Post-transcriptional gene regulation in Drosophila [Elektronische Ressource] : an investigation into the roles of RNA silencing and the DEAD-box helicase Belle / presented by Pavel Natalin

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Post-transcriptional gene regulation inDrosophila: an investigation into the roles ofRNA silencing and the DEAD-box helicaseBellePavel NatalinNovember 2008Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural SciencesPresented byDiploma biochemist Pavel Natalinborn in Khabarovsk, Russia.Oral-examination:Post-transcriptional gene regulation in Drosophila: an investigation into theroles of RNA silencing and the DEAD-box helicase BelleReferees:Prof. Dr. Matthias Hentze, EMBL, Heidelberg, Germany;Prof. Dr. Christine Clayton, ZMBH,erg,y.ContentsAcknowledgements ixSummary xiZusammenfassung xiiiAbbreviations xvI Introduction 11 Translational control in eukaryotes:cis-regulatory sequences and trans-acting factors. . . . . . . . 21.1 Origins of translational control . . . . . . . . . . . . . 21.2 General principles of translational control . . . . . . . 41.3 Translation initiation in eukaryotes . . . . . . . . . . . 51.4 Global mechanisms of control . . . . . . . 81.4.1 Phosphorylation of eIF2 . . . . . . . . . . . . 81.4.2 Phosphorylation of other translation factors . 91.5 Principles of selective translational control . . . . . . . 121.5.1 RNA operon . . . . . . . . . . . . . . . . . . 141.6 Selective control: mechanisms that afiect initiation oftranslation . . . . . . . . . . . . . . . . . . . . . . . . . 141.6.

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Post-transcriptional gene regulation in
Drosophila: an investigation into the roles of
RNA silencing and the DEAD-box helicase
Belle
Pavel Natalin
November 2008Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
Presented by
Diploma biochemist Pavel Natalin
born in Khabarovsk, Russia.
Oral-examination:Post-transcriptional gene regulation in Drosophila: an investigation into the
roles of RNA silencing and the DEAD-box helicase Belle
Referees:
Prof. Dr. Matthias Hentze, EMBL, Heidelberg, Germany;
Prof. Dr. Christine Clayton, ZMBH,erg,y.Contents
Acknowledgements ix
Summary xi
Zusammenfassung xiii
Abbreviations xv
I Introduction 1
1 Translational control in eukaryotes:
cis-regulatory sequences and trans-acting factors. . . . . . . . 2
1.1 Origins of translational control . . . . . . . . . . . . . 2
1.2 General principles of translational control . . . . . . . 4
1.3 Translation initiation in eukaryotes . . . . . . . . . . . 5
1.4 Global mechanisms of control . . . . . . . 8
1.4.1 Phosphorylation of eIF2 . . . . . . . . . . . . 8
1.4.2 Phosphorylation of other translation factors . 9
1.5 Principles of selective translational control . . . . . . . 12
1.5.1 RNA operon . . . . . . . . . . . . . . . . . . 14
1.6 Selective control: mechanisms that afiect initiation of
translation . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.6.1 Upstream AUGs and leaky scanning . . . . . . 16
1.6.2 ORFs and reinitiation . . . . . . . 16
1.6.3 Internal translation initiation: IRESs . . . . . 20
1.6.4 Translation initiation via PARS . . . . . . . . 23
01.6.5 Proteins targeted to the 5 UTR . . . . . . . 24
Iron-response proteins. . . . . . . . . . . . . . . 24
01.6.6 Proteins targeted to the 3 UTR . . . . . . . 25
CPEB and Maskin . . . . . . . . . . . . . . . . 25
Bicoid . . . . . . . . . . . . . . . . . . . . . . . 27
iContents ii
hnRNP K and E1 . . . . . . . . . . . . . . . . . 28
1.7 Selective control: complex mechanisms . . . . . . . . . 28
Bruno and regulation of oskar translation . . . . 29
Smaug and nanos translation . . . . . . . . . . 32
SXL and msl-2 . . . . . . . . . . . . 34
PUF domain proteins . . . . . . . . . . . . . . . 36
Fragile X mental retardation protein. . . . . . . 38
1.8 Selective control: regulation by miRNAs . . . . . . . . 40
1.8.1 miRNA and miRISC biogenesis . . . . . . . . 40
1.8.2 miRNA-mediated control of mRNA transla-
tion and decay . . . . . . . . . . . . . . . . . 43
mRNA decay . . . . . . . . . . . . . . . . . . . 43
Controversy over modes of translational control 44
II Results 48
2 Genome-wide analysis of mRNAs regulated by Drosha and
Argonaute proteins in Drosophila . . . . . . . . . . . . . . . . 49
2.1 Identiflcation of transcripts regulated by RNA
silencing pathways . . . . . . . . . . . . . . . . . . . . 50
2.2 DepletionofDroshaandAGO1leadstosimilarexpres-
sion proflles . . . . . . . . . . . . . . . . . . . . . . . . 52
