Translational Control in Plasmodium and Toxoplasma Parasites

The life cycles of apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii are complex, consisting of proliferative and latent stages within multiple hosts. Dramatic transformations take place during the cycles, and they demand precise control of gene expression at all levels, including translation. This review focuses on the mechanisms that regulate translational control in Plasmodium and Toxoplasma, with a particular emphasis on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α∼P) is a conserved mechanism that eukaryotic cells use to repress global protein synthesis while enhancing gene-specific translation of a subset of mRNAs. Elevated levels of eIF2α∼P have been observed during latent stages in both Toxoplasma and Plasmodium, indicating that translational control plays a role in maintaining dormancy. Parasite-specific eIF2α kinases and phosphatases are also required for proper developmental transitions and adaptation to cellular stresses encountered during the life cycle. Identification of small-molecule inhibitors of apicomplexan eIF2α kinases may selectively interfere with parasite translational control and lead to the development of new therapies to treat malaria and toxoplasmosis.     

Translational Control of the Picornavirus Phenotype

Picornaviruses are small animal viruses with positive-strand genomic RNA, which is translated using cap-independent internal translation initiation. The key role in this is played by ciselements of the 5"-untranslated region (5"-UTR) and, in particular, by the internal ribosome entry site (IRES). The function of translational ciselements requires both canonical translation initiation factors (eIFs) and additional IRES trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins which play a variety of functions in noninfected cells. Specific features of translational ciselements substantially affect the phenotype and, in particular, tissue tropism and pathogenic properties of picornaviruses. It is clear that, in some cases, the molecular mechanism involved is a change in interactions between viral ciselements and ITAFs. The properties and tissue distribution of ITAFs may determine the biological properties of other viruses that also use the IRES-dependent translation initiation. Since this mechanism is also involved in translation of several cell mRNAs, ITAF may contribute to the regulation of the most important aspects of the living activity in noninfected cells.     

DNA Transfection to Study Translational Control in Mammalian Cells

Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular mRNAs. To study the effect of a particular factor or its modification on the translational capacity of an mRNA, there are a number of potential approaches that includein vitrotranslation reactions as well asin vivoexperiments. Generally, experiments initially report a covalent modification that correlates with altered translational capacity of either a specific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are made at the modified residue to prevent modification (for example, a serine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translation factor on translation is the use of transient DNA transfection. However, in certain situations it is desirable to isolate stably transfected cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational control that involve either transient or stable DNA transfection.