Structural And Molecular Studies Of Nucleic Acid Chaperones

168 Pages·2012·5.71 MB·English

STRUCTURAL AND MOLECULAR STUDIES OF NUCLEIC ACID CHAPERONES

by

LEI DING

DAVID E. GRAVES, COMMITTEE CHAIR
CHING-YI CHEN
BINGDONG SHA
TIM M. TOWNES
DMITRY VASSYLYEV

A DISSERTATION

Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy

BIRMINGHAM, ALABAMA

2012
Copyright by
Lei Ding
2012
STRUCTURAL AND MOLECULAR STUDIES OF NUCLEIC ACID CHAPERONES

LEI DING

BIOCHEMISTRY AND MOLECULAR GENETICS

ABSTRACT

Nucleic acids are the storage of genetic information that needs to be expressed
and inherited. As abundant cellular macromolecules they are also major players of a wide
variety of biological processes. They usually undergo post-transcriptional modifications
and/or fold into higher order structures in order to accomplish their distinct functions.
Most biological events involving nucleic acids are achieved by proteins that
interact with them. The functionality requires specific recognition of nucleic acids by
proteins at molecular level. Nucleic acid chaperones are a group of nucleic acids binding
proteins that associate with structured RNAs or DNAs and assist correct folding of their
biological relevant conformations.
Recent studies have shown that protein hnRNP A1 regulates the processing of the
primary transcript of microRNA-18a. We demonstrated that hnRNP A1 recognizes the
pri-miR18a through binding to its terminal loop which contains binding motifs
recognized by two RRM domains of hnRNP A1 (UP1). We solved the crystal structure of
UP1 in the complex with a single stranded RNA sequence derived from the terminal loop
of pri-miR-18a, revealed the molecular basis for hnRNPA1-RNA recognition. Our result
from mutagenesis and in vitro binding studies suggest chaperone activity of hnRNP A1 in
the processing of pri-miRNA.
Human telomeric DNA can form higher order structures called G-quadruplex
which may be involved in telomere regulation. hnRNP A1 and its N-terminal region UP1
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have been previously found bind and unfold this structure efficiently. Mutagenesis
studies on the two conserved RRMs of UP1 demonstrated the distinct functions of RRM1
and RRM2; RRM1 is the key domain for initiating binding and unfolding of G-
quadruplex structural motif; RRM2 contributes to the synergistic efficacy with RRM1 in
the binding and unfolding process. We also identified the loop2 on the hTel22 G-
quadruplex DNA as critical site for initiating recognition and unfolding by UP1. A model
is proposed for how UP1 binds and unfolds telomeric G-quadruplex.
Many small RNAs, including small-interfering RNAs and piRNAs in Drosophila,
are methylated at 3' ends. The 3’ end 2’-O-methylated RNA is preferably recognized by
the PAZ domain of the Ago2. Here we present the crystal structure of the Drosophila
Ago2 PAZ domain bound with a 3’ end 2’-O-methylated RNA, providing a structural
basis for the specific recognition of the methyl modification. In vitro binding and
mutagenesis studies illustrate that 3’ end methylated small RNAs are preferably bound by
PAZ domains from Drosophila Ago2 and Piwi subfamily Argonaute proteins. We also
characterized RNA chaperone activity of the human Ago1 PAZ domain, implying roles
of PAZ domains in small RNA-target recognition.

Keywords: siRNA, piRNA, PAZ domain, PIWI domain, Argonaute (Ago), 3’ methylated
RNA, hnRNP A1, Unfolding Protein 1 (UP1), RRM, miR-18a, Terminal loop, Isothermal
Titration Calorimetry (ITC), Circular Dichroism (CD), Thermodynamics, Enthalpy, G-
quadruplex DNA, X-ray Crystallography, Mutagenesis
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DEDICATION

This work is dedicated to my parents, Zhiqiang Ding and Guiyun Zeng. My
achievements are a result of their patience, encouragement and unconditional support.
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ACKNOWLEGEMENTS

First of all, I would like to express my deepest gratitude to my advisor and
committee chair, Dr. David E. Graves for his guidance, patience, enthusiasm, and
providing me a great freedom of doing research that I was interested. The professional
and thorough reviewing of my manuscripts of his very busy schedule made important
suggestions and revisions which greatly enhanced this thesis.

I would also like to sincerely thank my previous advisor, Dr. Jin-biao Ma, who
recruited me to UAB and started my interest in the structural biology of protein-nucleic
acid interaction. The training under his guidance during the first four years of my
graduate study helped me to establish my experimental skills and his great vision has
guided and inspired my critical thinking in science.

My sincere appreciations also go to Dr. Ying Huang, for all the great time that we
were working together in Dr. Ma’s lab and the nice conversations in both science and life
topics we share. Working with Dr. Huang was like a breeze. Her great scientific
knowledge and laboratory skills helped me learnt a lot and solved numerous problems
during my study. Her enthusiasm attitude and fantastic management toward both
scientific and personal life also inspired me greatly and will continually benefit me in the
future.