2.3 Predicted miRNA targets are signiflcantly enriched
among up-regulated transcripts . . . . . . . . . . . . . 53
2.4 Identiflcation of a core set of transcripts regulated by
the miRNA pathway . . . . . . . . . . . . . . . . . . . 54
2.5 Core transcripts represent authentic miRNA targets . . 57
2.6 AGO2 associates with miRNAs . . . . . . . . . . . . . 59
2.7 A few transcripts are regulated exclusively in the indi-
vidual knockdowns . . . . . . . . . . . . . . . . . . . . 60
3 AroleforDrosophila RNA-helicaseBelleintranslationalcontrol 65
3.1 Belle is a cytoplasmic protein that does not localize to
P bodies . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2 Belle is required for cell viability . . . . . . . . . . . . 65
3.3 Protein synthesis is inhibited in cells depleted of Belle . 67
3.4 Belle represses translation of bound mRNA . . . . . . 69
3.5 Tethering of Belle induces the formation of heavy
mRNPs which are difierent from polysomes. . . . . . . 71
3.6 Tandema–nitypuriflcationofproteinsassociatedwith
Belle in Drosophila S2 cells . . . . . . . . . . . . . . . 74Contents iii
3.7 Characterization of proteins associated with Belle in
Drosophila S2 cells using Gene Ontology . . . . . . . . 74
3.8 Functional enrichment analysis . . . . . . . . . . . . . 81
3.9 Bioinformatic analysis of interactions of proteins asso-
ciated with Belle . . . . . . . . . . . . . . . . . . . . . 86
3.10 Belle interacts with translation factors and RBPs . . . 89
3.11 Belle associates with mRNAs that are regulated at the
level of translation . . . . . . . . . . . . . . . . . . . . 96
III Discussion 115
4 Genome-wide analysis of mRNAs regulated by Drosha and
Argonaute proteins
in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.1 Transcripts regulated by Drosha and Argonaute proteins116
4.2 Crosstalk between AGO1 and AGO2 . . . . . . . . . . 116
4.3 miRNAs afiect mRNA expression levels . . . . . . . . . 119
4.4 Drosha regulate mRNAs independently
of Argonaute proteins . . . . . . . . . . . . . . . . . . 121
5 A role for RNA-helicase Belle in translational control . . . . . 122
5.1 The subcellular localization of Belle . . . . . . . . . . . 123
5.2 Belle is required for cell viability and general
translation e–ciency . . . . . . . . . . . . . . . . . . . 124
5.3 Belle functions as a translational repressor . . . . . . . 126
5.4 TheassociationofBellewithvariousproteincomplexes
re ects its functional diversity . . . . . . . . . . . . . . 127
5.5 Concluding remarks. . . . . . . . . . . . . . . . . . . . 133
IV Materials and methods 135
6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
6.1 Chemicals and reagents. . . . . . . . . . . . . . . . . . 136
6.2 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 136
6.3 DNA oligonucleotides . . . . . . . . . . . . . . . . . . . 137
6.4 Membranes and fllter paper . . . . . . . . . . . . . . . 137
6.5 Chromatography resins and columns . . . . . . . . . . 137
6.6 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 138
6.7 DNA oligonucleotide microarrays . . . . . . . . . . . . 138
6.8 Bacterial strains. . . . . . . . . . . . . . . . . . . . . . 138
7 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.1 DNA cloning . . . . . . . . . . . . . . . . . . . . . . . 139Contents iv
7.1.1 Generation of cDNA library . . . . . . . . . . 139
7.1.2 Ampliflcation of DNA by PCR . . . . . . . . 139
7.1.3 DNA agarose gel electrophoresis. . . . . . . . 141
7.1.4 Puriflcation of DNA fragments . . . . . . . . 141
7.1.5 Restriction endonuclease digest of DNA . . . 141
7.1.6 Ligation of DNA fragments . . . . . . . . . . 142
7.1.7 Preparation of competent E. coli cells . . . . 142
7.1.8 Transformation of E. coli cells . . . . . . . . . 143
7.1.9 Isolation of plasmid DNA from E. coli cells . 143
7.1.10 PCR mutagenesis . . . . . . . . . . . . . . . . 144
7.1.11 Genetic constructs . . . . . . . . . . . . . . . 145
7.2 Cell culture and transfection . . . . . . . . . . . . . . . 145
7.2.1 Propagation of D. melanogaster S2 cells . . . 146
7.2.2 Transfection . . . . . . . . . . . . . . . . . . . 146
7.2.3 RNA interference . . . . . . . . . . . . . . . . 147
7.2.4 Generation of stable cell lines . . . . . . . . . 147
7.3 RNA isolation and analysis . . . . . . . . . . . . . . . 148
TM7.3.1 RNA extraction with TriFast reagent . . . 148
7.3.2 Phenol-chloroform-isoamyl alcohol extraction 148
7.3.3 DNase treatment of RNA preparations . . . . 149
7.3.4 Preparation of double-stranded RNA . . . . . 150
7.3.5 Northern blot . . . . . . . . . . . . . . . . . . 150
