Kinase-mediated assembly of the mRNA entry channel in 40S ribosomes

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 DESCRIPTION (provided by applicant): Rapidly dividing cells must produce 2,000 new ribosomes every minute and ensure that they are fully functional. The mRNA entry channel is formed on the beak structure of the small ribosomal subunit, and two out of the three proteins at the beak are associated with DBA, an inherited human disease, caused by haploinsufficiency of ribosomal proteins (r-proteins). As expected from loss of r-proteins, patients have low cell numbers in rapidly dividing tissues, but are also predisposed to cancer, suggesting that these patients fail in producing both the numbers and quality of ribosomes needed. Our recent data indicates that r- proteins at the mRNA entry channel are assembled late during maturation, and that assembly is regulated by release of the assembly factor Ltv1, which requires phosphorylation by the Hrr25 kinase. The overall goal of this project is to dissect the assembly of the mRNA entry channel using structural and biochemical methods. In Aim 1 we will dissect how Ltv1 dissociation affects the structure of pre-40S subunits, and the mRNA binding channel. We will also test how release of Ltv1 is linked to recruitment of the ribosomal protein Asc1, which is required for translation of IRES-containing viral messages. In Aim 2 we will use structural and yeast biochemical methods to define how incorporation of Rps10 at the beak is linked to 18S rRNA processing at the platform, via a network of proteins involved in DBA. These studies will provide insight into assembly events that are very rapid within cells and increase our understanding of the mechanisms by which defects in ribosome maturation can lead to tumorigenesis.