Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular Biophysics and Biochemistry
First Advisor
Baserga, Susan
Abstract
Ribosomes are the complex macromolecular machines which assemble proteins in all forms of life, and their production process is known as ribosome biogenesis (RB). In eukaryotes, RB begins in the nucleolus with the transcription of the pre-ribosomal (pre-r)RNA, which undergoes modification, processing, and assembly into mature ribosomes. Malfunctioning RB can cause severe human diseases including rare developmental disorders called ribosomopathies, as well as cancer. Therefore, better understanding of the human RB machinery and its regulation is imperative for pursuing more effective clinical diagnoses and therapies. In this dissertation, I present work which simultaneously advances the field of RB in the areas of methodology, basic science, and clinical molecular pathology. A key cell-based assay pioneered by the Baserga laboratory identifies novel candidate regulators of RB by monitoring for changes in nucleolar number. While robust, the nucleolar number assay does not directly quantify nucleolar production of pre-rRNA. To measure RB more directly, I adapted this pipeline into a high-throughput assay for nucleolar rRNA biogenesis, which simultaneously reports on synthesis and stability of the pre-rRNA. This new method uses 5-ethynyl uridine (5-EU) incorporation to establish direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease. Although screening campaigns have broadly revealed novel protein regulators of human RB, I argue that non-coding (nc)RNAs comprise the next frontier in understanding RB regulation. Small ncRNAs called microRNAs are particularly exciting potential regulators of the RB pathway because they control other complex processes like development and cellular metabolism. Furthermore, a handful of microRNAs are already known to regulate RB. To investigate which other microRNAs may serve as novel nodes of RB control, I conducted a systematic screen of 2,603 human mature microRNA mimics in human MCF10A breast epithelial cells using the nucleolar number assay developed by the Baserga laboratory. I discovered 72 novel microRNA negative regulators of RB, 64 of which decrease nucleolar number in MCF10A cells. Bioinformatic analyses support the conclusion that the novel microRNA hits preferentially target transcripts encoding cell cycle factors or nucleolar proteins. Strikingly, 51/72 microRNA mimics strongly inhibited nucleolar rRNA biogenesis as measured by nucleolar 5-EU incorporation. Rigorous selection and validation of a subset of 15 microRNA mimic hits revealed that these hits starkly impaired global protein synthesis and, unexpectedly, pre-rRNA processing. Consistent with my bioinformatic studies, most of these hits cause upregulation of the cell cycle inhibitor CDKN1A (p21). I also demonstrated that two microRNAs in the MIR-28 family, hsa-miR-28-5p and hsa-miR-708-5p, caused a severe defect in pre-18S rRNA processing by directly targeting the ribosomal protein transcript, RPS28. Additionally, I defined a role for the small oncogenic protein SPRR3 in promoting pre-rRNA transcription. Ultimately, my work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics. Together with an international team of medical researchers, I defined a new molecular basis for the ribosomopathy, alopecia, neurologic defects, and endocrinopathy (ANE) syndrome, which is caused by defects in the conserved ribosome assembly factor RBM28. Our team investigated a female pediatric ANE syndrome patient who presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike previous cases, this patient possessed biallelic splicing variants at the 5’ splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) in RBM28. My in silico analyses and minigene splicing experiments in cells indicated that each splice variant specifically causes skipping of its respective mutant exon. Using a yeast model, I demonstrated that the ΔE5 variant impairs overall growth and large subunit rRNA. In contrast, the ΔE8 variant is completely null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. My results define a new underlying pathology of ANE syndrome, further delineating an emerging class of assembly factor ribosomopathies and underscoring the importance of nucleolar processes in human health. Looking forward, work outlined in this dissertation sets the stage for further inquiry into the role of ncRNA regulators of RB, particularly by using more advanced genetic techniques, and underscores the importance of acquiring additional structural information about maturing human pre-ribosomes.
Recommended Citation
Bryant, Carson J., "Uncovering Novel Biological Regulators of Ribosome Biogenesis" (2023). Yale Graduate School of Arts and Sciences Dissertations. 872.
https://elischolar.library.yale.edu/gsas_dissertations/872
