TRANS-ACTING FACTORS AFFECTING RETROVIRAL RECODING

Lisa Green

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2012 © 2012 Lisa Green All Rights Reserved Thesis Abstract

Trans-Acting Factors Affecting Retroviral Recoding

Lisa Green

The production of retroviral enzymes requires a translational recoding event which subverts normal decoding, either by direct suppression of termination with the insertion of an amino acid at a stop codon (readthrough), or by an alteration of the reading frame of the mRNA

(frameshift). It has been determined that retroviral readthrough and frameshift require cis-acting factors in the mRNA to stimulate recoding on the eukaryotic ribosome. Here we investigate the affects of trans-acting factors on recoding, primarily in the context of the MoMLV gag-pol junction.

We report the effects of a host protein, Large Ribosomal Protein Four (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we show that transfection of cells with an RPL4 cDNA expression construct enhances recoding efficiency in a dose-dependent manner. The increase in the frequency of recoding can be more than 2-fold, adequate to disrupt normal viral production. This effect is cell line specific, and appears to be distinct to RPL4 among ribosomal proteins.

The RPL4 increase occurs with both retroviral readthrough and frameshift sequences, and even at other viral readthrough regions that do not involve RNA secondary structures. We show that RPL4 effects are negated by release factor over-expression, and that RPL4 will increase readthrough above the levels of a hyperactive mutant and in addition to G418. When co- transfected with Moloney murine leukemia provirus, the RPL4-mediated increase in readthrough reduces the amount of virus released.

We also examined the effects of aminoglycoside drugs and the small molecule PTC124 on readthrough of the MoMLV gag-pol junction. We show that G418, paromomycin and PTC124 increase readthrough of our MoMLV reporter in a dose dependent manner in 293A cells. These drugs reduce viral replication, as measured by a recombinant transducing virus assay. We further examine G418 and paromomycin in an in-vitro system; readthrough is increased to higher levels than those seen in vivo. G418 displays deleterious effects on cell viability and overall translation.

Paromomycin does not appear as toxic, suggesting differences in interactions by which these drugs enhance readthrough.

Table of Contents

List of Figures and Tables iii

Acknowledgements viii

Chapter 1: Introduction 1

Retroviruses 2

Translation 15

Recoding 30

Chapter 2: Materials and Methods 49

Chapter 3: RPL4 effects on retroviral recoding 72 mRPL4 enhances readthrough at the MoMLV gag:gag-pol junction 76

RPL4 effects on readthrough are cell line dependent 78

Mouse and human RPL4 both enhance MoMLV/XMRV gag-pol readthrough 81

RPL4 enhancement of readthrough is distinctive among ribosomal proteins 83

RPL4 mutants do not increase readthrough 91

RPL4 Time Course 99

RPL4 enhances translation of other functional viral recoding sequences 101

RPL4 effect on readthrough of non-recoding premature stop codons is undetermined 104

PseudoKnot mutants and RPL4 112

RPL4 increases readthrough at all three stop codons 115

RPL4 does not enhance readthrough in in-vitro translation reactions 119

Release factor over-expression negates RPL4 enhanced readthrough 124

RPL4 and G418 act additively to increase MoMLV readthrough 127

i RPL4 enhancement of readthrough is not due to ribosome biogenesis 130

RPL4 increase in gag-pol readthrough has deleterious effects on viral replication 135

Chapter 4: Readthrough Promoting Drugs and Retroviral Recoding 141

Aminoglycosides increase MoMLV readthrough in vivo 146

Readthrough promoting drugs can affect retroviral replication 154

Aminoglycosides and PTC124 increase Sindbis/Leaky stop codon readthrough in vivo 160

G418 and PTC124 may enhance HIV-1 frameshift in vivo 162

Aminoglycosides do not increase readthrough of MoMLV in drug-resistant cells 164

Aminoglycosides increase MoMLV/PTC readthrough in vitro 164

Chapter 5: Discussion 177

RPL4 177

Readthrough promoting drugs 191

Final thoughts 195

References 197

Appendix 207

Functional Pseudoknot FACS screen 207

Cryo-EM structure: The pseudoknot on the ribosome 219

ii Figures

Figure 1-1. Retroviral particle 3

Figure 1-2. Retroviral replication cycle 6

Figure 1-3. Simple and complex retroviral genome 7

Figure 1-4. Reverse Transcription 9

Figure 1-5. General Structure and Organization of the ribosome 14

Figure 1-6. A, P, and E Site occupancy during translation elongation 19

Figure 1-7. 80s ribosome 20

Figure 1-8. Cognate fit; normal elongation 23

Figure 1-9. Decoding center 26

Figure 1-10. eRF1 structure 28

Figure 1-11. Recoding: Frameshift 32

Figure 1-12. Recoding: Readthrough 33

Figure 1-13. Possible models of -1 programmed ribosomal frameshift 35

Figure 1-14. Proposed mechanics of frameshifting pseudoknot 38

Figure 1-15. Readthrough of the MoMLV gag-pol junction 41

Figure 1-16. Schematic of MoMLV pseudoknot 42

Figure 1-17. Functional effects on mutations to the MoMLV pseudoknot 44

Figure 1-18. Traditional and revised MoMLV pseudoknot structure 45

Figure 1-19. Inactive and active conformations of the MoMLV pseudoknot 47

iii Figure 3-1. 60S ribosome and RPL4 74

Figure 3-2. Schematic of MoMLV 13PK6 luciferase assay reporter constructs and calculation of readthrough 75

Figure 3-3. mRPL4 effects on readthrough of MoMLV gag-pol junction 77

Figure 3-4. RPL4 effects on MoMLV gag-pol readthrough: cell line survey 80

Figure 3-5. mRPL4 and hRPL4 induced increase of MoMLV gag-pol readthrough 82

Figure 3-6. Survey of effect of additional ribosomal proteins on MoMLV gag-pol readthrough. 84

Figure 3-7. Effect of myc-tagged ribosomal proteins on MoMLV gag-pol readthrough 85

Figure 3-8. Expression of myc-tagged ribosomal proteins 86

Figure 3-9. Expression of N-terminal flag-tagged RPL4 88

Figure 3-10. Expression of mRPL4 and hRLP4 89

Figure 3-11. qPCR analysis of RPL4 mRNA levels in 293 cells 90

Figure 3-12 RPL4-FS mutant 92

Figure 3-13. Effect of L4-FS on readthrough of the MoMLV gag-pol junction 93

Figure 3-14. C-terminal differences between mouse and human RPL4 and schematic of RPL4 c- terminal truncation mutants 95

Figure 3-15 RPL4 C-terminal truncation mutants effect on readthrough 96

Figure 3-16. Schematic of RPL4 helicase-binding domain mutants 97

Figure 3-17. Effect of RPL4 helicase-binding domain mutants on MoMLV gag-pol readthrough 98

Figure 3-18 Timecourse of RPL4 enhancement of readthrough and corresponding RPL4 expression 100

Figure 3-19. Effect of RPL4 on additional viral recoding sequences 103

Figure 3-20. Readthrough frequency of the 13ScrPK6 reporter 106

Figure 3-21. Linear range of the dual luciferase assay 107

iv Figure 3-22. UPF1 dominant negative mutant effects on readthrough of 13ScrPK6 109

Figure 3-23. Readthrough efficiency of 13ScrPK6_RSE reporter 111

Figure 3-24. Comparison of readthrough efficiency of reporter constructs 113

Figure 3-25. RPL4 effects on CollStop readthrough 114

Figure 3-26. Effects of RPL4 on MoMLV pseudoknot mutants 116

Figure 3-27. Effects of RPL4 on MoMLV hyperactive pseudoknot mutant 117

Figure 3-28. RPL4 enhancement of readthrough of alternate stop codons 118

Figure 3-29. RPL4 effect on readthrough in TnT IVT reaction 121

Figure 3-30. Effect of pre-translated RPL4 on MoMLV readthrough in TnT IVT reaction 122

Figure 3-31. Effect of RPL4‐GST tagged protein on readthrough in TnT IVT reaction 123

Figure 3-32. Release factor effect on MoMLV readthrough 125

Figure 3-33. Combined effects of Release Factors and RPL4 on MoMLV readthrough 126

Figure 3-34. Combined effects of RPL4 and RPL7 on MoMLV readthrough 128

Figure 3-35. eRF1 effects on RPL4 enhancement of readthrough 129

Figure 3-36. Combined effects of RPL4 and G418 on readthrough 131

Figure 3-37. Monoclonal antibody detection of endogenous mouse and human RPL4 133

Figure 3-38. Levels of endogenous RPL4 in responsive and non-responsive cells 134

Figure 3-39. RPL4 overexpression in responsive and non-responsive cell lines 136

Figure 3-40. RPL4 overexpression effects on L10a expression 137

Figure 3-41. Effects of RPL4 overexpression on 28S rRNA in responsive and non-responsive cell lines 138

Figure 3-42. Effects of RPL4 on viral replication 140

Figure 4-1. Aminoglycoside Structures 142

Figure 4-2. Substitutions and variations in aminoglycoside structure 143

Figure 4-3. Paromomycin bound to 16S rRNA 145

Figure 4-4. G418 effects on MoMLV readthrough in 293A cells 147

v Figure 4-5. G418 effects on viral production: transducing assay 149

Figure 4-6. G418 effects on cell viability and readthrough 150

Figure 4-7. Readthrough promoting drug effects on MoMLV readthrough 152

Figure 4-8. Readthrough promoting drug effects on MoMLV readthrough: fold change summary 153

Figure 4-9. Streptomycin effects on MoMLV readthrough in 293A cells 155

Figure 4-10. Drug effects on viral replication: transducing virus assay 156

Figure 4-11. Drug effects on viral replication:: detail, 1:4 virus dilution 157

Figure 4-12. Drug effects on viral replication: producer cells 158

Figure 4-13. Drug effects on Sindbis leaky stop codon 161

Figure 4-14: Drug effects on HIV-1 frameshift 163

Figure 4-15. Drug effects on MoMLV readthrough in 293T neo-resistant cells 165

Figure 4-16. G418 effects on MoMLV readthrough in vitro 167

Figure 4-17. G418 effects on MoMLV readthrough in vitro: fold change 168

Figure 4-18. G418 effects on CollStop readthrough in vitro 169

Figure 4-19. G418 effects on CollStop readthrough in vitro: fold change 170

Figure 4-20. Paromomycin effects on MoMLV readthrough in vitro 172

Figure 4-21. Paromomycin effects on MoMLV readthrough in vitro: fold change 173

Figure 4-22. Paromomycin effects on CollStop readthrough in vitro 174

Figure 4-23. Paromomycin effects on CollStop readthrough in vitro: fold change 175

