RESUMO
Isolated liver nuclei or whole lymph node lymphocytes stimulated with concanavalin A in culture were irradiated with ultraviolet light. The crosslinked structures of poly(A)+ heterogeneous nuclear RNA and protein were purified on oligo(dT)-cellulose after labelling irradiated nuclei in the presence of adenosine 5'-[gamma-32P]triphosphate and analysed by SDS-polyacrylamide gel electrophoresis. The liver and lymphocyte nuclear proteins included about 17-19 species of 35-150 kDa and were shown to produce quite similar electrophoretic band patterns. Two proteins of 110-120 and 40-42 kDa were phosphorylated. Using partial proteolytic digestion the large-size crosslinked phosphoprotein has been identified as the 110 kDa component described previously (Schweiger, A. and Kostka, G. (1984) Biochim. Biophys. Acta 782, 262-268). The 40-42 kDa band was presumably related to the group C species of main proteins associated with heterogeneous nuclear RNA. In crosslinked nuclear structures from rats treated with low doses of alpha-amanitin for 1 h the relative amount of the 110-120 kDa phosphoprotein was reduced while the labelling with [32P]ATP was almost abolished.
Assuntos
Fosfoproteínas/isolamento & purificação , Poli A/metabolismo , RNA Nuclear Heterogêneo/metabolismo , Ribonucleoproteínas/isolamento & purificação , Animais , Núcleo Celular/análise , Concanavalina A/farmacologia , Eletroforese em Gel de Poliacrilamida , Fígado/análise , Linfonodos , Linfócitos/análise , Linfócitos/efeitos dos fármacos , Fosfoproteínas/efeitos da radiação , Fosforilação , Poli A/efeitos da radiação , Proteínas Quinases/metabolismo , RNA Nuclear Heterogêneo/efeitos da radiação , Ratos , Ribonucleoproteínas/efeitos da radiação , Raios UltravioletaAssuntos
Nucleoproteínas/metabolismo , RNA Nuclear Heterogêneo/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa/metabolismo , Humanos , Peso Molecular , Poli A/metabolismo , RNA Nuclear Heterogêneo/efeitos da radiação , RNA Mensageiro/efeitos da radiação , Raios UltravioletaAssuntos
Proteínas de Transporte/efeitos da radiação , RNA Nuclear Heterogêneo/efeitos da radiação , RNA Mensageiro/efeitos da radiação , Ribonucleoproteínas/efeitos da radiação , Raios Ultravioleta , Adenosina/análogos & derivados , Adenosina/síntese química , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade/métodos , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida/métodos , Células HeLa/metabolismo , Humanos , Indicadores e Reagentes , RNA Nuclear Heterogêneo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteínas/isolamento & purificação , Vanadatos/síntese químicaRESUMO
The psoralen derivative 4'-hydroxymethyl-4, 5', 8-trimethylpsoralen (hydroxymethyltrioxsalen) has been used in experiments with isolated HeLa cell nuclei to photochemically cross-link double helical regions in heterogeneous nuclear RNA in situ. Although there are other self-complementary sequences in hnRNA that can form base-paired structures upon phenol deproteinization of annealing, the present in situ cross-linking results demonstrate that some double-stranded regions are an authentic component of native hnRNA structure. Moreover, these special regions of secondary structure are apparently highly accessible to chemical probes within the intact cell nucleus, despite the fact that hnRNA possesses a ribonucleoprotein organization.
Assuntos
Células HeLa/efeitos da radiação , RNA Nuclear Heterogêneo/efeitos da radiação , Raios Ultravioleta , Sequência de Bases , Núcleo Celular/metabolismo , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Conformação de Ácido Nucleico , Renaturação de Ácido Nucleico , RNA Nuclear Heterogêneo/metabolismo , Trioxsaleno/análogos & derivados , Trioxsaleno/farmacologiaRESUMO
RNA-protein interaction in the 30S subunits of rat liver hnRNP has been studied by crosslinking of informofer proteins to hnRNA induced by UV irradiation. Irradiation of 30S particles with 254 nm UV light in doses of 1 1 x 10(5) erg/mm2 leads to the extensive crosslinking hnRNA to informofer proteins. The crosslinked material was analyzed either by resedimentation in a 15-30% sucrose gradient in the presence of 3 M guanidine-HCl and 1 M NaCl or by centrifugation in a Cs2SO4 density gradient containing guanidine-HCl and sarkosyl. The crosslinked complexes sedimented at about 25S in the sucrose gradient and proved to be heterogeneous in isopycnic centrifugation experiments. The proteins of the crosslinked complexes were analyzed by polyacrylamide gel electrophoresis. Proteins with Mr values of 70 000, 58 000, 43 000 and 40 000 appeared to be crosslinked with hnRNAs of the 30S particles. In the unirradiated 30S particles after centrifugation in the CS2SO4 density gradient containing guanidine-HCl and sarkosyl two minor proteins were observed with Mr values of 70 000 and 58 000, banded in density zones characteristic for free RNA.
Assuntos
Nucleoproteínas/metabolismo , RNA Nuclear Heterogêneo/metabolismo , Ribonucleoproteínas/metabolismo , Raios Ultravioleta , Animais , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Densitometria , Ribonucleoproteínas Nucleares Heterogêneas , Técnicas In Vitro , Fígado/metabolismo , Peso Molecular , RNA Nuclear Heterogêneo/efeitos da radiação , Ratos , Ribonucleoproteínas/efeitos da radiaçãoRESUMO
A study was made of the content of hnRNA, nuclear poly(A) RNA and biosynthesis of rlnRNA in truncus cerebri of rats divided into 3 groups by the forced swimming test 6-8 min and 60 min after a short-term exposure to sparsely ionizing radiation of 100 Gy. The observed changes in the nuclear RNA metabolism can subsequently lead to the impairment of the synthesis of proteins required for normal functioning of CNS, and to the development of CNS syndrome.