I am also grateful to all the members of my graduate committee, Dr. Ching-Yi
Chen, Dr. Bingdong Sha, Dr. Tim M. Townes and Dr. Dmitry Vassylyev for taking the
time to serve on my committee and reviewing the research that I have worked with
during my graduate study.

A part of this work was in collaboration with Dr. Jason Hudson when he was a
graduate student in Dr. Graves’s lab. It was a great experience to work with him. In
addition, Vu Le and Dr. Edwin Lewis, at Mississippi State University helped me in the
data fitting and analyzing. I could not have done without them.

I thank all fellow labmates that I have been worked with in both the Dr. Ma’s
group and Dr. Graves’s group, especially Dr. Zhenhua Tian and Kate Hayden, for all the
inspiring time during the numerous discussions and brain storms with me.

Lastly, I have to thank my parents, it is impossible for me to finish all these work
without their patience and support in my life. I must thank my wife, Rongbing Xie, for
her understanding and support, her patience and endurance to taking care of our home
and kids when I have to work overtime.

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TABLE OF CONTENTS

Page

ABSTRACT ....................................................................................................................... iii

DEDICATION .....................................................................................................................v

ACKNOWLEDGMENTS ................................................................................................. vi

LIST OF TABLES ............................................................................................................. ix

LIST OF FIGURES .............................................................................................................x

CHAPTER

I. INTRODUCTION ....................................................................................................14

Nucleic Acids (DNA and RNA) ............................................................................16
RNA and its Folded Structures ..............................................................................17
G-quadruplex DNAs and their Topologies ............................................................18
The Nucleic Acid Chaperone Proteins ...................................................................21
Conserved Nucleic Acid Binding Domains in Chaperone Proteins ......................23
Biogenesis of Small RNAs ....................................................................................23
The miRNA pathway .................................................................................25
The siRNA pathway ...................................................................................25
The piRNA pathway ..................................................................................26
The Processing of Pri-miRNA ...............................................................................28
hnRNP A1 and UP1 ...............................................................................................28
hnRNP A1 as RNA Chaperon in Pri-miRNA Processing .....................................31
Argonaute Protein Family ......................................................................................31
PAZ Domain and the 3’ End RNA Recognition ....................................................32
Telomere and Telomere Length Maintenance .......................................................33
G-quadruplex Structure Formed by Human Telomeric DNA ...............................35
G-quadruplex Structure in the Regulation of Telomere Length ............................36
hnRNP A1/ UP1 Unfolds G-quadruplex structure motif .......................................37
Overview of Dissertation .......................................................................................37

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II. STRUCTURAL BASIS FOR THE RECOGNITION OF PRI-MIR-18A BY
HNRNP A1 ..............................................................................................................39

III. THE BINDING AND UNFOLDING OF THE G-QUADRUPLEX
STRUCTURAL MOTIF FORMED BY THE HUMAN TELOMERIC
SEQUENCE BY UP1 ............................................................................................85

IV. STRUCTURAL INSIGHTS INTO DISTINCT FUNCTIONS OF PAZ DOMAINS
BOUND TO 3’-TERMINAL 2’-O-METHYLATED SMALL RNAS .................123

V. CONCLUSIONS ...................................................................................................159

LIST OF REFERENCES .................................................................................................162

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LIST OF TABLES

Tables Page
INTRODUCTION
1 Small regulatory RNAs in animals ........................................................................24

STRUCTURAL BASIS FOR THE RECOGNITION OF PRI-MIR-18A BY HNRNP A1

1 Data collection and refinement statistics ...............................................................68

2 Summary of dissociation constants for UP1 mutants and pri-miR-18a Tloop RNA
binding measured by ITC.......................................................................................69

STRUCTURAL INSIGHTS INTO DISTINCT FUNCTIONS OF PAZ DOMAINS
BOUND TO 3’-TERMINAL 2’-O-METHYLATED SMALL RNAS

1 Crystallographic statistics ...................................................................................144
S1 The summary of isoelectric points and charges at pH 7 of selective PAZ domains
from Ago and Piwi subfamilies. ..........................................................................158

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LIST OF FIGURES

Figure Page

INTRODUCTION

1 Structural comparison between DNA and RNA nucleotide (Adenine) .................16

2 An example of RNA secondary structure of pre-miR-18a ....................................18

3 Schematic representation of G-quadruplex DNA structure. ..................................19
4 Strand orientations of different types of G-quadruplex structure motifs ...............20
5 Schematic representations of different loop arrangements in G-quadruplex
structures ................................................................................................................21
6 Biogenesis of small RNAs in Drosophila melanogaster. .......................................27
7 Schematic diagrams of conserved domains in hnRNP A1 and UP1 .....................29
8 X-ray crystal structure of UP1 complex with 12-nucleotide human telomeric DNA
“TTAGGGTTAGGG” ...........................................................................................30
9 Schematic representations of conserved domains of Argonaute proteins in
eukaryotes. .............................................................................................................32
10 X-ray crystal structure of human Ago1 PAZ domain complex with a short single
stranded RNA sequence 5’-CGUGACUCU-3’ .....................................................33
11 End of replication problem of DNA replication. ...................................................35
12 Solved structure of 22-mer human telomeric G-quadruplex DNA hTel22. ..........36

x