Figure 5-1: Potential Mechanisms for RPL4 enhancement of Recoding 188 x Figure A-1. Schematic of 2FP reporter construct and FACS screen overview 208

Figure A-2. MoMLV 2FP and single color controls 210

Figure A-3. Readthrough in MoMLV pool and clones. 211

Figure A-4. Readthrough in MoMLV 2FP pool and clones 212

Figure A-5. Effect of RPL4 and PABP on readthrough in MoMLV 2FP pool and clones 214

vi Figure A-6. Effect of RPL4 and PABP on readthrough in MoMLV 2FP clone 18 215

Figure A-7. Effects of RPL4 on 13PK6 readthrough in MoMLV 2FP clone 18 216

Figure A-8. RPL4 effect on readthrough in transient transfection of MoMLV 2FP 218

Figure A-9. Proposed experimental design for cryo-EM structure of MoMLV pseudoknot promoting readthrough on the ribosome 221

Figure A-10. Effects of eRF1 depletion on recoding in IVT lysate 223

Figure A-11. Translation in tRNA depleted IVT lysates 225

Figure A-12. MoMLV pseudoknot constructs for cryo-EM structure 227

Figure A-13. Translation in dialyzed TnT IVT lysate 228

Tables

Table 1-1. Classification of retroviruses 5

Table 1-2. Conservation of ribosomal proteins across kingdoms 16

Table 2-1. Cloning Inserts 57

Table 3-1. Cell lines used in dual luciferase assay for survey of RPL4 effects 79

vii Acknowledgments

I would like to thank my mentor, Steve Goff, for accepting me into his lab, and for letting me work on a project that I find truly fascinating. Steve is continually patient and brilliant, and allows all his proteges to work with independence and creativity. Being in the Goff lab has been a privilege and a great experience and one of the highlights of my life.

I would like to thank the Goff lab members who have been so generous with their knowledge and opinions. Thanks especially to Brian Houck-Loomis, who worked on the pseudoknot structure and started the RPL4 project, and graciously supplied me with reagents, plasmids and training.

Thanks to my bay-mate Bobby Hogg who has been an encouraging and non-judgmental voice of reason, and has listened to multitude of my theories and experimental concepts.

I would like to acknowledge my students whom I trained in the Goff lab for their questions

(which made me a better scientist!), their hard work, and their enthusiasm for RPL4 and all things readthrough-related, and for their assistance with the work that appears in this manuscript:

Andrew Kim (RPL4 time course, eRF1/eRF3/RPL4), Susie Tozier (drug IVTs), Sanna Bystrom

(streptomycin, kanamycin surveys), Kausik Regunath (flag-tagged RPL4 expression), Jason

Zhang and Chonticha.

I would also like to thank Chloe Bulinski and Ron Prywes for their encouragement and their help in reserving my place in the class of 2006 when I decided to postpone my entry into graduate

viii school after the birth of my daughter. Their kindness and assistance enabled me to stay at

Columbia without having to choose between a PhD and motherhood.

Thanks to my mother Christine Green, my father and step-mother John Green and Donna Busch my sister Melissa Green, and my mother-in-law Barbara Gregory for their confidence in me. I would like to especially thank my husband Will Bragger, who has endured my ‘going back to school’ for more than a decade, and has never doubted that I would succeed. He is the force that keeps me grounded and keeps me going.

This work is dedicated to my daughter, Giana Charlotte, who was born one month before I started graduated school (initially) in 2005. She is the light of my life, and her unbiased curiosity and unbridled creativity inspire me everyday.

ix Chapter 1: Introduction

Viruses are extraordinary bio-machines – parasitic entities assembled from biological components that are programmed to replicate. Efficient and compact, viral particles contain distinct genes and unique proteins that enable them to exploit the natural functions of higher organisms to produce more virus. A viral particle must utilize host cells’ innate functions in order to replicate and resultant pathology in the host is ultimately a means to ensure viral spread. Viruses are truly fascinating as they exist at the edge of life – they are not free-living themselves, but are bound by the dictates of biological organisms, and depend on the life processes of the cells they infect to exist.

Natural selection has put evolutionary pressure on the virus, and those particles that escape destruction by the host and maximize their replication are successful and survive.

Multiple viral genes and gene products inactivate facets of the immune system, and

‘exploit normal cellular processes’ to enable production of progeny virus. Perhaps most ubiquitous of this ‘borrowing’ of host services is the use of the host cell’s translational machinery to produce viral proteins. Ribosomes, tRNAs, elongation and termination factors are provided by the host cells. Many viruses have developed processes that capitalize on normal translational functions, and also abnormal functions, to allow production of viral proteins in a preferential or specific manner. The phenomenon of recoding – alternate interpretation of mRNAs on the ribosome during translation — is used by many viruses, and is the subject of this dissertation.

1 Retroviruses

A particular class of viruses that utilize recoding in production of their proteins is also unique in its type of infection. Retroviruses are enveloped viruses that bud from the host cells and carry their genome as two copies of a single RNA sequence (Figure 1-1). The viral genome is reverse transcribed into double-stranded DNA and stably incorporated into the genome of the host as a provirus, thereby establishing a permanent and potentially heritable infection. Thus the infected host is never free of the infection; the virus may lie dormant for a variety of reasons, but the integration of the viral genome is permanent and constitutes a mutation.

Many organisms contain endogenous retro-elements — archived sequences from previous viral infections. In humans, endogenous retroviruses (HERVs) have been implicated in autoimmune syndromes such as rheumatoid arthritis; patients test positive for HTLV antigens which are cross-reactive with proteins in afflicted tissues, but do not have antibodies against the virus, indicating expression of an incomplete viral sequence

(Balada, Vilardell-Tarres et al. 2010). Active infection by retroviruses is endemic to some species, and can combine with endogenous retro-elements. Combination of exo- and endogenous retroviral sequences can cause pathology and a high incidence of lethality.

The majority of mice, for example, carry a murine leukemia virus, while a substantial percentage of cats are infected by feline leukemia virus. However retro-elements are predominately innocuous, and some may even have acquired a positive role in the host

2 A

B

© ViralZone 2010, Swiss Institute of Bioinformatics

Figure 1-1. Viral Particles. (A) An example of immature and mature gammaretrovius, i.e. MoMLV. (B) An example of immature and mature lentivirus, HIV-1. HIV-1 has a distinctive truncated cone shaped core upon maturation (ViralZone:www.expasy.org/viralzone, Swiss Institute of Bioinformatics.)

3 physiology. In mice and humans, endogenous retro-elements appear to have a role in differentiation of early embryonic tissues in development (Jern and Coffin 2008)

(Macfarlan, Gifford et al. 2011).

Retroviruses were originally grouped by morphology, but now are classified into seven genera, based on similarities in nucleotide sequence (Table 1). The events of the retroviral replication cycle (Figure 2) are conventionally divided into two categories separated by the completion of the integration of viral DNA: the early events, which include infection through to integration of the provirus, and the late events, starting with transcription of the retroviral genes from the integrated provirus and concluding with the release and subsequent maturation of the nascent virions. A single retroviral particle contains two copies of single-stranded RNA, approximately 8.5 kb in length, which contain all the genes of the virus. All retroviruses share the basic simple genome organization with genes encoding the same proteins: structural proteins (capsid, matrix, nucleocapsid) from the gag gene, replication enzymes from pol gene, and surface proteins that mediate entry and fusion from the env gene. After transcription from the proviral DNA, full-length mRNAs act as both templates for translation of gag and pol genes and as genomes for packaging into new virions (Figure 1-3). Env proteins are translated from a spliced mRNA, and complex retroviruses produce a variety of additional proteins through multiple splicing events. These accessory proteins can promote infection by thwarting various aspects of the immune system, or otherwise aid in retroviral replication

(Kirchhoff 2010).

4 related Example Genera Morphology gag-pol recoding endogenous retrovirus

Avian, i.e. Rous Sarcoma Virus alpharetrovirus C pro-pol frameshift (RSV)

Mouse mammary gag-pro frameshift tumor virus betaretrovirus B Class II (MMTV) pro-pol frameshift

Human T-cell gag-pol frameshift leukemia virus deltaretrovirus C. D (HTLV) pro-pol frameshift

Fish viruses. i.e. Walleye Dermal epsilonretrovirus C unknown Sarcoma virus

Murine leukemia virus, i.e. suppression of gammaretrovirus Moloney Murine C termination/ Class I Leukemia Virus readthrough (MoMLV)

HIV-1 lentivirus cone-shaped core gag-pol frameshift

bud from ER, Simian foamy none- spliced pol spumavirus make dsDNA Class III virus (SFV) transcript prior to infection

Table 1-1: Classification of retroviruses and related spumavirus. Genera is based on sequence similarities in the pol gene. Morphology is divided into types based on surface and core.

5 Infectious virion with RNA genome

Adsorption to specific receptor Membrane fusion and endocytosis

Uncoating

Reverse transcription

Transcription Splicing Transport of dsDNA to nucleus Export of genomic RNA and mRNA Integration of viral DNA Generation of provirus Translation of viral proteins:

gag, gag-pol, env

Assembly

Budding

Release

Maturation

Figure 1-2: Retroviral replication cycle. Early events begin with adsorption and culminate at integration of viral DNA. Late events include transcription to maturation.

6 A

! !

B

© ViralZone 2010, Swiss Institute of Bioinformatics

Figure 1-3. Simple and complex retroviral genome. (A) MoMLV genome consisting of gag, pro/pol and env genes. (B) HIV-1 genome with gag, pro/pol and env plus accessory genes produced by splicing events (ViralZone:www.expasy.org/viralzone, Swiss Institute of Bioinformatics.)

