Research in the Browning laboratory seeks a molecular description of the initiation of translation in plants. Early events include the interaction of eIF4F, eIF4A and eIF4B to unwind secondary structure in the 5’ UTR of a mRNA in an ATP-dependent manner. Interaction of this mRNA·pre-initiation complex with the 40S ribosome involves additional initiation factors, eIF2, eIF3, eIF1A, eIF5 and others. We have focused our attention on eIF4F, eIF4A, eIF4B and more recently eIF3.

The eIF4F complex consists of two subunits, eIF4G and eIF4E. eIF4G serves as a “scaffold” for the assembly of other initiation factors, eIF4A, eIF3 and poly A binding protein (PABP). eIF4E binds the m7GpppG “cap” group on the 5’ ends of mRNAs, and is also known as “cap binding protein”. Plants have an isozyme form of eIF4F that is not present in other eukaryotes. We named this form eIF(iso)4F and it also has two subunits, eIF(iso)4G and eIF(iso)4E.

eIF4A is the prototype for the “DEAH/D box” helicases. These are ATP-dependent RNA helicases that presumably unwind secondary structures in RNA.

eIF4B is an RNA binding protein whose function is not clear. It has been shown to interact with eIF3, and potentially to have two RNA binding sites. These two RNA binding sites are speculated to interact with mRNA and with 18S rRNA to facilitate binding of the mRNA to 40S ribosomes. eIF4B amino acid sequences are the least conserved among eukaryotes suggesting that there may be multi-functional roles or alternative regulation of this protein.

eIF3 is the most complex initiation factor consisting of 13 non-identical subunits. The exact roles of all the subunits in initiation of translation are still largely unknown although recent advances in structure of mammalian eIF3 will help move this area forward.

Research Project: Do plant use redox as a method to regulate protein synthesis? We have shown that eIF4E (and probably eIF(iso)4E) have a disulfide bond that may affect the ability to bind to capped mRNAs. We are using various methods (including mutagenesis, knock out plants, NMR, redox stress inducers, etc) to show that redox is used for regulation of translation.

Research Project: How do mRNAs discriminate between eIF4F and eIF(iso)4F? We have shown that mRNAs utilize eIF4F and eIF(iso)4F differentially. Some mRNAs do not have a preference, others will not use eIF(iso)4F in their translation. What is the basis of this discrimination? Is at the mRNA or protein level or both. We have made “mixed” complexes of eIF4G/eIF(iso4E) and eIF(iso)4G/eIF4E that are functional. We will use these “mixed” complexes to elucidate the role of RNA-protein recognition to discriminate between eIF4F and eIF(iso)4F.

Research Project: Why are there two forms of eIF4F in plants? We are seeking an answer for the role of the isozyme form (eIF(iso)4F in higher plants. This form is present in all higher plants, yet its function is unknown. In vitro analysis of both native and recombinat forms of the protein indicate they have similar functions. However, we have shown that some mRNAs preferentially use eIF4F over eIF(iso)4F (see avove). We are using genomic approaches to seek clues to the function of this protein. The completion of the A. thaliana genome and the use of this plant as a model system make it ideal for a genomic approach. We are using T-DNA insertion mutagenesis to “knock-out” the genes for the subunits, eIF(iso)4G and eIF(iso)4E. Will a mutant lacking all the genes have a phenotype?

We are using DNA arrays to look at expression profiles in the knockouts for the subunits, eIF4G, eIF4E, eIF(iso)4G and eIF(iso)4E to determine which mRNAs appear to be lost from polysomes.

Research Project: How do you initiate translation in the 3’ UTR??? Most normal cellular mRNAs are believed to have the m7GpppG “cap” group on the 5’ end. This m7GpppG “cap” group is added during transcription in the nucleus and there are nuclear cap-binding proteins that may have a role in the export of mRNAs. It has also recently been reported that eIF4E may also have a role in the nucleus. There is a group of mammalian viruses, picornoviruses (e.g., polio, EMCV), that have a long 5’ untranslated region and lack a m7GpppG “cap” group. These viral RNAs recruit the 40S ribosome to an “internal ribosome entry site” or IRES. IRES containing RNAs do not need eIF4E for their initiation and many viruses that use this method have altered requirements for other initiation factors as well. This is often referred to as “cap-independent” translation. A number of cellular mRNAs are also thought to use a different type of IRES for translation as well. Plant viruses do not appear to use IRESes as frequently, but have evolved other cap-independent methods. We study, Satellite Tobacco Necrosis Virus (STNV) RNA which uses a cap-independent method of initiation, but does not contain an IRES. STNV RNA has evolved a ~100 nucleotide element in its 3’ UTR that specifically recruits eIF4E/eIF(iso)4E and its associated large subunit, eIF4G/eIF(iso)4G. We have shown through several types of experiments that this binding is specific for eIF4E/eIF(iso)4E.

Research Project: eIF3. One of the most complex initiation factors, eIF3, contains 13 non-identical subunits. Both wheat and Arabidopsis eIF3 more closely resemble mammalian eIF3 rather than S. cerevisiae. To be able to analyze this complex biochemically, it is necessary to be able to make mutants and produce a recombinant complex. The project for the Freshman Research Initiative that I lead is to clone using the "biobrick" method all the subunits for eIF3, assemble the subunits into operons and express the complex in E. coli. The current status (as of August 2008) is all the subunits have been biobricked, shown to express as single proteins and the operons are under construction.