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Rps10 Protein Contribution to Ribosomal mRNA Selectivity
Author(s) -
Bush Jessica A.,
Ferretti Max,
Karbstein Katrin
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.526.28
Subject(s) - ribosomal protein , biology , ribosome biogenesis , ribosomal rna , gene , plasmid , microbiology and biotechnology , messenger rna , luciferase , promoter , translation (biology) , ribosome , genetics , gene expression , rna , transfection
Previous work from the Karbstein Lab on ribosomal biogenesis has shown that interactions between the ribosomal protein Rps26 and the mRNA promotes mRNA selectivity, thereby shaping protein homeostasis. Importantly, cells lacking sufficient amounts of Rps26 follow a distinct translational program, which might explain in part the pathogenesis of Diamond‐Blackfan Anemia (DBA), a disease caused by lack of ribosomal proteins, including both Rps26 and Rps10. Both Rps26 and Rps10 are located near the translated mRNA within the entry channel and can be used to predict contact with mRNA downstream of the start and stop codon during translation initiation and termination, respectively. Initial tests were conducted to observe the effects from mutation of the putative Rps10‐contact region on translation. Five mutated plasmids were studied for their affect on Rps10 binding. Results from dual luciferase assays have shown a significant decrease in protein production as a result of mutations in the mRNA plasmid. This decrease appears be dependent on Rps10 levels when compared to the traditional Kozak sequence. To test this hypothesis three different yeast strains were used, WT, TEF and Dox yeast strains. Wild type yeast cells contain their own endogenous copies of the Rps10 genes. TEF and Dox are both genetically modified yeast strains in which the endogenous copies of Rps10 were removed from the genome and replaced by a newly introduced copy in a plasmid under control of either a TEF or Dox promoter. These promoters allow for controlled Rps10 production levels. Doxycycline can be added to cells to further repress their Rps10 production. Using western analysis, we observed that yeast cells with the Dox promoter and treated with doxycycline had significantly reduced levels of Rps10 in their ribosomes. Cells that were successfully depleted of Rps10 had significantly lower protein production then their Rps10 replete counterparts. This translational difference is believed to be a result of a decreased ability of the ribosome to bind mRNA as a result of diminished Rps10 levels. Support or Funding Information K. Karbstein is an HHMI Faculty Scholar This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .