin, which retains a farnesyl group. Farnesyltransferase inhibitors (FTI), pravastatin and zoledronic acid have been used in clinical trials to target the mevalonate pathway in HGPS patients to inhibit farnesylation of progerin, in order to reduce its toxicity. Some other compounds that have been suggested as treatments include rapamycin, IGF1 and N-acetyl cysteine (NAC). We have analysed the distribution of prelamin A, lamin A, lamin A/C, progerin, lamin B1 and B2 in nuclei of HGPS cells before and after treatments with these drugs, an FTI and a geranylgeranyltransferase inhibitor (GGTI) and FTI with pravastatin and zoledronic acid in combination. Confirming other studies prelamin A, lamin A, progerin and lamin B2 staining was different between control and HGPS fibroblasts. The drugs that reduced progerin staining were FTI, pravastatin, zoledronic acid and rapamycin. However, drugs affecting the mevalonate pathway increased prelamin A, with only FTI reducing internal prelamin A foci. The distribution of lamin A in HGPS cells was improved with treatments of FTI, pravastatin and FTI + GGTI. All treatments reduced the number of cells displaying internal speckles of lamin A/C and lamin B2. Drugs targeting the mevalonate pathway worked best for progerin reduction, with zoledronic acid removing internal progerin speckles. Rapamycin and NAC, which impact on the MTOR pathway, both reduced both pools of progerin without increasing prelamin A in HGPS cell nuclei.


Asunto(s)
Lamina Tipo A/metabolismo , Ácido Mevalónico/metabolismo , Progeria/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Progeria/patología
4.
Nucleic Acids Res ; 40(6): 2639-52, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22110043

RESUMEN

Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs.


Asunto(s)
Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas del Complejo SMN/metabolismo , Empalmosomas/metabolismo , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Empalme del ARN
5.
Cell Commun Signal ; 11: 88, 2013 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-24245560

RESUMEN

BACKGROUND: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies. RESULTS: This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells. CONCLUSIONS: The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy.


Asunto(s)
Exosomas , Técnicas de Transferencia de Gen , ARN Interferente Pequeño/administración & dosificación , Líquido Ascítico/citología , Línea Celular Tumoral , Humanos , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética
6.
J Med Genet ; 47(3): 176-81, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19797196

RESUMEN

BACKGROUND: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. AIM: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. METHODS: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and gamma-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. RESULTS: Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. CONCLUSION: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.


Asunto(s)
Proteína Quinasa Activada por ADN/fisiología , Neoplasias de Cabeza y Cuello/radioterapia , Hemangiosarcoma/radioterapia , Proteínas Nucleares/fisiología , Tolerancia a Radiación/genética , Neoplasias Cutáneas/radioterapia , Xerodermia Pigmentosa/genética , Empalme Alternativo/fisiología , Secuencia de Aminoácidos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Proteína Quinasa Activada por ADN/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Hemangiosarcoma/genética , Hemangiosarcoma/patología , Humanos , Isoenzimas/genética , Isoenzimas/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Traumatismos por Radiación/genética , Cuero Cabelludo , Homología de Secuencia de Aminoácido , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Xerodermia Pigmentosa/patología
7.
Open Biol ; 11(12): 210276, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34847775

RESUMEN

Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs.


Asunto(s)
Proteínas Portadoras/metabolismo , Vesículas Extracelulares/enzimología , Hexoquinasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Proteómica/métodos , Hormonas Tiroideas/metabolismo , Proteínas Portadoras/sangre , Línea Celular Tumoral , Proliferación Celular , Niño , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glucólisis , Hexoquinasa/sangre , Humanos , Espectrometría de Masas , Proteínas de la Membrana/sangre , Neuroblastoma/sangre , Fosforilación , Hormonas Tiroideas/sangre , Proteínas de Unión a Hormona Tiroide
8.
Nat Struct Mol Biol ; 11(5): 463-8, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15098019

RESUMEN

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated B Delta U1) at a stage after U4/U6.U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of B Delta U1 by single-particle electron cryomicroscopy at a resolution of approximately 40 A. The overall size of the complex is about 370 x 270 x 170 A. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6.U5 tri-snRNP. This provides initial insight into the structural organization of complex B.


