Structural Basis for Translation Initiation in Leishmania Major

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ABSTRACT About 20 species of the protozoan parasite Leishmania are distributed across the globe and cause roughly 1 million new cases of leishmaniasis annually. The most severe form of leishmaniasis (e.g., kala-azar or visceral leishmaniasis) is lethal if untreated. Few therapies are available, and significant side effects and parasite resistance limit their effectiveness. The World Health Organization lists Leishmaniasis as a neglected tropical disease for which the development of new treatments is a high priority. Most of the gene expression in Leishmania is regulated at the level of translation. In mammalian cells, translation initiation is well characterized. A crucial step in this process includes the recognition by eIF4E, a cap-binding protein, of an m7GTP moiety located at the 5’ end of messenger RNAs (mRNAs). eIF4E, through its interaction with other translation initiation factors, ultimately coordinates the recruitment of the small ribosomal subunit. To date, six Leishmania mRNA cap-binding protein isoforms (LIF4E-1 through -6) have been identified. The Leishmania mRNA cap structure is unique and consists of the eukaryotic m7GTP cap followed by four nucleotides that are hypermethylated (“cap-4”). A conserved mini-exon spliced leader RNA (SL RNA) of 39 nucleotides is also added to each transcript through trans-splicing. We previously determined the X-ray crystal structure of LIF4E-1, the only cap-binding isoform that is expressed in Leishmania amastigotes (human infective stage), bound to an interacting partner that represses its cap-binding activity (L4E-IP1). LIF4E-1 was also recently shown to interact directly with the subunit “a” of Leishmania initiation factor 3 (LIF3), a large (~ 800 kDa) multiprotein complex that binds the small ribosomal subunit. An interaction between an IF4E cap-binding protein and an IF3 subunit has never been observed in other systems, and the details of the LIF4E-1/LIF3a molecular interaction are unknown. We hypothesize that a LIF4E-1/cap-4 SL RNA/LIF3a interaction influences the organization of LIF3 and how it assembles a competent pre-initiation complex (PIC) in amastigotes. The LIF4E-1/LIF3a interaction would bypass the need for an eIF4G-like scaffolding protein. In this proposal, we will pursue two specific aims: 1) To define the molecular basis for LIF3a interaction with the cap-binding protein LIF4E-1. 2) To determine a high-resolution cryo-EM structure of LIF4E-1/cap-4 SL RNA bound to LIF3a or assembled with LIF3 on the small ribosomal subunit. Our work will reveal the molecular basis for unique protein- protein interactions in Leishmania parasites, and this information, in turn, could guide the development of specific translation initiation inhibitors against these parasites.