ABSTRACT
BACKGROUND: Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. RESULTS: As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5-7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. CONCLUSION: The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds.
ABSTRACT
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.
Subject(s)
Exons , RNA Splice Sites , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Crystallography, X-Ray , Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression , Humans , Models, Molecular , Negative Staining , Neoplasm Proteins/chemistry , Neoplasm Proteins/ultrastructure , Nuclear Proteins/chemistry , Nuclear Proteins/ultrastructure , Protein Binding , RNA-Binding Proteins , Ribonucleoproteins/isolation & purification , Ribonucleoproteins/ultrastructureABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-4A/genetics , Mutation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Codon, Nonsense , Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4A/metabolism , Exons , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , In Vitro Techniques , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA Stability , RNA, Messenger/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Carrier Proteins , Exons , Nuclear Proteins , RNA Splicing , Ribonucleoproteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromatography, Liquid , Co-Repressor Proteins , Cytoplasm/chemistry , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression , HeLa Cells , Humans , Mass Spectrometry , Models, Biological , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/geneticsABSTRACT
Over the past decade many studies have revealed a complex web of interconnections between the numerous steps required for eukaryotic gene expression. One set of interconnections link nuclear pre-mRNA splicing and the subsequent metabolism of the spliced mRNAs. It is now apparent that the means of connection is a set of proteins, collectively called the exon junction complex, which are deposited as a consequence of splicing upstream of mRNA exon-exon junctions.
Subject(s)
Exons , RNA Splicing/genetics , RNA, Messenger/genetics , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , Humans , Protein Biosynthesis , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/metabolism , Spliceosomes/genetics , Spliceosomes/metabolismABSTRACT
The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs.
Subject(s)
Eukaryotic Initiation Factor-4A/metabolism , Exons/genetics , RNA Splicing , RNA, Messenger/metabolism , Binding Sites , Codon, Nonsense , Genes, Reporter , Glutathione Transferase/genetics , Humans , Kinetics , Polymerase Chain Reaction , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208-735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors.