Asunto(s)
Proteínas/química , Empalmosomas/química , Catálisis , Humanos , Microscopía Electrónica , Conformación Proteica , Proteínas/ultraestructura , Empalmosomas/ultraestructura
9.
Cancer Rep (Hoboken) ; 2(5): e1207, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-32721124

RESUMEN

BACKGROUND: Haematological malignancies harbouring rearrangements of the KMT2A gene represent a unique subtype of leukaemia, with biphenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t(4;11)(q21;q23) producing the KMT2A-AFF1 chimera. AIM: The aim of this study was to confirm and review the cytogenetic and molecular features of the KMT2A-rearranged RS4;11 cell line and put those in context with other reports of cell lines also harbouring a t(4;11) rearrangement. METHODS AND RESULTS: The main chromosomal rearrangements t(4;11)(q21;q23) and i(7q), described when the cell line was first established, were confirmed by fluorescence in situ hybridisation (FISH) and 24-colour karyotyping by M-FISH. Additional cytogenetic abnormalities were investigated by further FISH experiments, including the presence of trisomy 18 as a clonal abnormality and the discovery of one chromosome 8 being an i(8q), which indicates a duplication of the oncogene MYC. A homozygous deletion of 9p21 containing the tumour-suppressor genes CDKN2A and CDKN2B was also revealed by FISH. The production of the fusion transcript KMT2A-AFF1 arising from the der(11)t(4;11) was confirmed by RT-PCR, but sequencing of the amplified fragment revealed the presence of multiple isoforms. Two transcript variants, resulting from alternative splicing, were identified differing in one glutamine residue in the translated protein. CONCLUSION: As karyotype evolution is a common issue in cell lines, we highlight the need to monitor cell lines in order to re-confirm their characteristics over time. We also reviewed the literature to provide a comparison of key features of several cell lines harbouring a t(4;11). This would guide scientists in selecting the most suitable research model for this particular type of KMT2A-leukaemia.


Asunto(s)
Proteínas de Unión al ADN/genética , N-Metiltransferasa de Histona-Lisina/genética , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Factores de Elongación Transcripcional/genética , Línea Celular Tumoral , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 4/genética , Humanos , Cariotipificación , Leucemia/patología , Eliminación de Secuencia , Translocación Genética
10.
Immunobiology ; 224(3): 408-418, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30954271

RESUMEN

Hydrophilic lung surfactant proteins have emerged as key immunomodulators which are potent at the recognition and clearance of pulmonary pathogens. Surfactant protein A (SP-A) is a surfactant-associated innate immune molecule, which is known to interact with a variety of pathogens, and display anti-microbial effects. SP-A, being a carbohydrate pattern recognition molecule, has a wide range of innate immune functions against respiratory pathogens, including influenza A virus (IAV). Some pandemic pH1N1 strains resist neutralization by SP-A due to differences in the N-glycosylation of viral hemagglutinin (HA). Here, we provide evidence, for the first time, that a recombinant form of human SP-A (rfhSP-A), composed of α-helical neck and carbohydrate recognition domains, can actually promote the IAV replication, as observed by an upregulation of M1 expression in lung epithelial cell line, A549, when challenged with pH1N1 and H3N2 IAV subtypes. rfhSP-A (10 µg/ml) bound neuraminidase (NA) (∼60 kDa), matrix protein 1 (M1) (∼25 kDa) and M2 (∼17 kDa) in a calcium dependent manner, as revealed by far western blotting, and direct binding ELISA. However, human full length native SP-A downregulated mRNA expression levels of M1 in A549 cells challenged with IAV subtypes. Furthermore, qPCR analysis showed that transcriptional levels of TNF-α, IL-12, IL-6, IFN-α and RANTES were enhanced following rfhSP-A treatment by both IAV subtypes at 6 h post-IAV infection of A549 lung epithelial cells. In the case of full length SP-A treatment, mRNA expression levels of TNF-α and IL-6 were downregulated during the mid-to-late stage of IAV infection of A549 cells. Multiplex cytokine/chemokine array revealed enhanced levels of both IL-6 and TNF-α due to rfhSP-A treatment in the case of both IAV subtypes tested, while no significant effect was seen in the case of IL-12. Enhancement of IAV infection of pH1N1 and H3N2 subtypes by truncated rfhSP-A, concomitant with infection inhibition by full-length SP-A, appears to suggest that a complete SP-A molecule is required for protection against IAV. This is in contrast to a recombinant form of trimeric lectin domains of human SP-D (rfhSP-D) that acts as an entry inhibitor of IAV.