Denaturing agarose gel electrophoresis. . . . . . 150
Transfer of RNA to membranes . . . . . . . . . 151
32Preparation of [ P]-labeled DNA probes . . . . 152
Northern hybridization . . . . . . . . . . . . . . 153
7.3.6 Preparation of RNA for microarray analysis . 153
7.4 Immunoprecipitation . . . . . . . . . . . . . . . . . . . 154
7.5 Protein gel electrophoresis and Western blot . . . . . . 155
7.5.1 Polyacrylamide gel electrophoresis . . . . . . 155
7.5.2 Transfer of proteins to nitrocellulose mem-
branes . . . . . . . . . . . . . . . . . . . . . . 156
7.5.3 Western blot . . . . . . . . . . . . . . . . . . 156
7.6 In vivo metabolic labeling of cells . . . . . . . . . . . . 157
7.7 Analysisofpolysomesbysedimentationinsucrosegra-
dients . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
7.8 Tandem a–nity puriflcation and mass spectrometry
analysis of protein complexes . . . . . . . . . . . . . . 160
7.8.1 Tandem a–nity puriflcation . . . . . . . . . . 160
7.8.2 Silver staining of protein gels . . . . . . . . . 161Contents v
7.8.3 Destaining the gel and preparing samples for
mass spectrometry analysis . . . . . . . . . . 162
7.8.4 Mass sp of purifled proteins162
7.9 Immuno uorescence and confocal microscopy. . . . . . 163
7.9.1 Immuno uorescence . . . . . . . . . . . . . . 163
7.9.2 Confocal uorescence microscopy . . . . . . . 164
References 171List of Tables
1 Enrichment of predicted miRNA targets amongst core tran-
scripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2 Biochemical activities of eIF4A mutants. . . . . . . . . . . . . 70
3 InterProtermssigniflcantlyenrichedinannotationsofproteins
associated with Belle. . . . . . . . . . . . . . . . . . . . . . . . 85
4 Clusters identifled using MCODE-Plugin in the network of
proteins associated with Belle. . . . . . . . . . . . . . . . . . . 89
5 GO terms signiflcantly enriched in annotations of proteins as-
sociated with Belle. . . . . . . . . . . . . . . . . . . . . . . . . 99
6 Proteins associated with Belle in Drosophila S2 cells. . . . . . 103
7 Resolving gels for denaturing SDS-PAGE . . . . . . . . . . . . 155
8 Antibodies used for Western blot . . . . . . . . . . . . . . . . 157
9 Antibodies used for immuno uorescence . . . . . . . . . . . . 164
10 Oligonucleotide sequences used for cloning, mutagenesis and
RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
11 Oligonucleotide sequences used for dsRNA preparation . . . . 167
12 Details of plasmid construction . . . . . . . . . . . . . . . . . 168
viList of Figures
1 Translation initiation pathway . . . . . . . . . . . . . . . . . . 6
2 Schematic overview of the eIF2fi-phosphorylation-dependent
integrated stress response. . . . . . . . . . . . . . . . . . . . . 10
3 mRNA-speciflc cis-acting regulatory sequences which partici-
pate in translational control. . . . . . . . . . . . . . . . . . . . 13
4 Post-transcriptional RNA operons. . . . . . . . . . . . . . . . 15
5 Examples of leaky scanning. . . . . . . . . . . . . . . . . . . . 17
6 uORFs function in various ways to modulate translation. . . . 19
0 07 Inhibition of translation initiation by 5- and 3-UTR-binding
proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
8 Inhibition of 60S subunit joining by hnRNP K and E. . . . . . 29
9 Bruno-mediated translational repression of oskar mRNA. . . . 31
10 Smaug-mediated of nanos . . 33
11 SXL-mediated control of msl-2 mRNA . . . . . . 35
12 PUF-protein-mediated translational control mechanisms. . . . 37
13 miRNA-mediated silencing pathway in Drosophila. . . . . . . . 42
14 Mechanisms of translational repression by miRNAs . . . . . . 45
15 Expression proflles of Drosophila S2 cells depleted of Drosha
or Argonaute proteins. . . . . . . . . . . . . . . . . . . . . . . 51
16 RNAs regulated by Drosha, AGO1 or AGO2. . . . . . . . . . 53
17 Core transcripts regulated by the miRNA-pathway. . . . . . . 55
18 Core represent authentic miRNA targets. . . . . . 58
19 Depletion of AGO2 inhibits siRNA-guided, but not miRNA-
guided gene silencing. . . . . . . . . . . . . . . . . . . . . . . . 60
20 AGO2 associates with miRNAs. . . . . . . . . . . . . . . . . . 61
21 RNAs exclusively regulated in the individual knockdowns. . . 62
22ely in the Drosha knockdown. . . . . 63
23 Argonaute proteins regulate expression of transposons in S2
cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
24 Subcellular localization of Belle in S2 cells. . . . . . . . . . . . 66
25 Belle is required for S2 cells proliferation. . . . . . . . . . . . . 67
vii