RESUMO
We describe a defective HeLa nuclear extract which is particularly deficient in step 2 of splicing reaction. With this extract we have studied the conservation of a second-step activity from yeast to human cells. We detected a S. cerevisiae second-step splicing activity that allows restoration of step 2 of the defective HeLa nuclear extract, which indicates that yeast purified fraction has a second-step activity that is conserved from yeast to human cells. The activity is a yeast UsnRNP protein(s) since it is purified with anti-trimethylguanosine by immunoaffinity columns.
Assuntos
Evolução Biológica , Splicing de RNA , Cromatografia de Afinidade , Teste de Complementação Genética , Globinas/genética , Células HeLa , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Ribonucleoproteínas Nucleares Pequenas/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Two regions common to all UsnRNP core polypeptides have been described: Sm motif 1 and Sm motif 2. Rabbits were immunized with a 22 amino-acid peptide containing one segment of Sm motif 1 (YRGTLVSTDNYFNLQLNEAEEF, corresponding to residues 11-32) from yeast F protein. After immunization, the rabbit sera contained antibodies that not only reacted specifically with the peptide from yeast F protein but also cross-reacted with Sm polypeptides from mammals; that is, with purified human U1snRNPs. The results suggest that the peptide used and human Sm polypeptides contain a common feature recognized by the polyclonal antibodies. A large collection of human systemic lupus erythematosus sera was assayed using the yeast peptide as an antigen source. Seventy per cent of systemic lupus erythematosus sera contain an antibody specificity that cross-reacts with the yeast peptide.
Assuntos
Anticorpos Antifúngicos/metabolismo , Autoantígenos/imunologia , Proteínas Fúngicas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ribonucleoproteínas Nucleares Pequenas/imunologia , Sequência de Aminoácidos , Reações Cruzadas , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas Centrais de snRNPRESUMO
2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG-binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.