Asunto(s)
Antivirales/metabolismo , Células Epiteliales/fisiología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Pulmón/patología , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Células Epiteliales/virología , Glicosilación , Humanos , Mediadores de Inflamación/metabolismo , Unión Proteica , Dominios Proteicos/genética , Proteína A Asociada a Surfactante Pulmonar/genética , Proteínas Recombinantes/genética , Virulencia , Replicación Viral
11.
Front Immunol ; 9: 533, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29867915

RESUMEN

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen-macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1ß, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-ß) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1ß, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host-pathogen interactions in tuberculosis.


Asunto(s)
Macrófagos/fisiología , Mycobacterium bovis/fisiología , Properdina/fisiología , Trombospondinas/fisiología , Citocinas/genética , Humanos , Células THP-1
12.
Mol Cell Biol ; 22(10): 3219-29, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11971955

RESUMEN

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.


Asunto(s)
ARN Nuclear Pequeño/química , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Empalmosomas/metabolismo , Anticuerpos/metabolismo , Núcleo Celular/química , Células HeLa , Humanos , Conformación de Ácido Nucleico , Pruebas de Precipitina , Subunidades de Proteína , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo
13.
Mol Cell Biol ; 22(14): 5141-56, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12077342

RESUMEN

A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.


Asunto(s)
Proteínas de Drosophila , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Clonación Molecular , ADN Helicasas , Proteínas de Unión al ADN , Genes de Helminto , Histona Desacetilasas/genética , Histona Desacetilasas/aislamiento & purificación , Histona Desacetilasas/metabolismo , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Co-Represor 1 de Receptor Nuclear , Proteínas Serina-Treonina Quinasas/genética , Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , Empalme del ARN , Factores de Empalme de ARN , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Schizosaccharomyces/enzimología , Factores de Transcripción/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos Híbridos
14.
J Chromatogr A ; 1150(1-2): 117-23, 2007 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-16996525

RESUMEN

Known methods of quantitative chromatographic analysis (calibration, external standard, internal standard and standard addition) require the application of sample preparation techniques without significant losses of analytes. If this condition cannot be satisfied, the compensation of these losses should be provided. The modification of known method of quantitative chromatographic analysis (double internal standard), implying the addition of two homologues (previous and following) of target analytes as internal standards into initial samples is considered. This approach permits us to compensate significant losses both analytes and standards at all stages of sample preparation. The advantages of this method are demonstrated on the examples of liquid-liquid extraction, head space analysis (HSA), distillation of volatile compounds with volatile solvents (concentration in condensates) and evaporation of volatile solvents (concentrating in the residues of solvents). In all cases the application of two homologues as internal standards provides accurate results (the typical relative errors are within 1-6%) at the values of a factor of composition distortion of initial samples (K', the definition is suggested) from 0.2 up to 4. These results are in accordance with general relationships between variations in any physicochemical properties of organic compounds within homologous series. The single found exception was the evaporation of volatile solvents (the open phase transition process) when to get the results with relative errors not more then +10% requires the minimal changes in the composition of initial samples (K' values should not be more then approximately 1.5).


Asunto(s)
Cromatografía/métodos , Cromatografía/normas , Calibración , Cinética , Modelos Químicos , Estándares de Referencia , Reproducibilidad de los Resultados
15.
PLoS One ; 12(9): e0185126, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28961258

RESUMEN

P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126. The mutation eliminates the recognition site for the restriction endonuclease Sca I that allowed us to carry out RFLP analysis of DNA extracted from the clinical samples and suggests that this mutation is more frequent than is documented in the p53 databases. Both the ECV-304 and EJ cell lines, that probably originate from the bladder carcinoma T24 cell line, were confirmed to contain the homozygous 378C>G mutation but were shown to produce the p53 protein of expected full-length size detected by Western blotting. We provide evidence that the 378C>G mutation generates an alternative 3' splice site (ss) which is more often used instead of the authentic upstream 3' ss, driving the production of mRNA encoding the protein with the single amino acid deletion (p53ΔY126). Using endogenous expression, we demonstrated that the p53ΔY126 protein is nearly as active as the wild type protein in inducing the p21/Waf1 expression and apoptosis.


