RESUMO
To accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2H-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2H-seq to investigate enzymes involved in protein methylation, a largely unexplored post-translational modification. The reported network of 523 interactions involving 22 methyltransferases or demethylases is comprehensively annotated and validated through coimmunoprecipitation experiments and defines previously undiscovered cellular roles of nonhistone protein methylation.
Assuntos
Metiltransferases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Cromatografia Líquida , Escherichia coli , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Metiltransferases/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Espectrometria de Massas em TandemRESUMO
More than 200 proteins copurify with spliceosomes, the compositionally dynamic RNPs catalyzing pre-mRNA splicing. To better understand protein - protein interactions governing splicing, we systematically investigated interactions between human spliceosomal proteins. A comprehensive Y2H interaction matrix screen generated a protein interaction map comprising 632 interactions between 196 proteins. Among these, 242 interactions were found between spliceosomal core proteins and largely validated by coimmunoprecipitation. To reveal dynamic changes in protein interactions, we integrated spliceosomal complex purification information with our interaction data and performed link clustering. These data, together with interaction competition experiments, suggest that during step 1 of splicing, hPRP8 interactions with SF3b proteins are replaced by hSLU7, positioning this second step factor close to the active site, and that the DEAH-box helicases hPRP2 and hPRP16 cooperate through ordered interactions with GPKOW. Our data provide extensive information about the spliceosomal protein interaction network and its dynamics.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Ligação Competitiva , Proteínas de Transporte/metabolismo , Análise por Conglomerados , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/fisiologia , Humanos , Mapas de Interação de Proteínas , Proteômica , RNA Helicases/metabolismo , RNA Helicases/fisiologia , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
The yeast two-hybrid (Y2H) system is currently one of the most important techniques for protein-protein interaction (PPI) discovery. Here, we describe a stringent three-step Y2H matrix interaction approach that is suitable for systematic PPI screening on a proteome scale. We start with the identification and elimination of autoactivating strains that would lead to false-positive signals and prevent the identification of interactions. Nonautoactivating strains are used for the primary PPI screen that is carried out in quadruplicate with arrayed preys. Interacting pairs of baits and preys are identified in a pairwise retest step. Only PPI pairs that pass the retest step are regarded as potentially biologically relevant interactions and are considered for further analysis.
