Generation of Export-Competent mRNPs Öffentlichkeit
Soucek, Sharon (2014)
Published
Abstract
Gene expression is regulated to produce a protein expression profile that dictates the function of each cell. Messenger RNA (mRNA) serves as an intermediate between the genome and translation of proteins. Mature mRNA requires processing events including the addition of a 5'-cap, removal of introns, and 3'-end processing. These maturation steps are mediated by RNA binding proteins that associate with the mRNA throughout its post-transcriptional journey. Given that mRNA biogenesis is essential for proper gene expression, mutations within RNA binding proteins impair mRNA maturation and consequently lead to numerous human diseases. Of relevance to this work, mutations in the zinc finger RNA binding protein gene, ZC3H14, lead to a non-syndromic form of autosomal intellectual disability. The goal of this study is to understand how RNA binding proteins ensure proper processing of pre-mRNA to give rise to export-competent mRNA/protein complexes (mRNPs) within the nucleus. To address this goal, we focused our efforts on the Nuclear Poly(A) RNA Binding protein, Nab2, which is the putative budding yeast orthologue of ZC3H14. As a first step to define how RNA binding proteins regulate target mRNAs, we used a structure-function approach to reveal how Nab2 binds to polyadenosine RNA. We describe the structure of Nab2 zinc fingers 5-7, which are necessary and sufficient for high affinity polyadenosine RNA binding, and identify key residues involved. Structure-guided variants show that Nab2 modulates poly(A) tail length and functions in the generation of export-competent mRNPs. We extend this analysis to understand how Nab coordinates splicing, 3'-end processing, and mRNA decay. We identified several genes that genetically interact with NAB2 including other poly(A) RNA binding proteins, components of the splicing machinery, and the nuclear exosome. To add to our understanding of how RNA binding proteins modulate specific classes of RNA, we provide an initial characterization of the Mmi1 protein, which is essential for the regulation of meiotic transcripts. These findings contribute to an understanding of how multiple steps in nuclear mRNA processing are coordinated and lay the groundwork for probing key functions of ZC3H14 in the brain that lead to intellectual disability.
Table of Contents
Chapter 1:Introduction 1
Regulation of gene expression 2
Post-transcriptional processing 4
mRNA biogenesis is coupled by RNA binding proteins 13
Consequences of coupling RNA processing events: transcript specificity 19
Proteins implicated in multiple steps of post-transcriptional regulation may couple RNA processing and surveillance 21
Nuclear poly(A) RNA binding protein, Nab2, is a zinc finger RNA binding protein that is implicated in multiple steps of mRNA biogenesis 22
Nab2 and the regulation of poly(A) tail length 25
RNA binding proteins and disease 27
Scope and significance of dissertation 28
Figures 33
Chapter 2:Structural and Functional Basis for Polyadenosine Binding by Nab2 41
Introduction 42
Results 44
Discussion 53
Figures 57
Experimental Procedures 70
Chapter 3:Nab2 Plays a Role in mRNA Splicing and Surveillance 77
Introduction 78
Results 82
Discussion 97
Figures 104
Experimental Procedures 124
Chapter 4:The Interplay between RNA Binding Proteins and mRNA Processing 129
Introduction 130
Results 134
Discussion 148
Figures 156
Experimental Procedures 167
Chapter 5: Characterization of the RNA Binding Protein, Mmi1: Regulation of Meiotic Transcripts 172
Introduction 173
Results 176
Discussion 178
Figures 181
Experimental Procedures 183
Chapter 6: Discussion 185
A model for Nab2 at the crux of mRNP assembly 187
Molecular recognition of polyadenosine RNA 188
Coupling splicing, 3'-end processing, export, and RNA surveillance 192
The implications of coupling RNA processing events: control of poly(A) tail length 199
Future directions: implications for ZC3H14 in human disease 202
Final conclusions 206
Figures 207
References 209
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