7 The early phase of the retroviral life cycle begins with recognition of receptor on the outside of a host cell by virally-encoded envelope proteins that stud the surface of the virion. The initial interaction between the virion and host cell may be non-specific, but the interaction between cellular receptor and viral surface (SU) proteins result in stabilization of the two that allows entry of the virion into the host cell.

Entry of the viral components into the host cell occurs via fusion of viral and cellular membranes mediated by a conformational change in viral transmembrane proteins that pull the lipids membranes of the cell and virion together. Conventionally it has been understood that fusion occurs at the cell surface. However, some viruses such as MLV enter via a pH-dependent pathway, presumably involving acidification in the endosome.

Furthermore, recently it has been demonstrated that HIV may sometimes enter cells through an endosomic pathway (Miyauchi, Kim et al. 2009; Miyauchi, Marin et al. 2011) or even via a viral/immunological synapse between cells (Haller and Fackler 2008).

After viral attachment and entry, uncoating occurs. The viral genome and virally encoded enzymes are separated from the lipid bilayer of the virus stripped, to some extent, of the structural proteins matrix and capsid. The RNA genome is then converted to proviral

DNA by the viral reverse transcriptase enzyme. This process requires a cellular tRNA bound to the viral RNA to function as a primer; the specific tRNA chosen to serve as

8 tRNA 5’ AAA 3’ R U5 PBS PPT U3 R ↓ -sssDNA R U5 PBS PPT U3 R AAA ↓

PBS PPT U3 R AAA ↓ R U5 first

PBS PPT U3 R AAA strand transfer ! PPT U3 R U5

PBS PPT

PBS PPT U3 R U5

PPT PBS +sssDNA

PBS PPT U3 R U5

U3 R U5 PBS ↓ PBS U5 PBS R U5 R second

U3 U3 strand transfer

U5 R U3 PBS ↓ PPT U3 R U5

U5 R U3 PBS PPT U3 R U5

Figure 1-4. Reverse Transcription. DNA synthesis begins with (-) strand DNA initiated at the primer binding site (PBS) with a cellular tRNA. The retroviral reverse transcriptase enzyme (RT), which is both a DNA polymerase and an RNA nuclease, creates a short DNA segment the minus strand strong stop (-sssDNA). The R sequence of the -sssDNA anneals to the R sequence at the 3’ end of the RNA (first strand transfer). -sssDNA acts as the primer for the rest of minus strand DNA. RT degrades the RNA template as DNA is polymerized, except for the PPT, which primes transcription of the first portion of plus strand DNA at 3’ end of the minus strand DNA. This produces another short fragment of DNA, the plus strand strong stop (+sssDNA). RT degrades the PPT RNA after the +sssDNA has been primed, and the +sssDNA is transcribed using the -sssDNA and the tRNA primer as a template, allowing reconstitution of the PBS on the plus strand DNA. The minus and plus strands are annealed (second strand transfer) through the PBS sequences, and circularization of the minus strand DNA allows transcription of the remainder of the plus strand DNA.

9 primer varies with the retrovirus (MoMLV requires tRNApro and HIV, tRNALys3). Reverse transcription produces a double-stranded DNA molecule that is not identical to the viral

RNA; the process of transcription generates 5’ and 3’ terminal sequences that serve as regulatory sequences for the DNA provirus once it is integrated (Figure 1-4). The reverse transcription reaction is also notable because it allows genetic recombination. Because the virion carries two copies of the genome, it can harbor genomes of two separate retroviruses that have infected the cell. Cross-over during transcription allows swapping of sequences, much like inter-strand exchange during mitosis. Deficient sequences can complement each other to produce infectious and/or pathological virus, as described in the case of endogenous retroelements.

When reverse transcription is complete, the double stranded DNA provirus enters the nucleus. This process is also different among retroviruses: some, such as MoMLV, require that the cell undergo division so there is a breakdown of the nuclear lamina. HIV however can infect non-dividing cells. Entry into the nucleus is a function of the pre- integration complex (PIC) containing the provirus, viral integrase, and perhaps cellular chaperones. The exact identity of the components of the PIC is unknown at this time, although some mediators of nuclear entry have been tentatively identified. After entry into the nucleus, the provirus is inserted into the host genome via the viral integrase.

Insertion site preferences also vary according to retrovirus; gammaretroviruses integrate into transcription start sites, lentiviruses tend to integrate non-preferentially into all regions of active transcription, and deltaretroviruses display no trends in integration sites

10 (reviewed in (Ciuffi 2008). Insertion next to genes that control the cell life cycle can cause cancer due to the active promoter of the retrovirus. Transcription of viral mRNA is mediated by the LTR, and the RNA is 5’ capped and poly-adenylated, and exported to the cytoplasm.

Once in the cytoplasm, viral mRNA is translated by the host ribosomes. During the replication of retroviruses translation of the pol genes is dependent on translation of the upstream gag proteins; pol lacks a start codon and is only translated as a fusion protein with the upstream gag protein. This process requires a recoding event to subvert the normal termination that occurs at the end of gag with an efficiency of about 5%, and is explored in the work described in this thesis. The env proteins are translated from a spliced mRNA and are cleaved by both viral and cellular proteases (Coffin 1997). Gag, gag-pol and env proteins are transported to the cell membrane where they assemble. The

Psi (ψ) packaging signal in the 5’ non-coding region of the mRNA mediates the association between the RNA genome and the structural components of the core. The stoichiometry of viral components is critical for proper assembly and RNA genome recruitment; proper regulation of the ratio of translation of gag and gag-pol products is a necessary factor in viral replication. After assembly, nascent virions bud from the plasma membrane. The virus matures only after release from the cell; the viral protease cleaves the precursor gag and gag-pol to allow the mature core to form.

11 Viral infections can cause pathogenesis as virions replicate and disseminate. Many viruses kill their host cells through lytic release of progeny, and cause disease to the organism as a whole by specific disregulation of immune response and disruption of normal function. In retroviral infections, release of virus occurs continuously over the life of the cell as most retroviruses generally do not cause lysis or cell death through their replication cycle. In many organisms, the ability of the retrovirus to integrate into the genome and disrupt the normal sequence of DNA causes disease in the host, not the actual production of virus. The discovery and study of retroviruses lead to the understanding of cancer as a result of genetic lesions. Discovery of cancer-causing infectious agents, the avian sarcoma/leukosis viruses in the early 1900s, lead to understanding that cancer was a genetic —DNA encoded— disease, and to the eventual establishment of the field of molecular biology. Later, in the 1980s, HTLV and HIV were identified as the first known retroviruses to infect humans and cause pathology.

Through integration retroviruses are able to disrupt normal cellular growth regulation and affect proliferation, causing oncogenesis. Retroviruses may also acquire a portion of a gene in the process of integration. The cellular gene becomes part of the genome of progeny virus, and is transmitted to newly infective cells. If the cellular gene has oncogenic potential, the virus can become acutely transforming. Studies of Abelson

Mouse Murine Leukemia virus lead to the identification of the gene v-Abl, which is carried by the virus and upon integration, causes leukemia in mice. In the 1980s it was discovered that the viral gene had a cellular homolog (Goff, Gilboa et al. 1980) (Davis,

12 Konopka et al. 1985), and c-Abl was cloned, which allowed further study. It was revealed that a single cellular event, a chromosome break and translocation, resulted in the causative agent for chronic myologenous leukemia in humans, (Shtivelman, Lifshitz et al.

1985), (Ben-Neriah, Daley et al. 1986) . The Philadelphia chromosome, as this product of this recombination is called, is the fusion of the Abl promoter and the BCR tyrosine kinase, a pro-growth regulatory gene, which results in a powerful recombinant oncogene.

This recombination is not unlike a scenario of retroviral infection in its ultimate result; the linear sequence of the host DNA is changed permanently, and the regulation that depends on proximity of adjacent sequences is lost. Thus, as a genetic mutation, infection with a retrovirus leaves an indelible mark on the host; the provirus, providing the blueprint of the virus, remains for as long as the cell it infected lives and divides.

Due to permanent presence of the retrovirus, those that cause pathology result in omnipresent danger to the host. In addition, initial infection is often silent and non- symptomatic, with disease developing only after the virus has established a widespread infection in the host. After integration, viral genes are transcribed and translated using the host cell machinery. Just as the study of transforming retroviruses lead to understanding of cellular causes of oncogenesis, investigation of translation of viral proteins is a tool for understanding normal regulation of protein synthesis.

13 GTPase associated peptidyl center tranferase small subunit A-site tRNA center decoding center

P-site tRNA small subunit large subunit large subunit

Figure 1-5: General structure and organization of the ribosome. Cyrogenic electron microscope map of the 70S (prokayrotic) ribosome, the 30S subunit and the 50S subunit. The P- and A- site are depicted in green and magenta respectively. The position of the decoding center (DC) active site the GTPase associated center (GAC) and the peptidyltransferase center (PTC) are also denoted. Figure and text adapted from (Frank and Gonzalez 2010).

14 Translation

Translation of proteins is a complex and highly regulated process. The ribosome has evolved to interpret the genetic code (via messenger RNA) and produce proteins, which perform the majority of functions of the cell. Multiple additional factors regulate ribosome biogenesis, initiation, elongation and termination steps the translation, and ribosome recycling. Translational processes take up the majority of the ‘life’ of the cell, both in energy resources and gene products, thus highlighting the importance and intricacy of the translation machinery.

The ribosome is a composite macromolecule of proteins and RNA segments which reads messenger RNA and conducts assembly of amino acids into proteins. The ribosome is a physical arena for production of proteins, and is responsible for selection of the correct amino acid in the correct order and for promoting the linkage of amino acids through creation of the peptide bond. In all organisms, the ribosome consists of two subunits, small and large, and multiple separate RNA segments. The completed prokaryotic ribosome consists of 3 rRNA segments and 57 proteins, with a combined sedimentation value of 70S. Individually, the 30S subunit contains the 16S rRNA, and 23 proteins, and the 50s subunit contains 5S and 23S rRNA and 34 proteins. The eukaryotic ribosome is larger, and the whole ensemble is measured at 80S; the 40S subunit contains a longer 18s rRNA and 33 proteins, the 60S subunit has 5S and 28S rRNAs, plus the eukaryotic specific 5.8S rRNA, and 46 proteins. The small subunit houses the decoding center,

15 Table 1-2. Conservation of Ribosomal Proteins across kingdoms. Lecompte et al, Comparative analysis of ribosomal proteins in complete genomes: and example of reductive evolution at the domain scale, Nucleic Acids Research, 2002, Vol. 30, Issue 24, p. 5384 by permission of Oxford University Press. (Lecompte, Ripp et al. 2002)

16 where the coding sequence dictated by mRNA is translated into the corresponding peptide, and the large subunit contains the peptidyl transferase center (PTC) where the acceptor arm of the tRNA is accommodated and peptide bonds are formed (Figure 1-5).

