Investigating the structure and function of a prion-like domain in the Nrd1-Nab3-Sen1 transcription termination system translation missing: zh.hyrax.visibility.files_restricted.text

Loya, Travis (Fall 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/kp78gh20s?locale=zh
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Abstract

From a single genome, yeast must adapt their transcriptional profile to respond to or initiate changes in the cell. A key step in this process is transcriptional termination, which in yeast is carried out by two systems for RNA polymerase II (pol II) transcripts. This work focuses on the Nrd1-Nab3-Sen1 (NNS) complex responsible for regulating several classes of small, non-coding RNAs. The complex consists of two RNA recognition motif (RRM)-containing RNA-binding proteins, Nrd1 and Nab3, and a helicase Sen1. Nrd1 and Nab3 interact with pol II, nascent transcripts, and processing factors to coordinate termination and processing of transcripts. While the RNA recognition motifs and other structured interaction domains have been studied in these proteins, we recently discovered mutations in a previously uncharacterized region of Nab3 that cause termination defects and in some cases death. This thesis describes the analysis of this domain. 

           Interestingly, outside of a small α-helix at the very C-terminus, much of the domain has no predicted structure, as expected from its low complexity sequence. Biochemical characterization of the region revealed it contains a prion-like domain enriched for glutamine and proline that forms amyloid-like fibers in vitro. This finding is consistent with a growing body of research showing that prion-like domains (PrLDs) are overrepresented within RNA-binding proteins and are responsible for mediating homo- and heterotypic protein-protein interactions. From this finding, we explored the ability of heterologous prion-like domains to complement Nab3’s prion-like domain and found subsets that rescue function.                        

           Prion-like domains are also implicated in facilitating formation of various RNA granules in the cell. These granules are thought to be membrane free compartments where machinery involved in RNA metabolism is sequestered to perform its cellular function. Examples include the nucleolus, stress granules, P-bodies, intranuclear quality control/ juxta nuclear quality control (INQ/JUNQ), and insoluble protein deposit (IPOD). In response to glucose starvation, Nab3 and Nrd1 are localized to a novel nuclear granule. This dissertation describes how Nab3’s localization is dependent upon the amyloid forming properties of the prion-like domain, is reversible upon refeeding the yeast, and function in mediating granule assembly. This work deepens our understanding of the regulation and function of prion-like domains in RNA metabolism across eukarya.  

Table of Contents

Table of ContentsChapter 1 Background and significance…………………………………………………….…… 1

1.1 Transcription regulation in Saccharomyces cerevisiae ……………………………………… 11.1.1      

An overview of Nrd1-Nab3-Sen1 (NNS) termination ………………………………….. 31.1.2      

Factors involved in NNS termination …………………………………………………… 4

1.1.3      NNS recruitment to target transcripts …………………………………………………… 8

1.1.4      NNS regulation at sites of transcriptional attenuation ………………………………...… 9

1.2 Prions, Prion-like domains (PrLD), and RNA-binding proteins ………………………….... 10

1.3  RNP Granules, RNA and PrLDs …………………………………………………………... 12

1.4 NNS, PrLDs, and a novel granule ………………………………………………………….. 15

Chapter 2 Amyloid-like Assembly of the Low Complexity Domain of Yeast Nab3 ………..… 17

2.1 Abstract …..……………………………………………………………………………….... 18

2.2 Introduction ..……………………………………………………………………………….. 19

2.3 Materials and Methods …..…………………………………………………………………. 20

2.4 Results …..………………………………………………………………………………….. 25

2.5 Discussion ..…………………………………………………………………………….…... 28

Chapter 3: The hnRNP-like Nab3 termination factor can employ heterologous prion-like domains in place of its own essential low complexity domain ……….………………………... 42

3.1 Abstract …………………………………………………………..……………………….... 43

3.2 Introduction ………………………………………………………..……………………….. 44

3.3 Materials and Methods ……………………………………………..………………………. 46

3.4 Results ………………………………………………………………..…………………….. 50

3.5 Discussion ……………………………………………………………..………………….... 58

Chapter 4: Nab3’s localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain …………………………….………………...…. 81

4.1 Abstract …………………………………………………………………………………..… 82

4.2 Introduction ……………………………………………………………………………..….. 83

4.3 Materials and Methods …………………………………………………………………..…. 86

4.4 Results …………………………………………………………………………………….... 92

4.5 Discussion ……………………………………………………………………………….... 101

Chapter 5 Conclusions ....…………………………………………………………………...… 123

5.1 Discovery of an essential assembly domain in the C-terminus of Nab3 ……………….… 124

5.2 Exploring the interoperability of PrLDs in RNA-binding proteins …………………….… 125

5.3 Nab3’s RRM and PrLD are both required for optimal granule recruitment ……………… 128

5.4 Future directions ……………………………………………………………………….......130

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