Mitochondrial deficiencies influence variable penetrance of nuclear Nab3 granule accumulation Público

Abstract

Yeast regularly withstand changes in their environment and they must adapt their genome in order to survive. From their genome, yeast generate different types of RNA which they use to regulate protein expression. A critical step in generating these RNAs is transcription termination, which is performed through two different mechanisms in yeast depending on the type of RNA transcribed. For protein coding messenger RNA, yeast employ transcription termination through a set of factors brought in by the cleavage/ polyadenylation complex. In the case of non-coding RNAs such as small nuclear RNA, small nucleolar RNA and cryptic unstable transcripts, transcription termination is performed by the Nrd1-Nab3-Sen1 transcription termination complex (NNS), which this thesis focuses on. The choice of transcription termination mechanism is a highly specific and regulated process. RNA polymerase II, which is the enzyme that transcribes the DNA into RNA, contains a C-terminal domain (CTD) on its largest subunit which different termination complex components interact with. Early in transcription, the CTD is heavily phosphorylated at serine 5 of a seven amino acid repeat. This in turn allows Nrd1 to preferentially bind to activate transcription termination. As Pol II progresses down the template, the phosphorylation is removed from serine 5 and serine 2 becomes the predominant form of phosphorylation. This event signals for the binding of cleavage/ polyadenylation factors to deploy polyadenylation-coupled transcription termination.

This thesis focuses primarily on one of the three NNS subunits, nuclear polyadenylated RNA-binding protein 3 (Nab3). Nab3 is a hnRNP-like protein that contains an aspartate/glutamate rich near its N-terminus, an RNA recognition motif and a proline/glutamine rich, prion-like domain at its C-terminus. The C-terminus is essential for viability, for proper transcription termination, for the ability to form amyloid filaments in vitro and to assemble a membraneless subcellular compartment in vivo known as the Nrd1-Nab3 granule (NNG). The work in this thesis establishes that the assembly and accumulation of the Nrd1-Nab3 granule displays variable penetrance across various yeast strains. Once this variation was discovered and established, this work aimed to explain the cause for variation.

Interestingly, this thesis reports that mitochondrial function and respiratory capacity are key contributors to the variance seen for Nrd1-Nab3 granule accumulation. The data herein demonstrate that NNG accumulation is sensitive to oxidative phosphorylation under carbon restricted conditions and can be reduced by the addition of extracellular ATP, suggesting an ATP-dependent mode of disassembly. Additionally, a serine-arginine protein kinase implicated in the disassembly of other subcellular compartments is shown to be important for Nrd1-Nab3 granule disassembly through mitochondrial function. This work deepens our understanding of the cause for Nrd1-Nab3 granule accumulation variation as well as our understanding of key factors in granule biogenesis.

Table of Contents

1.0 Transcription termination and RNA-binding proteins…………………………………... 1

1.1   An introduction to eukaryotic transcription……………………………………………… 1

1.1.1  Classes of RNA transcripts…………………………………………………………. 2

1.1.2  Transcription termination in Saccharomyces cerevisiae…………………………… 3

1.1.3  Messenger RNA transcription termination in Saccharomyces cerevisiae……………………………………………………………………………. 4

1.1.4  Non-coding transcription termination in Saccharomyces cerevisiae……………………………………………………………………………. 5

1.2   Components of the Nrd1-Nab3-Sen1 (NNS) transcription termination complex…………………………………………………………………………………...7

1.2.1  Nrd1: a hnRNP-like protein…………………………………………………………7

1.2.2  Nab3: another hnRNP-like protein………………………………………………….8

1.2.3  Sen1: a helicase protein…………………………………………………………......9

1.3   An introduction to RNA-binding proteins………………………………………...…….10

1.3.1  Domains found in RNA-binding proteins…………………………...…………….10

1.3.2  Biological roles of RNA-binding proteins…………………………...……………11

1.4   An introduction to prions & prion-like proteins…………………………………...……11

1.4.1 Yeast prions and prion-like proteins……………………………………...………...12

1.4.2 Yeast as a model for human neurodegenerative diseases…………………………..14

1.5   Nab3: a prion-like protein………………………………………………………….……15

1.6   Glucose deprivation…………………………………………………………..…………16

1.6.1  Transcriptome and proteome changes in response to glucose deprivation……..…17

1.6.2  Nrd1-Nab3 transcript binding changes in response to glucose deprivation……….19

1.7   Research focus…………………………………………………………………………..20

1.8   Figures………………………………………………………………………………..…22

 

2.0 Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis……………………………………………...……25

2.1   Abstract……………………………………………………………………………….…26

2.2   Introduction…………………………………………………………………………...…27

2.3   Materials and methods………………………………………………………………..…30

2.4   Results…………………………………………………………………………...………34

2.4.1 Rationale……………………………………………………………………………35

2.4.2 Quantification of the dramatic rearrangement of GFP-Nab3 in yeast nuclei………36

2.4.3  The computational tool enables a detailed analysis of fluorescent protein distribution………………………………………………………………………….37

2.4.4  Measurement of strain-to-strain differences in GFP-Nab3 granule accumulation……………………………………………………………..…………39

2.4.5  The granule accumulation phenotype correlates with growth and respiration defects………………………………………………………………………………40

2.4.6 Quantification of filament formation by IMP dehydrogenase in cultured human cells…………………………………………………………………………………41

2.5   Discussion………………………………………………………………………….……43

2.6   Acknowledgements………………………………………………………………...……45

2.7   Figures & tables…………………………………………………………………………46

3.0 Nab3 nuclear granule accumulation is driven by respiratory capacity…………………62

3.1   Abstract……………………………………………………….…………………………63

3.2   Introduction…………………………………………………………...…………………64

3.3   Materials and methods………………………………………………..…………………67

3.4   Results……………………………………………………………………………...……70

3.4.1  Mitochondrial manipulations in a low granule accumulating yeast strain increase granule accumulation………………………………………………………….……70

3.4.2  Enrichment for a respiratory competent cell population results in a reduction of Nab3 granule accumulation…………………………………………………...……72

3.4.3  Nab3 granule accumulation is lost by the addition of extracellular ATP…….……73

3.4.4  SKY1 knockout results in mitochondrial defects and increased Nab3 granule accumulation………………………………………………………………..………73

3.5   Discussion………………………………………………………………………….……74

3.6   Acknowledgements………………………………………………………………...……77

3.7   Figures & tables…………………………………………………………………………78

4.0 Discussion and future directions……………………………………………………..……89

4.1   Summary……………………………………………………………………………...…89

4.2   The importance of a computational tool to quantify Nab3 granule accumulation in various yeast strains………...………………………………………………………...…90

4.3   The importance of mitochondrial function on Nab3 granule accumulation………….…90

4.4   Future directions for this research project…………………………………...……….…92

4.4.1  Breaking down the composition of the Nab3 granule………………………..……92

4.4.2  Identifying key players in assembly and disassembly of the Nab3 granule…….…96

4.4.3  Discovering other factors that influence Nab3 granule accumulation…….………98

4.5   Implications of mitochondrial function in neurodegenerative diseases…………...…..100

4.6   Final remarks………………………………………………………………..…………101

4.7   Figures…………………………………………………………………………………103

5.0 References……………………………………………………………………………..……105

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Cell
Palavra-chave	Granule Nab3 Mitochondria Yeast
Committee Chair / Thesis Advisor	Reines, Daniel, Emory University
Committee Members	Bassell, Gary, Emory University Ghalei, Homa, Emory University Conn, Graeme, Emory University Seyfried, Nicholas, Emory University

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