The ribosomal subunits move in relation to each other in a motion termed ratcheting to advance the tRNA and mRNA through the active site of translation.

In all ribosomes, the processes of proofreading and peptide bond formation are mediated by the rRNA. Initiation and termination events are carried out by extra-ribosomal proteins which are not homologous among kingdoms, although there is homology among the elongation factors. Alignment of prokaryotic and eukaryotic ribosomal protein sequences and comparison of their predicted structures have lead to a designation of some proteins being considered conserved among all organism, while others are unique to one or a subset of kingdoms (Table 1-2) (Lecompte, Ripp et al. 2002). Homologous regions of rRNA between kingdoms have also suggested that the reactions mediated by rRNA work by universal mechanisms.

Ribosomes can accommodate three tRNAs at a time; each tRNA moves through the A, P and E sites sequentially as the tRNA is accepted as a cognate match to the mRNA codon and peptide bond formation occurs. An amino-acylated tRNA enters the ribosome at the

A (amino-acyl) site, after the match between the codon of the mRNA and the anticodon of the tRNA are verified in the decoding center. This assures that the amino acid carried

17 by the tRNA is the one specified by the mRNA. Next, peptide bond formation occurs between the amino acid on the tRNA in the adjacent P (peptidyl) site, with the newly added amino acid at the C terminal end of the nascent peptide. The tRNA in the A site, now attached to the nascent peptide, moves to the P site, and the empty tRNA in the P site moves to the last site, the E (exit) site (Figure 1-6).

Most of the understanding of the basic mechanisms of translation elongation and termination comes from study of prokaryotic organisms. Structural information was obtained primarily from bacterial ribosome; the structure of an E. coli 70S ribosome was obtained in 1998 (Malhotra, Penczek et al. 1998) through cryo-electron microscopy. The ease of manipulation of genetics in single-cell organisms has allowed for mutational studies of conserved regions of rRNA and proteins. Reconstitution of bacterial translational systems has enabled mechanistic studies into functions of initiation, elongation and termination factors. The intersection of crystallography and cryo-EM has allowed reconstruction of eukaryotic ribosomes (Taylor, Devkota et al. 2009). However recent extensive biochemical work has allowed the reconstitution of the more complex eukaryotic translation system, with a 3 Å crystal structure of a yeast ribosome recently reported (Ben-Shem, Jenner et al. 2010; Ben-Shem, Garreau de Loubresse et al. 2011)

(Figure 1-7). This work has allowed insight into the specific workings of eukaryotic translation, revealing distinct differences from bacterial translation.

18 A B C

tRNAs aa-tRNA large subunit selection peptide translocation mRNA (decoding & bond formation small subunit proofreading) E P A E P A E P A E P A

Figure 1-6. A, P and E site occupancy during translation elongation. (A) amino- acyl tRNA (aa-tRNA) is accommodated at the A site, and after proof-reading, peptide bond formation occurs (B), transferring the nascent peptide to the A site. The tRNA moves to the P site during translocation (C), leaving the A site empty to allow a new aa-tRNA to be accommodated. After the next peptide bond formation, the tRNA at the P site moves to the E site, and is released after the subsequent peptide bond formation. Figure and text adapted from (Frank and Gonzalez 2010)

19 Figure 1-7. 80S ribosome. Architecture of the 80S ribosome (A) Interface or “front view” of 60S and 40S subunits. Landmarks include head, body (Bd) and platform (Pt) of 40S as well as central protuberance (CP), L1-stalk and P-stalk of 60S. (B) Solvent- side or “back” view of the 60S and 40S subunits. From (Ben-Shem, Garreau de Loubresse et al. 2011) http://www.sciencemag.org/content/334/6062/1524.full. Reprinted with permission from AAAS.

20 Translation begins with initiation, the identification of an mRNA by ribosomal components and extra-ribosomal proteins called initiation factors (eIF1-5). In eukaryotes, inhibitory factors prevent association of the large and small subunits until the 40S subunit locates a AUG start codon. The small subunit forms the 43S pre-initiation complex with tRNAmet and eIF1 and eIF2 and GTP, and the inhibitory factors eIF1a and eIF3. eIF4 subunits A and G also join, and this complex interacts with eIF4E, which independently recognizes the 5’ cap present on most mRNAs. eIF4E homes the pre-initiation complex to the mRNA; it is released from the complex, and the pre-initiation complex then scans the mRNA for the start codon. Upon interaction with the AUG, the initiation factors are released via GTPase action of eIF5, and the 60s subunit joins the small subunit, placing the tRNAmet in the position (P site) to form a peptide bond with the next amino acid accepted into the ribosome (at the A site) (reviewed in (Marintchev and Wagner 2005).

Elongation, the process of joining amino acids to create a peptide, commences once the start codon is in the P site. Elongation requires functions of both subunits, as well as additional cytoplasmic elongation factors, the multi-subunit eEF1 and the translocase eEF2. eEF1 is a complex of subunits α, β, and γ. It interacts with all charged tRNAs and facilitates their interaction with the ribosome as a ternary complex with GTP. eEF1 stays associated with the tRNA until the decoding process in the A site is complete. The small subunit has a global conformational change, going from an open to closed position upon decoding, implying a role for specific ribosomal proteins in the progression of elongation.

Once the tRNA has been recognized as a match for the codon on the message, eEF1 is

21 released, and eEF2 promotes the ratcheting of the ribosome, where the small subunit moves relative to the large subunit. This movement advances the mRNA:tRNA through the ribosome and places the next codon in the A site (Figure 1-8).

Proofreading occurs in the decoding center in the small subunit, and is mediated by the rRNA of the small subunit. The process provides quality control, assuring that the proper sequences of amino acids for the message being translated. The general mechanism is conserved among the kingdoms, and the basic mechanism has been illustrated in bacteria.

The same principles are assumed to extend to eukaryotic ribosomes, although there is evidence that eukaryotic ribosomes, especially those of higher organisms, have additional elements to provide higher fidelity (Dresios, Panopoulos et al. 2006; Ben-Shem, Jenner et al. 2010; Ben-Shem, Garreau de Loubresse et al. 2011)

The decoding process has several discriminatory steps, and passage through these steps is irreversible (reviewed in (Zaher and Green 2009). This was initially proposed as a kinetic mechanism, mediated by the change in free energy associated with the codon:anticodon interaction and the subsequent rate of hydrolysis of GTP by elongation factor 1 (eEF1 in eukaryotes, Ef-Tu in prokaryotes). Here the rate of GTP hydrolysis is regulated by the appropriateness of the fit between the codon and the anti-codon. A perfect complementarity of all 3 nucleotides between the codon and anticodon results in the lowest change in free energy, as measured by GTP hydrolysis. A near cognate match,

22 !"# p-tRNA

A #$! mRNA

!"# aa-tRNA

!"# #$! B

!"#

!"# C #$!

!"#

!"# D #$!

!"#

$"# E

Figure 1-8. Cognate fit; normal elongation. During normal translation elongation, (A) aa-tRNA selection occurs as the mRNA in the A site is decoded. Selection of a cognate aa-tRNA allows its accomodation into the A site of the ribosome (B). Peptide bond formation occurs, transferring the nascent peptide (orange line) from the P-site tRNA to the A site tRNA (C). (D) Translocation occurs, moving the A site tRNA to the P site. (E) When a stop codon enters the E site, it is not decoded by an aa-tRNA. A class I release factor (black oval) interacts with the mRNA and the ribosome and the peptide is released.

23 where one base pair of the tRNA anti-codon is not a normal complement to the codon, requires more energy to be accommodated fully into the A site. A non-cognate tRNA, where there is more than one non-complementary base pair, will fail to stimulate GTP hydrolysis and will not linger at the A site. Cognate and near-cognate interactions induce a structural change in EF-Tu (and likely eEF1) which controls the rate of GTP hydrolysis; decreasing the rate of GTP hydrolysis has been shown to increase the accurateness of proofreading (Thompson and Karim 1982; Zaher and Green 2009). GTP hydrolysis stimulates the release of EF-Tu/eEF1 and proofreading occurs to discriminate between cognate and near-cognate fits. The off-rates — disassociation of tRNAs from the A site

— are different for cognate vs. near cognate fits; the off-rate of near-cognate tRNAs after

EF-Tu release is higher than cognate tRNAs.

Proofreading is performed in the decoding center, formed by portions of the 16S/18S rRNA. A conserved set of nucleotides, G529 and G530, A1492 and A1493, are responsible for measuring the fit of the codon:anticodon pairing as measured by the minor groove of the helix formed first two bases of the codon:anticodon nucleotides. A proper Waston-Crick pairing of these nucleotides results in conformational changes in the decoding center. A1492 and A1493 and G530 change orientation and extend into the minor groove of codon:anticodon helix to “inspect” the fit. It has been observed that mismatch of the first base pair results in a less compact fit in the decoding center (Zaher and Green 2009). This results in improper hydrogen bonding with A1492, and exclusion of water needed to solvate polar groups (Ogle, Carter et al. 2003), which results in higher

24 energy losses and enhanced off-rates. The combined actions of A1492, A1493 and G530 have been called a ‘molecular caliper’ (Figure 1-9). The steric dependence of all the elements is like the completion of a puzzle with fitting pieces. The proper fit in the decoding center induces the closed conformation of the small subunit, which in turn affects the large subunit where the tRNAs are accommodated in the A, P and E sites.

Some variation in third base pair of the codon is permitted, resulting in third position wobble associated with many tRNA species for the same amino acid. After decoding, if the tRNA is not readily disassociated from the A site, the peptide bond is formed, and the nascent peptide is transferred from the tRNA in the P site to the newly accommodated tRNA in the A site.

