Recognition of Polyadenosine RNA Open Access

Kelly, Seth Michael (2009)

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Throughout their lifecycles, mRNA transcripts are coated by a collection of RNA binding proteins. These RNA binding proteins function in a diverse set of processes, but collectively they dictate the fate of the transcripts to which they bind. While some RNA binding proteins recognize sequences found in only a handful of transcripts, others, such as those proteins that bind the poly(A) tail of mRNA transcripts, recognize sequences ubiquitous to all mRNA transcripts. These RNA binding protein binds post-transcriptionally regulate gene expression. Therefore, in order to completely comprehend the regulation of gene expression, it is essential to understand the molecular mechanisms of RNA recognition by RNA binding proteins.

In this dissertation I present data demonstrating that Cys-Cys-Cys-His (CCCH) zinc fingers of the S. cerevisiae protein, Nuclear poly (A) Binding protein 2 (Nab2) and its putative human orthologue, ZC3H14, specifically recognize polyadenosine RNA with high affinity. All previously characterized poly(A) RNA binding proteins bind polyadenosine RNA via at least one RNA Recognition Motif. Hence, recognition of polyadenosine RNA via CCCH zinc fingers is a novel mechanism of poly(A) RNA recognition. Genetic and biochemical studies provide compelling evidence that ZnF 5-7 mediate high affinity binding to polyadenosine RNA. To gain further understanding of the mechanism of poly(A) RNA recognition by CCCH zinc fingers, the atomic resolution structure of Nab2 zinc fingers 5 - 7 was solved using NMR. Using this structural data, we identified several conserved positively charged and aromatic residues that could potentially interact with polyadenosine RNA. Changing these amino acids to alanine resulted in loss of binding to polyadenosine RNA in vitro and conferred cold-sensitive growth defects in vivo. We have also identified several genes that genetically interact with NAB2 including components of the mRNA 3'-end processing machinery, a component of the nuclear exosome, and components necessary for the transcriptional termination of the RNA polymerase II. Together, these findings define a novel evolutionarily conserved family of polyadenosine RNA binding proteins. These findings also further demonstrate that polyadenosine RNA binding proteins are key players involved in the post-transcriptional regulation of gene expression.

Table of Contents

Table of Contents

Chapter 1: A general Introduction 1

Introduction 2

The evolutionary conservation of mRNA processing and export factors 3

The budding yeast Saccharomyces cerevisiae as a model system 7

The mRNA "assembly line" 8

Coupling transcription to mRNA export from the nucleus 11

Coupling 3'-end formation and mRNA export 18

A link between 3'-end formation and nuclear export: Poly(A)

RNA binding proteins 20

Coupling splicing and mRNA export from the nucleus 21

Translocation through the nuclear pore complex 22

A molecular wardrobe change completes nuclear export 23

The exosome contains both quality control and processing

functions 26

Molecular recognition of RNA via multiple conserved domains 27

The relevance of RNA binding proteins to disease 31

A brief summary of information known about Nab2 prior to

this dissertation 33

Scope and significance of the dissertation 34

Chapter 2: Recognition of polyadenosine RNA by a conserved

family of zinc finger proteins 38

Introduction 39 Results 41 Discussion 63

Experimental procedures 65

Chapter 3: Poly(A) RNA binding by Nab2 is required for correct

3'-end formation 76

Introduction 77 Results 80 Discussion 112

Experimental procedures 119

Chapter 4: Nab2 genetically interacts with RNA processing components 130

Introduction 131 Results 137 Discussion 154

Experimental Procedures 156

Chapter 5: Conclusion and Discussion 163

A brief review 164

The specificity of CCCH zinc fingers 166

The implications of coupling between mRNA 3'-end

processing and nuclear export 169

Distinguishing poly(A) tails from one another 170

A putative cytoplasmic function of Nab2 172

Final conclusions and future directions 174

References 175

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