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Date of Award

Summer 8-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. These modifications are critical for a variety of cellular processes, including ribosomal biogenesis and splicing of RNAs. Recent studies have suggested an expanded role for snoRNAs beyond ribosomal biogenesis and splicing, including, regulation of chromatin structure, metabolism, and neoplastic transformation. The contribution of snoRNAs to the regulation of normal and malignant hematopoiesis is largely unknown. The lack of a method to accurately and comprehensively assess snoRNA expression has limited research in this area. In particular, array-based methods only interrogate a subset of snoRNAs and cannot distinguish between mature and precursor snoRNAs. To better characterize the role of snoRNAs in the regulation of hematopoiesis, we therefore developed a next-generation sequencing technique and bioinformatic approaches to quantify snoRNAs accurately. We used our snoRNA sequencing pipeline to characterize snoRNA expression in acute myeloid leukemia (AML). We show that snoRNAs are regulated in a lineage- and development-specific fashion in normal hematopoiesis. Surprisingly, RNA splicing did not appear to be a major determinant of expression for most snoRNAs. Expression of most snoRNAs in AML cells was similar to that observed in normal CD34+ cells. In contrast to a prior study, no increased in C/D box snoRNAs was observed in core binding factor AML. On the other hand, certain snoRNAs appear to be dysregulated in specific genetic subtypes of AML. In particular, snoRNAs in the imprinted DLK1-DIO3 locus are markedly overexpressed in acute promyelocytic leukemia. We also observed reduced expression of SNORA21 in several genetic subtypes of AML, most notably AML carrying spliceosome gene mutations. SNORA21 mediates pseudouridylation of key nucleotides in the peptidyl transfer center region of rRNA. To better understand the impact of reduced SNORA21 expression in AML, we generated SNORA21 null K562 cells using CRISPR gene editing. Loss of SNORA21 in K562 cells is associated with impaired ribosome biogenesis and reduced global translation. However, ribosome profiling/RNASeq suggests that only a few genes show significant changes in translational efficiency. Loss of SNORA21 in K562 cells results in reduced cellular proliferation due to an increase in non-apoptotic cell death. Electron microscopy and mitochondrial assays of SNORA21-/- K562 cells suggest that reduced mitochondrial function may contribute to the increase in cell death. Collectively these data provide compelling evidence for snoRNA involvement in leukemogenesis and highlights the importance of ribosomal biogenesis as a basis to better understand leukemogenesis.

Language

English (en)

Chair and Committee

Daniel C. Link

Committee Members

Timothy Ley, Matthew Walter, David Spencer, Hani Zaher,

Available for download on Wednesday, November 04, 2020

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