Study of eukaryotic translation has suggested that although the basic mechanisms of decoding are the same, proof-reading may be more rigorous in higher organisms.

Miscoding, the insertion of the wrong amino acid at a codon, occurs very infrequently in normal eukaryotic translation (approximately 1 error per 104 - 105 elongation steps)

(Luce, Tschanz et al. 1985). Eukaryotic proofreading utilizes a pair of adenines (A1755 and A1756) and a guanine residue (G530) but other nucleotides in the decoding sequences differ. This variation appears to confirm resistance to aminoglycoside antibiotics. The recent structural data from models of eukaryotic ribosomes also indicate protein-mediated mechanisms to increase fidelity and maintain quality control.

Eukaryotic rRNA segments are longer than their prokaryotic counterpart, and these additional sections, termed expansion sequences, form extra bridges between the

25 A

Figure 1-9. Decoding Center (A) Schematic of 16S decoding center with tRNAs in A and P site, and mRNA Shown are the A- and P-site tRNAs as green and red strand ribbons, the mRNA fragment as space filled, and two segments of the 16S RNA in backbone (the self-folding long helix 1402–1498 (1GIX.pdb) containing the conserved A-1492 and A-1493 as black dots and the short segment containing G-530 shown as a black dot. The proteins: S9 is displayed as yellow, S12 as cyan, and S13 as gray. (Smith, Lee et al. 2008).

26 subunits. This implies extra coordination and control between the decoding process occurring in the small subunit and the actions of the peptidyl transfer center in the large subunits. Eukaryotic specific protein moieties are also involved in the intersubunit interactions. For example, a eukaryotic specific ridge is formed between the P site tRNA and the E site by a region of the 18S rRNA, which is shifted into the E site upon ratcheting. There is no corresponding structure in the prokaryotic ribosome. It is thought that this mechanism prevents back-ratcheting to ensure proper progression of elongation and maintenance of the reading frame (Ben-Shem, Jenner et al. 2010).

Termination of the elongation reaction occurs when a stop codon (UAG, UGA or UAA) enters the A site. There are no tRNAs which normally decode these codons. In the absence of a cognate tRNA, a termination factor recognizes the stop codon, and stimulates a hydrolysis reaction which allows for the release of the peptide from the peptidyl tRNA. Termination requires two types of release factors. Class 1 factors are those which interact with the mRNA and recognize the stop codon. Prokaryotes utilize two Class I factors, RF1 and RF2. Both recognize the UAA stop codon, but RF1 also recognizes UAG; RF2 recognizes UGA. Eukaryotes have a single Class I factor, eRF1, which recognizes all three stop codons. All Class I factors share a GGQ motif that is necessary for promoting hydrolysis in the peptidyl transferase center (Figure 1-10).

Another domain interacts with the codon of the mRNA in the decoding site. In this way, release factors are similar to tRNAs. However, unlike the relatively conserved tRNA sequences, eukaryotic and prokaryotic Class I release factors lack homology. Because

27 Figure 1-10. eRF1 structure. (A) Movement of the GGQ motif of eRF1 (lime green, free state) upon binding to eRF3 (dark green, bound state). The GGQ motif is responsible for hydrolysis of the peptide bond and release of the nascent peptide. (B) Comparison of tRNA and eRF3-bound eRF1. eRF3 binding causes a bend in Domain M of eRF1 resulting in an eRF1 conformation similar to that of a tRNA. Adapted legend and figure (Cheng, Saito et al. 2009).

28 termination is the result of a protein:mRNA reaction, it is thought that different regions of the decoding center are involved in termination; it is not merely a mimic of the elongation reaction (Youngman, He et al. 2007).

Eukaryotes and prokaryotes have a single Class II release factor each, eRF3 and F3 respectively. Both are GTPases, but their roles in termination are very different. F3 promotes the disassociation from the ribosome and the recycling of RF1/2, but does not directly affect termination of the elongation process. In contrast, eRF3 makes eRF1 work better. Although eRF1 alone is sufficient to catalyze termination, eRF1 and eRF3 form a ternary complex with GTP which increases the rate of termination. Interestingly, the binding of eRF1/eRF3/GTP appears to affect the shape of the ribosome. The portion of the ribosome which protects the mRNA is pulled back, indicating a conformational change in the entry site and underscoring the plasticity of the ribosome as whole. eRF3 does not appear to contribute to the recycling of eRF1 (Alkalaeva, Pisarev et al. 2006).

After termination, the newly translated peptide is released from the ribosome and is folded, modified and transported as necessary. For ribosome recycling prokaryotes utilize two other factors, RRF, which is essential for disassembly, and EIF3, which associates with the small subunit to prevent interaction with the large subunit until initiation. EIF3 also mediates release of old tRNA and mRNAs from the small subunit (Hirokawa,

Nijman et al. 2005; Singh, Das et al. 2005; Seshadri and Varshney 2006). Recycling of

29 the 80S ribosome is less well understood. Eukaryotes do not express RRF or an homologous protein. Rather, the 80S ribosome is disassembled by ABCE1, which possesses general NTP hydrolysis capabilities and requires the presence of eRF1. ABCE1 binds to the ribosome after peptide release and results in separation of the 60S subunit from the tRNA and mRNA bound 40S subunit (Pisarev, Hellen et al. 2007; Pisarev,

Skabkin et al. 2010).

Recoding

Viral translation capitalizes on nuances of translation to promote viral protein synthesis, sometimes at the expense of the host translation. So far, adaptations to many aspects of initiation and elongation have been discovered, and many of these have been studied in depth. Cap-snatching (Mir, Duran et al. 2008)and the use of an IRES (Hellen and Sarnow

2001; Hellen 2007; Hellen 2009) maximize initiation on viral mRNAs and multiple translation tools such as recoding, ribosome shunting (Ryabova, Pooggin et al. 2002;

Racine and Duncan 2010), and “hiccuping” caused by the foot and mouth virus 2A peptide (Donnelly, Luke et al. 2001) give flexibility to a compact genome.

All retroviral translation depends on a recoding event to translate viral enzymes. These products are encoded by the pol gene, which lacks its own promoter and a start codon.

The viral enzymes are only produced as a fusion product with the gag structural protein precursor. The gag protein sequence terminates with a stop codon, and for translation to

30 continue into the pol region the stop codon must be hidden or misidentified by the translating ribosome. This can occur by either of two mechanisms. During a frameshift event the ribosome re-adjusts its position on the mRNA in response to stimulus on the message itself, and the stop codon in essence disappears (Figure 1-11). It also occurs via a readthrough event, where the reading frame is the same, but an amino acid is placed at the site of termination (Figure 1-12). Both processes happen at a low frequency, which remarkably corresponds with the proportion of fusion product to structural components needed for viral replication, and alteration of this ratio can disrupt the virus’ ability to replicate.

The most extensively studied retroviral recoding event is frameshifting. A frameshift event alters the reading frame of the mRNA within the ribosome. The shift may adjust the reading frame forward to skip a base, i.e. the +1 frameshift of Epstein-Barr virus IL-10

(Yoon and Walter 2007) or back to include an already decoded nucleotide as in the -1 frameshift of HIV-1. The frameshift event occurs on a portion of mRNA containing nucleotides XXX YYY Z (X is any nucleotide, Y is an A or U, and Z is not G). This motif, designated the slippery sequence, is required, but not wholly sufficient, to promote frameshift. The event also requires the presence of a secondary structure downstream of the slippery sequence. This can be a pseudoknot, as in HTLV or a stemloop, as in HIV-1.

The spacer region between the slippery sequence and the secondary structure varies slightly among viruses, but deviation from the wild-type also affects frameshifting.

31 MMTV Normal translation ! GCU GAA AAU UCA AAA AAC UUG UAA AGG GGC AGU CCC CUA GCC CCA CUC AAA AGG GGG AUA AA

MMTV Framshift

GCU GAA AAU UCA AAA AAC UUG UAA AGG GGC AGU CCC CUA GCC CCA CUC AAA AGG GGG AUA AA GCU GAA AAU UCA AAA AAC CUU GUA AAG GGG CAG UCC CCU AGC CCC ACU CAA AAG GGG GAU AAA

Figure 1-11. Recoding: Frameshift. The translating ribosome encounters a slippery sequence upstream of a stop codon, and in response to a stimulus from a secondary mRNA structure, re-aligns on the mRNA in an alternate reading frame. This results in a nucleotide being skipped, or re-decoding as part of a new codon. In the new reading frame, the stop codon is re-aligned into a sense codon, and translation continues.

Monday, October 24, 2011

32 MoMLV Normal translation

G ACC CUA GAU GAC UAG! GGA GGU CAG GGU CAG GAG CCC CCC CCU GAA CC CAG GAU AAC CCU CAA AGU CGG GGG GCA ACC CGU C

MoMLV Readthrough (suppression of termination) ? G ACC CUA GAU GAC “CAG” GGA GGU CAG GGU CAG GAG CCC CCC CCU GAA CC CAG GAU AAC CCU CAA AGU CGG GGG GCA ACC CGU C

Figure 1-12. Recoding: Readthrough. The translating ribosome encounters a stop codon upstream of a secondary mRNA structure, and allows insertion of an amino acid instead of termination. Translation continues in the same reading frame.

Monday, October 24, 2011

33 Lentiviruses contain one frameshifting sequence at the end of gag to promote translation of the gag-pol fusion protein. The stop codon is not a stimulatory factor in frameshifting; in HIV-1, the stop codon for gag occurs about 130 bp downstream of the slippery sequence/stem loop and the frameshift event. Other retroviruses, such as the deltaretroviruses (MMTV) and betaretroviruses (HTLV) contain two separate frameshift stimulatory sequences. One occurs at the junction of gag and the coding region for the first of the pol products, PR, the viral protease. The second occurs at the end of the pro sequence and the start of the reverse transcriptase sequence. Thus, the proportions of the viral enzymes to structural components and to each other are more controlled than in the single frameshift sequences.

Conflicting evidence about when during elongation the actual slippage of the mRNA occurs has resulted in the proposal of three general models of frameshift (Figure 1-13).

