RESUMO
The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.
Assuntos
Sequência Conservada , Proteínas Nucleares/genética , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos , Transporte Biológico , Clonagem Molecular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The exon junction complex (EJC) is a key regulator of posttranscriptional mRNA fate and binds to mRNA during splicing. Although the composition of EJCs is well understood, the mechanism mediating splicing-dependent EJC assembly and the factor(s) recruiting the EJC remain elusive. Here, we identify CWC22 as an essential splicing factor that is required for EJC assembly. In CWC22-depleted cells, pre-mRNA splicing is impaired but is rescued by a central fragment of CWC22. We show that the MIF4G domain of CWC22 initiates EJC assembly via a direct interaction with the EJC core protein eIF4A3, and we characterize mutations in eIF4A3 that abolish binding to CWC22. These eIF4A3 mutants efficiently nucleate splicing-independent recombinant EJC core complexes, but they fail to support splicing-dependent EJC deposition. Our work establishes a direct link between the splicing machinery and the EJC, hence uncovering a molecular interaction at the center of a posttranscriptional gene regulation network.