Asunto(s)
Empalme Alternativo , Apoptosis , Codón sin Sentido , Proteína p53 Supresora de Tumor/genética , Western Blotting , Línea Celular Tumoral , ADN Complementario/genética , Citometría de Flujo , Humanos , Polimorfismo de Longitud del Fragmento de Restricción
16.
PLoS One ; 10(5): e0128430, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26020933

RESUMEN

Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing.


Asunto(s)
Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Empalmosomas/genética , Exones , Células HeLa , Humanos , Intrones , Unión Proteica , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Empalmosomas/química , Empalmosomas/metabolismo
17.
Nat Commun ; 3: 994, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22871813

RESUMEN

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.


Asunto(s)
Núcleo Celular/metabolismo , Empalme del ARN/fisiología , Empalmosomas/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Microscopía Fluorescente , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/genética , Fosforilación/fisiología , Empalme del ARN/genética , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo
18.
Nucleus ; 2(6): 517-22, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22064469

RESUMEN

Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe premature aging syndrome that affects children. These children display characteristics associated with normal aging and die young usually from cardiovascular problems or stroke. Classical HGPS is caused by mutations in the gene encoding the nuclear structural protein lamin A. This mutation leads to a novel version of lamin A that retains a farnesyl group from its processing. This protein is called Progerin and is toxic to cellular function. Pre-lamin A is an immature version of lamin A and also has a farnesylation modification, which is cleaved in the maturation process to create lamin A.


Asunto(s)
Investigación Biomédica , Mutación , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Precursores de Proteínas/metabolismo , Universidades , Inglaterra , Humanos , Lamina Tipo A , Proteínas Nucleares/genética , Progeria/genética , Precursores de Proteínas/genética
19.
PLoS One ; 4(9): e7202, 2009 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-19784376

RESUMEN

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.


Asunto(s)
ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/química , Empalmosomas/metabolismo , Catálisis , Células HeLa , Humanos , Espectrometría de Masas/métodos , Conformación Molecular , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Proteómica/métodos , ARN/metabolismo , Proteínas Recombinantes/química , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos
20.
Invest Ophthalmol Vis Sci ; 50(12): 5927-33, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19578015

RESUMEN

PURPOSE: Pre-mRNA processing factor 31 (PRPF31) is a ubiquitous protein needed for the assembly of the pre-mRNA splicing machinery. It has been shown that mutations in this gene cause autosomal dominant retinitis pigmentosa 11 (RP11), which is characterized by rod-cell degeneration. Interestingly, mutations in this ubiquitously expressed gene do not lead to phenotypes other than retinal malfunction. Furthermore, the dominant inheritance pattern has shown incomplete penetrance, which poses interesting questions about the disease mechanism of RP11. METHODS: To characterize PRPF31 function in the rod cells, two animal models have been generated. One was a heterozygous knock-in mouse (Prpf31(A216P/+)) carrying a point mutation p.A216P, which has previously been identified in RP11 patients. The second was a heterozygous knockout mouse (Prpf31(+/-)). Retinal degeneration in RP11 mouse models was monitored by electroretinography and histology. RESULTS: Generation of the mouse models is presented, as are results of ERGs and retinal morphology. No degenerative phenotype on fundus examination was found in Prpf31(A216P/+) and Prpf31(+/-) mice. Prpf31(A216P/A216P) and Prpf31(-/-) genotypes were embryonic lethal. CONCLUSIONS: The results imply that Prpf31 is necessary for survival, and there is no compensation mechanism in mouse for the lack of this splicing factor. The authors suggest that p.A216P mutation in Prpf31 does not exert a dominant negative effect and that one Prpf31 wild-type allele is sufficient for maintenance of the healthy retina in mice.


Asunto(s)
Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Genes Dominantes , Retinitis Pigmentosa/genética , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Electrorretinografía , Femenino , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Marcación de Gen , Genotipo , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oftalmoscopía , Mutación Puntual , Retina/fisiopatología , Retinitis Pigmentosa/fisiopatología
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