RESUMO
We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.
Assuntos
Processamento Alternativo/fisiologia , Antígenos de Neoplasias , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Animais , Apoptose/genética , Química Encefálica/genética , Tronco Encefálico/citologia , Tronco Encefálico/embriologia , Sobrevivência Celular/genética , Éxons/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/química , Antígeno Neuro-Oncológico Ventral , Ligação Proteica/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Ribonucleoproteínas/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.
Assuntos
Processamento Alternativo , Encéfalo/fisiopatologia , Doenças do Sistema Nervoso/genética , Neurônios/fisiologia , Animais , Encéfalo/fisiologia , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.
Assuntos
Genes Bacterianos/genética , Lipopolissacarídeos/biossíntese , Vibrio cholerae/genética , Sequência de Aminoácidos , Antígenos de Bactérias/biossíntese , Argentina , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Sequência de Bases , Carboidratos Epimerases/genética , Clonagem Molecular , Elementos de DNA Transponíveis/genética , Teste de Complementação Genética , Índia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Vibrio cholerae/imunologiaRESUMO
The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.