Structure-function analysis of the role of rRNA modifications during translation initiation on the human ribosome – CRYOMETH

Cellular mRNAs bind to the ribosome during translation initiation and are involved in the regulation of protein synthesis in different manners. While translation initiation in eukaryotes mainly takes place through a cap-dependent process, 10% of human mRNAs, coding for key regulators of cell physiology use an alternative element called internal ribosome entry site (IRES). The structure and molecular function of human IRESes when bound to the human ribosome are not known. Chemical modifications such as 2’-O methylation of the ribosomal RNA were shown to regulate IRES-dependent translation of various mRNAs. In this collaborative project, we want to address the function of a key cellular IRES mRNA, IGF-1R, and the regulatory role of 2’-O methylations in its translation initiation activity. The partners of this proposal are both experts in chemical modifications and can join forces efficiently through a synergy of functional and structural approaches, including RiboMethSeq analysis, "hybrid" in vitro translation and high-resolution cryo-EM analysis of the human ribosome as illustrated by their recent research (Nature 2015; Nature 2017; PNAS 2017; NAR Cancer 2020). The aim is to provide a detailed description of molecular interactions between an IRES and the human ribosome, identify and knock-out selected 2’-O-Me sites in a selective manner and determine the functional role of 2'-O-methylation in IGF-1R IRES translation initiation. The novelty and originality of CRYOMETH lies in providing the first 3D structure of a human IRES element bound to a human ribosome and in addressing the role of rRNA 2'-O methylation in the human ribosome during the translation initiation step. These data will open new venue of translational research and will lay the basis for applications in human health as several human pathologies are associated with ribosomal composition alteration, including human syndromes and cancer.