Each scenario involves different states of occupancy of the A, P and E sites, and accommodation status of the A-site tRNA. In addition, the role of the E site tRNA is variable. It has been recently shown that all three models of frameshift are kinetically possible, and may vary according to the exact nature of the stimulatory RNA structure

(Liao, Choi et al. 2011). However, for HIV-1, it appears that the slippage occurs because of incomplete translocation, and that the forces of decoding in the A site allow for accommodation of a tRNA that overrides the misalignment of the codon:anticodon

34 Figure 1-13. Proposed mechanisms of -1 programmed ribosomal frameshift. Two translation elongation cycles are depicted at the top: the ribosome undergoes decoding (DC), aa-tRNA accomodation (AA), peptidyl transfer (PT) and translocation twice to add two amino acids into the polypeptide sequences. A shift in reading frame may occur at the first TL step and the ribosome decodes a -1 frame A-site codon at the recoding site. Additionally, a -1 PRF may occur at the second AA stop, in which the ribosome has decoded the zero frame A-site codon. Incorporation of the -1 reading frame aa-tRNA starts at the following cycle. Moreover, the shift in reading frame may occur at the second TL and incorporation of the -1 reading frame aa starts at the following cycle. (Liao, Choi et al. 2011), Laio et al, The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting, Nucleic Acids Research, 2011, Vol. 39, No.1, p. 301, by permission of Oxford University Press.

35 interactions in the P and E sites. The kinetic models were performed using 70S ribosomes from E coli; this mechanism may be slightly different for eukaryotic 80S ribosomes (in cryo-EM samples, it is common to see promiscuous binding of any tRNA in the E site.)

This may indicate universal non-specificity or may be a prokaryotic specific event.

Mutational studies of the coronavirus infectious bronchitis virus (IBV) frameshift pseudoknot revealed an additional kinetic element involved in frameshifting (Kontos,

Napthine et al. 2001). Like HIV-1, IBV utilizes a -1 frameshift event to control translation of proteins (Brierley and Dos Ramos 2006). Pausing of translation occurs in the IBV and many other frameshift sequences and this coincides with placement of the 3’ portion of the slippery sequence in the A site of the ribosome (Kontos, Napthine et al.

2001) . The pause occurs regardless of the presence of the slippery sequence and is thought to be dependent on secondary structure downstream of the slippery sequence.

The rate of translation may be slowed as the ribosomal helicase takes longer to dissolve the secondary structure. However, pausing occurs with mutated secondary structures that do not support frameshift as well (Kontos, Napthine et al. 2001). Those secondary structures that induce frameshift must provide a specific stimulus or condition for frameshift to occur.

The placement of the pseudoknot at the entry of the mRNA channel on the ribosome results in a torsion of the mRNA:tRNA in the P site. A cryo-EM reconstruction of the

36 IBV pseudoknot on an 80S mammalian ribosome gives perhaps the most relevant information on the mechanisms involved (Figure 1-14) (Namy, Moran et al. 2006).

During pausing, the edge of the pseudoknot sequence is at the beginning of the mRNA entry channel of the ribosome; this is the putative location of the ribosomal helicase. The reconstruction captures eEF2, the translocase, in the A site, and shows a significant bend in the P site tRNA toward the A site. This conformational change indicates stress on the P site tRNA as eEF2 is promoting translocation and the unmelted PK is preventing full complementary movement of the mRNA. This global change in the shape of the P site tRNA affects the interaction of its anticodon with the codon of the mRNA. The codon:anticodon interaction is broken and the mRNA readjusts in a new reading frame.

Theoretically, because of the nature of the slippery sequence, certain non-cognate interactions are tolerated.

Gammaretroviruses such as Moloney Murine Leukemia Virus (MoMLV) and

Xenotropoic Murine Leukemia virus related virus (XMRV) and epsilonretroviruses do not produce the pol genes by frameshifting. Instead, these viruses utilize a readthrough event, where an amino acid is inserted at a stop codon (Figure 1-15). This event is distinct from frameshift in that the mRNA remains in the same reading frame. Readthrough in viruses does not seem to require a sequence in the decoding site analogous to the slippery sequences, as readthrough can occur with all three stop codon (though at different frequencies) and despite the identity of the nucleotides immediately upstream. A pseudoknot is absolutely required for retroviral readthrough, and particular nucleotide

37 Figure 1-14. Proposed mechanics of frameshifting pseudoknot. Three different states of the small subunit translating an mRNA containing a pseudoknot that induces -1 frameshifting are shown. a. The elongating ribosome approaching the pseudoknot in the zero reading frame. b. Engagement with the pseudoknot, generating a frameshifting intermediate in which the small subunit is stalled during translocation with eEF2 bound, causing tension in the mRNA that bends the P-site tRNA in a (+) sense direction. As a result the anticodon-codon interaction breaks over the slippery sequence, allowing a spring-like relaxation of the tRNA in a (-) sense direction. c. Re-engagement of the tRNA with the mRNA, leaving the ribosome translation in the -1 reading frame. Reprinted by permission from Macmillan Publishers Ltd: Nature, (Namy, Moran et al. 2006), copyright 2006. http://www.nature.com/nature/index.html

38 substitutions can have drastic affects on the incidence of readthrough.

Readthrough is also referred to as stop codon suppression, and can occur in yeast and bacteria with a suppressor tRNA . Suppressor tRNAs contain anti-codons that align to a stop codon; thus they cause normal termination to be suppressed by interacting with a termination signal and allowing an insertion of an amino acid which propagates elongation. There is evidence to support the notion that suppressor tRNAs play a role in cellular readthrough events, and it was hypothesized that the readthrough of the gag termination codon in MoMLV was a result of a glutamine charged suppressor tRNA

(Kuchino, Beier et al. 1987; Kuchino, Nishimura et al. 1988). Likewise, the concentration of tRNAglu suppressor was increased in certain infected cell lines. However, further examination refuted the role of the suppressor tRNA. Although glutamine is the predominant amino acid inserted during the readthrough event, other amino acids can also be inserted (Houck-Loomis 2009). Suppression works in viruses that utilize UGA which would not be decoded by the glutamine suppressor, and also with UAA (Feng,

Levin et al. 1989; Jones, Nemoto et al. 1989; Feng, Copeland et al. 1990). Most conclusively, readthrough can occur in in vitro lysates and in cells in the absence of any suppressor tRNAs (Feng, Hatfield et al. 1989). Thus the readthrough event in MoMLV likely involves interactions with the ribosome and/or perturbations of termination events.

39 The MoMLV pseudoknot structure (Figure 1-16) and spacing requirements have been extensively studied via mutational analysis (Feng, Yuan et al. 1992). Early analysis of the sequences required for the expression of gag-pol established the importance of the placement of the stop codon within the sequence. Movement of the stop codon one codon

3’ of its original position abolished readthrough. Likewise, changing the preceding CAG glutamine codon (the 9th codon upstream of the normal gag stop codon) to a UAG stop codon also does not support readthrough (Felsenstein and Goff 1992). This indicated that readthrough was entirely context dependent. Comparison of amino acid substitutions from mutated virus lacking the gag-pol protein revealed that a mutation that should allow for production of gag-pol with alternate codon usage was not viable; thus it was the nucleotide sequence and not the proteins encoded that prevented viral replication and production of gag-pol (Felsenstein and Goff 1988). The nature of the necessary nucleotides suggested the presence of stable stems in the sequence downstream of the stop codon.

Additional studies introduced the likelihood of a pseudoknot structure forming during translation of viral mRNA (Odawara, Yoshikura et al. 1991; Wills, Gesteland et al. 1991;

Feng, Yuan et al. 1992). Mutational analysis of the sequences downstream of the gag-pol junction illustrated the sequence involved in readthrough. It was determined that a stem loop is present downstream of the UAG stop codon. Double compensatory mutants that shuffled the nucleotide sequence but retained the putative stem loops retained the ability to readthrough. Careful design of mutants and analysis allowed a specific putative

40 A

95%

5%

MoMLV PK B UAGUAG

UAGUAG UAGUAG

GUCGUC

eRF1 CAGC AG tRNAglu

Termination Readthrough

Figure 1-15. Readthrough of the MoMLV gag-pol junction. (A) The pol gene lacks a start codon, and is produced only as a fusion protein with the upstream gag gene products. The gag gene contains a stop codon, and 95% of the time, normal termination occurs. Suppression of termination happens at a frequency of about 5%, and allows the translation of the pol genes at a specific ratio to the gag only product. (B) At the UAG stop codon of gag, the ribosome usually undergoes normal termination events resulting in the release of the gag protein. During the readthrough event, termination is suppressed, allowing the accommodation of a near cognate CAG tRNA at the UAG stop codon, and translation continues to produce the gag:pol fusion protein.

41 Stem 2

$% Loop 1

Loop 2 Stem 1

! " #

Figure 1-16. Schematic of MoMLV pseudoknot structure. The original proposed structure of the MoMLV pseudoknot downstream of the gag:pol junction, based on mutation analysis. The pseudoknot consists of two stems, and two loops, with an 8 nucleotide spacer between the UAG stop codon and the start of stem 1.

42 pseudoknot structure to be proposed. Interestingly, certain mutations that should have had little impact structurally were found to have effects on the proportion of readthrough

(summarized in Figure 1-17) (Felsenstein and Goff 1992; Alam, Wills et al. 1999) .

Our lab has recently published an NMR structure of the MoMLV gag-pol junction and downstream sequence which verifies several predictions made about its structure as well as suggests a mechanism for its action (Houck-Loomis, Durney et al. 2011). NMR data confirms that the necessary nucleotides 3’ to the stop codon are capable of forming a classic H-type pseudoknot. The two stems are co-axial stacked, as confirmed by proton walking in the spectrum. Stem 1 contains a 1X2 internal loop and is formed from bases

G9 to C33, while A29 is an unpaired bulged nucleotide. Stem 2 contains 7 base pairs, and

Loop 2 is made of nucleotides A34 thru C51. Interestingly, the NMR structure predicts that stem 1 may begin with nucleotide G5, instead of G9 as inferred from mutational studies

(Figure 1-18). This extends stem one to include bases formerly considered part of the

Loop 2 structure. However the traditional proposed structure allows for placement of the stop codon in the A site when the edge of Stem 1 is at the entry of the ribosome. The alternative NMR structure places the stop codon outside of the decoding center when the pseudoknot would be at the entry of the mRNA tunnel; this would suggest that the readthrough process starts upstream of the stop codon. Further biochemical work and/or structural studies will be needed to clarify the placement of the stop codon and the pseudoknot in the context of translation.

