Friday, November 22, 2024
11/22/2024
PROJECT SUMMARY Protein production is absolutely essential and faulty translation has been linked to a wide-range of diseases including cancer, neurodegeneration and a class of predominantly developmental disorders known as ribosomopathies. In order to ensure faithful translation of the genetic code, cells need to monitor the integrity of all components of the translation apparatus, including mRNAs, tRNAs, and ribosomes. Although the quality control mechanisms that monitor mRNAs and tRNAs are relatively well characterized, very little is known about quality control of the ribosome itself. The eukaryotic ribosome is a complex molecular machine consisting of 4 ribosomal RNAs (rRNA) and 80 proteins, and all these components are subjected to mutations, oxidative stress, and UV radiation. Notably, the rRNA carries out the core enzymatic functions of the ribosome during translation. Despite the importance of rRNA to ribosomal function and human health, the quality control mechanisms that cope with the presence of defective rRNA have not been studied in mammalian cells. Previous work in yeast has discovered that rRNAs harboring mutations are preferentially degraded, confirming the existence of quality control mechanisms targeting faulty rRNA. However, the components involved in the detection and degradation of these rRNAs remain poorly characterized. This knowledge gap is primarily due to technological constraints, including limited rRNA detection methods and appropriate reporter systems geared for genome-wide screens. Recent technological advances have primed the field for unbiased and mechanistic studies. Due to the critical nature of ribosomal quality control to human health, I seek to define the quality control mechanisms that mammalian cells impart on rRNA. I have engineered a mammalian fluorescent rRNA reporter that can be paired with both unbiased genetic screening and hypothesis-driven approaches. Taking advantage of this unique tool, I will determine how human cells cope with defective ribosomes harboring mutant 18S rRNA, the rRNA component of the small ribosomal subunit. I will first characterize how mutations in the 18S rRNAs interfere with translation. Second, I will determine the fate of defective 18S rRNA in mammalian cells. Lastly, I will identify the mammalian factors coping with defective rRNA. Together, the proposed studies will for the first time delineate mammalian rRNA quality control mechanisms, and provide the foundation for understanding how dysregulation of this process leads to human disease.
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