Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Tuschl Laboratory

Keywords

posttranscriptional regulation, RNA-binding proteins, ribonucleoprotein complexes, 28S rRNA maturation, NEF-sp exonuclease, tissue-specific expression

Abstract

Posttranscriptional gene regulation (PTGR) concerns all processes acting directly upon coding and non-coding RNAs, regulating and executing their maturation, ribonucleoprotein assembly, transport, stability and translation. RNA-binding proteins (RBPs) and ribonucleoprotein (RNP) complexes coordinate these processes. RBPs are central to cellular metabolism and their role in human diseases has been widely studied. Recent large-scale quantitative methods such as nextgeneration sequencing and modern protein mass spectrometry enabled new approaches to dissect PTGR networks and renewed interest in investigations of factors involved in PTGR at a genomewide level. A census of all coding and noncoding RBPs has previously not been readily available and the number of RBPs was estimated based on few selected protein classes. However, for system-wide analyses of PTGR a comprehensive account of the RBPs is necessary. To address this need, I developed a census of 1,542 manually curated RBPs and categorized their interactions with different classes of RNA, defined the number of factors in different regulatory pathways, and investigated their evolutionary patterns, abundance, and tissue-specific expression. Coregulated gene expression during developmental processes often gives novel insights into regulatory pathways and components. Furthermore, I showed that by classifying RBPs into their main regulatory RNA pathways we can start to understand the disease phenotypes of proteins involved in the same RNA metabolic pathways. These insights are useful for dissecting dysregulated PTGR pathways in human diseases and finding new therapeutic targets. Finally, I showed in this chapter that by careful domain analysis, novel RBPs can be predicted and characterized the previously unknown RG/GG-rich RBP FAM98A. Overall, this analysis provides a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism. In the second part of the thesis I focused on the uncharacterized, highly tissue-specific RNA exonuclease NEF-sp and characterized its function in pre-28S ribosomal RNA processing. Ribosomal RNA biogenesis requires a series of endo- and exonucleolytic processing steps for the production of mature rRNAs. Although the mechanism of 28S 3’ end rRNA maturation remains largely unknown in higher eukaryotes, it is thought that the 3’ external transcribed spacer (3’ETS) of the large 47S rRNA precursor, containing 18S, 5.8S, and 28S rRNA, is removed in a precise endonucleolytic cleavage reaction, guided by U8 snoRNA. Here I show instead that the 3’ETS is exonucleolytically trimmed by the DEDDh RNA exonuclease NEF-sp in Drosophila melanogaster. I characterize for the first time in higher eukaryotes a nuclease that is involved in the removal of the 3’ ETS. Interestingly, NEF-sp shows high tissue-specific expression in gonads. Gonad development is arrested in dNEF-sp mutants. Our results demonstrate that exonucleolytic trimming is essential for 28S rRNA maturation in higher eukaryotes and, counterintuitively, the expression of a factor involved in a core RNA metabolic process can be highly regulated. Our findings suggest an additional level of posttranscriptional gene regulation in the maturation of 28S rRNA, mediated by the regulated expression of RNA exonucleases.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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