43 C20A A A C G G C C21A G57C G C A17C G C A A17G A17U G C G C C24A G15C:C26G G C G16C:C25G G C C U G C A U G G15A:C26U C G A A G11C:C31G U A A G C A A29C G11C G C C G C A A U G U6A C G C U A C C43G C G U A G G A G A

U38A A40C U38C A39U

Figure 3­16. Summary of MoMLV pseudoknot mutations. Mutations are mapped to their locations in the high pH structure. Mutations in green stimulate an increase in readthrough activity; mutations in blue show near­wild type readthrough levels; muta­ Figuretions in orange display a mild to moderate decrease in readthrough; and mutations in red 1-17. Mutations to the MoMLV Pseudoknot and their effects on readthrougheffectively abolish the readthrough­stimulating activity of the pseudoknot.. Mutations are mapped to their locations in the high pH structure (see Figure 1-18 and text). Mutations in green stimulate an increase in readthrough activity; mutations in blue show near-wild type readthrough levels; mutations in orange display a mild to moderate decrease in readthrough, and mutations in red effectively abolish the readthrough-stimulating activity of the ribosome. Text and modified figure adapted from (Houck-Loomis 2009).

44 !"!#$%&'%()*+,)+*-.%/*012%3"+,45#""60(

Figure 1-18. Traditional and revised MoMLV pseudoknot structure. (A) Traditional schematic of MoMLV structure based on mutational analysis. (B) Revised schematic of MoMLV pseudoknot based on NMR and CD data. The spacer between the UAG stop codon and the start of stem 1 is shorter, with bases G5 to A9 becoming part of the stem 1. G5 to C7 pair with U38 to G36 respecitively to extend Stem 1 and reduce Loop 2. A8, A34 and G35 are unpaired bulges in stem 1, while the terminal base pair G5:U38 is a non- canonical base pair. (Figure and text adapted from (Houck-Loomis 2009).

45 The NMR studies reveal that the pseudoknot is capable of undergoing a dramatic conformational shift based on two or more protonation events, and this alternate conformation increases readthrough (Figure 1-19). Protonation of A17 is verified through the NMR data; protonation induces an alteration of the coaxial stacking of stem 2 and a downward shift which allow interactions between loop 2 and stem 1. This results in a kinked RNA motif that could putatively interact with the ribosome itself or cause a shift in the upstream mRNA in the decoding site to allow miscoding. Readthrough is pH dependent, as lower pH in IVT reactions confirms. Mutants that raise the pKa of the active form of the pseudoknot show increased readthrough at higher pH levels. Therefore, variations in the local pH of the cell are able to induce the change in pseudoknot structure, and the frequency of this change is directly linked to the proportion of readthrough of the gag-pol junction.

A model of the MoMLV pseudoknot gives insight into the role of the mRNA in inducing readthrough. But several key issues remain. The NMR structure proposes an elegant riboswitch phenomenon, but interactions with the ribosome and other host factors are still unclear. It seems likely that many other layers of regulation exist. The following work describes our attempts to investigate trans-acting factors that affect readthrough. Chapter

3 documents work done on the ribosomal protein RPL4 which is able to increase recoding in a dose-dependent manner. Chapter 4 details the affects of aminoglycoside

46 A B

Figure 1-19. Inactive and active conformation of the MoMLV pseudoknot. The MoMLV pseudoknot exists in at least two conformations. In the proposed inactive state (A), A17 is unpaired and composes loop 1 and loop 2 is unstructured. In the active state (B), resulting from the protonation of A17 and possibly other nucleotides, A17 shifts, and loop 2 tucks into stem 1, resulting in a more compact structure and a downward shift in stem 2 (Houck-Loomis 2009).

47 antibiotics on readthrough of the MoMLV gag-pol junction. The appendix includes two additional projects, work towards a FACS based screen to find proteins which increase readthrough, and preliminary experiments for a proposed cryo-EM structure of the

MoMLV PK on the ribosome.

48 Chapter 2: Materials and Methods

Tissue culture: 293A, 293T, Rat2, Hela, Te671, A549, Sirc and BHK cells were grown in DMEM with 10% FBS supplemented with L-glutamine plus pencillin/streptomycin.

NIH3T3 were grown in DMEM suppplemented with 10% BCS supplemented with L- glutamine plus pencillin/streptomycin. Cells were maintained in a 5% CO2 atmosphere at

37°C, and passaged at approximately 70% confluency.

Transfections:

Cells were plated at densities of 10,000 cells per well for 96 well plates or 250,000 cells per well for 6 well plates the day before transfection. Cells were transfected with Fugene

6 Transfection reagent (Roche) at a ratio of 3 ul Fugene:1 ug DNA, and harvested for dual luciferase assays, q-PCR, or western blotting 24 post transfection. Assays conducted in 96 well plates were transfected with 100 ng experimental or reporter and 60, 120, or

240 ng of ribosomal protein or release factor expression vector, 1.02 ul Fugene 6 and

Optimem Serum free media to 15 ul total volume, and 3 wells were transfected with each transfection mix. Assays conducted in 6 well plates were transfected with 1 ug experimental or control reporter, and 1, 3, or 6 ug ribosomal protein or release factor expression vector with 21 ul Fugene 6 and 72 ul Optimem serum free media. All transfections were adjusted with empty vector to contain the same total DNA per well per experiment. Cells were lysed with Promega Passive Lysis buffer.

49 Dual Luciferase Assay:

Cell lysates from transfections were transferred to opaque 96 well plates. For transfections done in 96 well format, cells were lysed with 20 ul Passive Lysis Buffer, and 15 ul from each well was transferred. Assays performed in 6 well plates were lysed with 150 ul Passive Lysis Buffer, and 20 ul from each well was plated in triplicate. Dual luciferase assays were performed using Promega Dual Luciferase kit with buffers diluted

1:4 or 1:2 with dH20. Assays were read on Berthold or Omega plate reader for a 24 second interval per well, with readings taken every half second for a total of 48 reading.

One hundred microliters of firefly luciferase reagent was dispensed for the first 12 seconds. Firefly signal was quenched by renilla luciferase reagent addition at 12. 5 seconds. Values from each interval of firefly and renilla luciferase were summed in the

Omega Mars Data Analysis program and exported to a spreadsheet program (Excel or

Numbers) for analysis.

Plasmids: 13PK6 and HIV-1 experimental and control reporter constructs in p2luc, mouse RPL4 and RPS3a in pcDNA 4, myc-tagged ribosomal proteins and MoMLV 2FP pQCXIP were generous gifts from former Goff lab member Brian Houck-Loomis. UPF1 dominant negative mutants and the RSE C fragment insert were generous gifts from Goff lab member Bobby Hogg. Sindbis, 13ScrPK6, 13ScrPK6_RSE and CollStop dual luciferase reporter constructs were cloned into p2luc. Mouse Rpl4 and Rps3a cloned into pcDNA4 were used for cell line assay, and into pcDNA3.1 for IVT reactions. Ribosomal proteins without c-terminal myc tags, human Rpl4, RPL4 mutants, PABP, eRF1 and eRF3

50 were cloned into pcDNA3.1. Cloning was completed using Sequence and Ligation

Independent Cloning (SLIC) protocols adapted from the Elledge lab (Li and Elledge

2007). A complete list of inserts is located at the end of the Materials and Methods section. All cloning products were verified with DNA sequencing.

q-PCR: RPL4 expression: Primers were designed specifically against mouse L4, at the junction of exons 3 and 4 using Primer3 software. 293A cells were transfected with the

RPL4 expression vector and mRNA was extracted using Trizol according to manufacturer’s instructions, and reverse-transcribed into cDNA with Superscript III First

Strand Synthesis for RT-PCR (Invitrogen.) cDNA was mixed with Fast Start SYBR green

Master (ROX) (Roche) according to manufacturer’s instructions and analyzed for levels of RPL4 and actin mRNA. Assay was run on a 7500 Fast Real time PCR System

(Applied Biosystems).

28S rRNA expression: Primers to 28s rRNA were designed using Primer3 software. Cells were transfected with 6 ug RPL4 expression vector, and cDNA was produced from endogenous and transfected cells as described above. cDNAs were analyzed for RPL4 and actin mRNA and 28S rRNA. Assays were run on an epGradient S Mastercycler

(Eppendorf) and analyzed with Realplex software.

51 Western Blots

For ribosomal expression assays, cells were plated at 250,000 cells per well the day before transfection and transfected with expression constructs and/or empty vector as described in text. Cells were lysed 6, 10, 24 and 48 hours post transfection for time course, and 24 hours for other proteins with RIPA buffer and protein levels were normalized by Bradford assay. Lysates were boiled for 5 minutes with 1X sample buffer and run on a 12% bis-acrylamide gel in protein electrophoresis buffer. Gels were transferred to PVDF membranes activated with methanol and equilibrated in phosphate transfer buffer. Membranes were probed with anti-RPL4 rabbit polyclonal (11302-1-AP

ProteinTech Group) or mouse monoclonal antibody (Sigma 4A3), anti-RPL10 mouse antibody (Abcam ab-55544), anti-actin mouse antibody (Sigma A1978), anti-tubulin mouse antibody (Sigma T6199), anti-mouse myc antibody (Santa Cruz 9E10) or anti-flag mouse antibody (Sigma M2), anti-GST mouse antibody (Covance MNS-112P) and species appropriate near-infrared antibodies from Licor. Blots were visualized on the

Licor Odyssey Imaging system. Quantification of actin and RPL4 signals was performed using Odyssey imaging software and Excel.

