Ribosomopathies are diseases that result from the haploinsufficiency of ribosomal proteins (RPs) or mutations in ribosome assembly factors {3,14}. Since these diseases involve the ribosome, a complex that is vital to every cell, they tend to preferentially impact tissues with high rates of proliferation, as is seen in the well- studied ribosomopathy, Diamond-Blackfan Anemia (DBA) {15}. In an apparent paradox, ribosomopathies carry a ~30-fold increased risk of cancer even though most symptoms appear to be caused by insufficient proliferation {14}. One resolution to this discrepancy is that RP haploinsufficiency leads to accumulation of ribosomes lacking a full complement of proteins. If the missing protein recognizes mRNA features that enhance their translation, these incomplete ribosomes will perturb the fine-tuned cellular control of translation. In investigating the maturation pathway of the small ribosomal subunit, the Karbstein Lab has identified a population of ribosomes (?Rps26 ribosomes) that lack Rps26, the last ribosomal protein to bind to the maturing small (40S) subunit and one of the proteins whose loss is implicated in DBA {16,17}. This population of ribosomes is capable of binding mRNA {18}, but in preliminary data I have shown that they prefer a different population of transcripts than their canonical (WT) counterparts. I hypothesize that Rps26 allows ribosomes to preferentially bind mRNA transcripts with strong Kozak sequences, and that ribosomes lacking this protein are blinded to the translational program of an actively growing cell.
Aim 1 : Identify the population of mRNAs bound by ?Rps26 ribosomes. I will sequence the mRNAs that co-purify with affinity-purified ?Rps26 ribosomes and identify the transcripts that are differentially represented when compared to WT ribosomes.
Aim 2 : Determine if Rps26 enhances the binding of mRNAs with strong Kozak sequences A strong Kozak sequence increases translatability of an mRNA {23}, but ?Rps26 ribosomes may not follow this pattern. I will test the ability of ?Rps26 ribosomes to recognize and bind mRNAs containing a strong Kozak sequence using a combination of biochemical and in vivo experiments.
Aim 3 : Investigate the biological relevance of Rps26 insufficiency Positive regulators of the High Osmolarity Glycerol (HOG), a MAP-kinase pathway, and the Rim101 high pH response pathway are enriched in the pool of mRNA bound to ?Rps26 ribosomes. In vivo data show that Rps26-insufficient yeast are more resistant to these forms of stress, validating the biological relevance of ?Rps26 ribosomes. The Ras/PKA and Tor pathways, both implicated in oncogenesis {57-60}, are also enriched in ?Rps26 ribosomes. My long-term goal is to generate an Rps26 deficient human cell line and test my findings from yeast that may explain the DBA cancer phenotype.
Ribosomes are the molecular machines that construct every protein within our cells. Deficiencies in ribosome construction cause diseases that, among other things, predispose people to cancer. By studying ribosomes that lack an essential protein, we will better understand how healthy ribosomes work and how defective ones contribute to human disease.
| Ferretti, Max Berman; Barre, Jennifer Louise; Karbstein, Katrin (2018) Translational Reprogramming Provides a Blueprint for Cellular Adaptation. Cell Chem Biol 25:1372-1379.e3 |
| Ferretti, Max B; Ghalei, Homa; Ward, Ethan A et al. (2017) Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements. Nat Struct Mol Biol 24:700-707 |
