Self-cleaving peptides: Mechanisms and Use in Diverse Eukaryotic Species

总结

SUMMARY Giardia lamblia is a single-cell eukaryote that infects hundreds of millions of people every year. Because Giardia has many molecular pathways that are simplified compared to other eukaryotes, it has potential as a nontraditional model system for studying the diversity and evolution of key biological processes. We recently serendipitously discovered that the 2A ‘self-cleaving’ peptide sequences work very poorly in Giardia, surprising because 2A peptides are thought to work universally in eukaryotes. Found in picornaviruses like foot-and-mouth disease virus and poliovirus, 2A peptides are an essential part of the viral life cycle because they enable two polypeptides to be produced from one open reading frame. Although often referred to as ‘self- cleaving,’ 2A peptides operate by causing the ribosome to skip a peptide bond. The mechanism of this is unknown but must involve specific interactions between the 2A nascent peptide chain and the exit tunnel of the ribosome. Thus, our discovery that 2A peptides work poorly in Giardia points at fundamental differences in its ribosomes compared to other eukaryotes and can be exploited to understand the mechanism of 2A action. Examination of our recently solved structure of the Giardia 80S ribosome reveals a compelling difference in the structure of ribosome protein uL4 in the exit channel: Giardia lacks a specific loop in uL4. We hypothesize that this loop is important for the peptide bond-skipping mechanism of 2A peptides, and its absences can partially explain why 2A peptides operate poorly in Giardia. Here, we will test this hypothesis and in so doing (1) define the mechanism by which 2A peptides induce bond skipping and (2) determine why it fails in Giardia. We will combine genetic, biochemical, and structural approaches in two aims. In the first aim, we will determine the extent to which 2A sequence variants can function in Giardia, with the goal of finding novel efficient and functional sequences that will serve as powerful tools for Giardia researchers. In the second aim, we will directly test the functional role of the uL4 loop and solve the structure of a T2A-ribosome complex by cryo-EM with the goal of describing the mechanism of peptide bond skipping in eukaryotes. Overall, this work will provide critical knowledge about the function of the Giardia ribosome, the fundamental workings of the eukaryotic translational machinery, and the mechanism of 2A peptide function. Our discoveries will facilitate the development of Giardia as a model organism and help lay the foundation for new anti-viral therapeutics that block 2A peptide activity.