RESUMO
The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII's RNA binding.
Assuntos
Éxons , Sítios de Splice de RNA , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Cristalografia por Raios X , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Coloração Negativa , Proteínas de Neoplasias/química , Proteínas de Neoplasias/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Ligação Proteica , Proteínas de Ligação a RNA , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/ultraestruturaRESUMO
The exon junction complex (EJC) is deposited on mRNAs by the process of pre-mRNA splicing and is a key effector of downstream mRNA metabolism. We previously demonstrated that human eIF4AIII, which is essential for nonsense-mediated mRNA decay (NMD), constitutes at least part of the RNA-binding platform anchoring other EJC components to the spliced mRNA. To determine the regions of eIF4AIII that are functionally important for EJC formation, for binding to other EJC components, and for NMD, we now report results of an extensive mutational analysis of human eIF4AIII. Using GFP-, GST- or Flag-fusions of eIF4AIII versions containing site-specific mutations or truncations, we analyzed subcellular localizations, protein-protein interactions, and EJC formation in vivo and in vitro. We also tested whether mutant proteins could rescue NMD inhibition resulting from RNAi depletion of endogenous eIF4AIII. Motifs Ia and VI, which are conserved among the eIF4A family of RNA helicases (DEAD-box proteins), are crucial for EJC formation and NMD, as is one eIF4AIII-specific region. An additional eIF4AIII-specific motif forms part of the binding site for MLN51, another EJC core component. Mutations in the canonical Walker A and B motifs that eliminate RNA-dependent ATP hydrolysis by eIF4AIII in vitro are of no detectable consequence for EJC formation and NMD activation. Implications of these findings are discussed in the context of other recent results and a new structural model for human eIF4AIII based on the known crystal structure of Saccharomyces cerevisiae eIF4AI.
Assuntos
Fator de Iniciação 4A em Eucariotos/genética , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Códon sem Sentido , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The multiprotein exon junction complex (EJC) is deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. In mammalian cells, this complex serves as a key modulator of spliced mRNA metabolism. To date, neither the complete composition nor the exact assembly pathway of the EJC has been entirely elucidated. Using in vitro splicing and a two-step chromatography procedure, we have purified the EJC and analyzed its components by mass spectrometry. In addition to finding most of the known EJC factors, we identified two novel EJC components, Acinus and SAP18. Heterokaryon analysis revealed that SAP18 is a shuttling protein whereas Acinus is restricted to the nucleus. In MS2 tethering assays Acinus stimulated gene expression at the RNA level, while MLN51, another EJC factor, stimulated mRNA translational efficiency. Using tandem affinity purification (TAP) of proteins overexpressed in HeLa cells, we demonstrated that Acinus binds directly to another EJC component, RNPS1, while stable association of SAP18 to form the trimeric apoptosis and splicing associated protein (ASAP) complex requires both Acinus and RNPS1. Using the same methodology, we further identified what appears to be the minimal stable EJC core, a heterotetrameric complex consisting of eIF4AIII, Magoh, Y14, and MLN51.