For viral replication assays 293T cells were transfected were transfected with 1 ug

MoMLV provirus pNCS and 1, 3, or 5 ugs mL4 or S3a and lysed with RIPA buffer with protease inhibitors 48 hours post transfection. Supernatants of transfected cells were clarified through a 45 micron filter. Virus was pelleted from clarified supernatants by

52 ultracentrifugation through 20% sucrose PBS at 25K in a Beckman Coulter SW-55 rotor for 2 hours, and recovered in 40 ul SDS containing sample buffer. Protein levels of cell lysates were normalized by Bradford assay. Samples were run on 12% gels and transferred onto Immobilon-FL PVDF membranes (Millipore). Western blots were probed with (R187) rat anti-capsid monoclonal antibody, and mouse anti-actin (Sigma

1978) for loading controls for cell lysates, and species appropriate fluorescent antibodies from LI-COR. Bots were visualized on the Odyssey Imaging system (LI-COR).

For detection of small peptides from CEMPK constructs, TnT reaction mixes of CEMPK constructs were loaded in BioRd Criterion 16.5% Tris-Tricine gels, and run with MES buffer at 50 mA for 3.5 hours. Lysates run with Transcend tRNA lys were imaged on the

Typhoon Scanner.

In Vitro Translation Reactions: mRNAs of 13PK6 and HIV-1 experimental and reporter constructs, RPL4, RPS3a, and CEMPK were transcribed using mMessage mMachine T7 kit and Poly (A) Tailing kit according manufacturer’s instructions. mRNA was recovered using a MegaClear kit (Ambion). The integrity of the mRNA was checked on an 1% agarose gel prepared with Northern Max buffer with SYBR safe dye; samples and marker were treated with glyoxal for 30 minutes at 50° C prior to loading. For RPL4 effects and

CEMPK samples, mRNA was translated in Ambion Retic Lysate IVT using 0.5 ul 20X translation mix (-) methionine, 0.2 ul 50X methionine, 6.8 ul retic lysate, and 1 ug total

53 mRNA with nuclease free water to make volume up to 10 ul. Lysates were incubated at

30° C for 75 minutes. For dual luciferase analysis, lysates were diluted with 30 ul passive lysis buffer after the translation reaction and plated at 10 ul, three times, in 96 well plates and read on the Omeg plate reader as per standard dual luciferase assay.

In vitro reaction using TnT (Transcription and Translation) reagents (Promega) were assembled as follows: 12.5 ul lysate, 1.0 ul TnT buffer, 0.5 ul TnT T7 RNA polymerase,

0.5 ul amino acid mix (equal parts (-) met, (-) lys, (-) - cys mixes), 1.0 ul reporter DNA,

1.0 additional DNA if need (for RPL4 tests), nuclease free water up to volume of 25 ul.

Reactions were incubated for 2 hours at 30° C. Dual luciferase assays. For reactions using Transcend tRNA (Promega), 0.5 ul, Transend tRNAlys was added and water volume was adjusted.

For in-vitro drug experiments, TnT reagents were combined as follows: 6.25 ul lysate, 0.5 ul TnT buffer, 0.25 ul TnT T7 polymerase, 0.25 ul amino acid mix, 0.25 reporter DNA,

4.5 ul nuclease-free water, 0.5 ug drug. Reactions were incubated for 2 hours at 30° C.

Reactions were diluted with Passive Lysis Buffer (Promega) and assayed by dual luciferase protocols as described.

54 GST-tagged Protein Synthesis: RPL4, RPS3a, Gluthaminyl Synthetase, Methionyl

Synthetase, and Lysyl Synthetase were cloned in the pGEX N-terminal GST containing expression vector. E Coli strain BL21 was transformed with each plasmid (1 ug added to

100 ul bacteria, incubated on ice for 30’) and cultures were grown overnight in ampicillin media at 37° C. Cultures were diluted 1:10 and grown until O.D. of 200. ITPG was added to final concentration of 0.1 mM and cultures were grown for 4 hours at 30° C on a shaker. Bacteria was pelleted, and resuspended in TBS with 0.5 mM DTT and protease inhibitors. Solution was sonicated on ice, for 15 secs, 4 times, and centrifuged, and supernatant was collected for soluble proteins. Supernatant was combined with glutathione-agarose beads, and incubated on a rocker overnight at 4°C.

eRF1 immunodepletion of IVT lysates: Sigma (E8156) and Abcam (ab30928) antibodies to eRF1 were added in 1:25 (1.6 ul) and 1:50 (0.8 ul) concentations to 40 ul retic lysate and incubated overnight at 4° C on a rocker. Lysates were added to 40 ul PBS washed Protein A sepharose beads, and incubated on a rocker for 2 houts at 4° C. Lysate was recovered after 3 mins spin at 4° C and frozen at -80° if not used immediately.

tRNA depletion of IVT lysates: Ethanolamine blocked epoxy-activated Sepharose column was prepared as follows: 2 ml epoxy-activated sepharose beads were swollen in dH20 for 15 minutes, and washed 5X with dH20 to remove preservative. Beads were resuspended in 1M ethanolamine and incubated overnight at room temperature on a

55 rocker. Beads were washed until wash was at a pH of 7. For tRNA depletion of IVT translation mix, ethanolamine blocked beads were washed 2X with 10X volume Buffer A supplemented wiht 80 mM KOAc and 0.5 mM Mg(OAC)2. IVT lysate was diluted to

70% original concentration, and mixed 1:1.3 with bead slurry, and mixed on rocker for 1 hr at 4°. Lysate was recovered and used for TnT reactions.

Immunoprecipitation of flag-tagged RPL4: Cells were transfected with 6 ug RPL4 expression vectors as described. 24 hours after transfection, cells were lysed with IP lysis buffer or Passive Lysis buffer, and 75 ul lysate was combined with 50 ul washed and pre- cleared protein G beads and 1.5 ul Sigma anti-flag F3145, and incubated overnight.

Alternately, lysates were incubated with anti-Flag M2 beads (Sigma). Beads were boiled with 5X sample buffer and analyzed via Western blotting as described.

56 Table 2-1: Cloning Inserts

Insert name Sequence

13PK6 GACCCTAGATGACTAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGATAACCCTCAAAGTCGGGGGGCAACCCGTC

13PK6-C GACCCTAGATGACCAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGATAACCCTCAAAGTCGGGGGGCAACCCGTC

HIV-1 GAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAG GGAAGGCCAGGGAATTTTCTTCAGAGCAGACCATAGCC

HIV-1-C GAGACAGGCTAACTTCTTAAGGGAAGATCTGGCCTTCCCACAA GGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCATAGCC

Sindbis CCAGTTGACTAGGATCC

Sindbis-C CCAGTCGACTAGGATCC

ScrPK ATGTCGACCTAGACCGGACGCCGGGCAGCGCGCAGCGGTCAG CACGTGAGCAATGCACTCCTGACAGAGCATACC

ScrPK-C ATGTCGACCCAGACCGGACGCCGGGCAGCGCGCAGCGGTCAG CACGTGAGCAATGCACTCCTGACAGAGCATACC

13ScrPK6 GACCCTAGATGACTAGGCCAGCGCGCAGCGGTCAGCACGTGA GCAATGCACTCCTGACAGAGCATACCCCCCGTC

13ScrPK-C GACCCTAGATGACCAGGCCAGCGCGCAGCGGTCAGCACGTGA GCAATGCACTCCTGACAGAGCATACCCCCCGTC

13ScrPK6_RSE GACCCTAGATGACTAGGCCAGCGCGCAGCGGTCAGCACGTGA GCAATGCACTCCTGACAGAGCATACCCCCCGTCGATCCTCTTCA ACTTCCCTGAGCTCGGGAGGGCCACTGTTCTCACTGTTGCGCTA CATCTGGCTATTCCGCTCAAATGGAAGCCAGACCACACGCCTGT GTGGATTGACCAGTGGCCCCTCCCTGAAGGTAAACTTGTAGCG CTAACGCAATTAGTGGAAAAAGAATTACAGTTAGGACATATAGA ACCTTCACTTAGTTGTTGGAACACACCTGTCTTCGTGATCCGGA AGGCTTCCGGGTCTTACCGCTTACTGCATGATTTGCGCGCTGTT AACGCCAAGCTTGTTCCTTTTGGGGCCGTCCAACAGGGGGCGC CAGTTCTCTCCGCGCTCCCGCGTGGCTGGCCCCTGATGGTCTTA GACCTCAAGGATTGCTTCTTTTCTATCCCTCTTGCGGAACAAGA TCGCGAAGCTTTTGCATTTACG

57 Insert name Sequence

13ScrPK6_RSE-C GACCCTAGATGACCAGGCCAGCGCGCAGCGGTCAGCACGTGA GCAATGCACTCCTGACAGAGCATACCCCCCGTCGATCCTCTTCA ACTTCCCTGAGCTCGGGAGGGCCACTGTTCTCACTGTTGCGCTA CATCTGGCTATTCCGCTCAAATGGAAGCCAGACCACACGCCTGT GTGGATTGACCAGTGGCCCCTCCCTGAAGGTAAACTTGTAGCG CTAACGCAATTAGTGGAAAAAGAATTACAGTTAGGACATATAGA ACCTTCACTTAGTTGTTGGAACACACCTGTCTTCGTGATCCGGA AGGCTTCCGGGTCTTACCGCTTACTGCATGATTTGCGCGCTGTT AACGCCAAGCTTGTTCCTTTTGGGGCCGTCCAACAGGGGGCGC CAGTTCTCTCCGCGCTCCCGCGTGGCTGGCCCCTGATGGTCTTA GACCTCAAGGATTGCTTCTTTTCTATCCCTCTTGCGGAACAAGA TCGCGAAGCTTTTGCATTTACG

CollStop GACCCTAGATGACTAGGGCAAGCCTGTGGATATTGAACTTCACC AACTTAAAATCTACTGCAGTGCAAACCTCATAGCTC

CollStop-C GACCCTAGATGACCAGGGCAAGCCTGTGGATATTGAACTTCAC CAACTTAAAATCTACTGCAGTGCAAACCTCATAGCTC mRPL4* ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGA GCACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATC CACTGAAGAACCTGAGAATCATGTTGAAGCTGAACCCTTACGC CAAGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGAAATC ACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACTGCACT GGCAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGGACTGC AGACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAAGGTGG ATGCCAAGAAGCAGAAGCCTGCAGGGAAAAAGGTGGTTGCCA AGAAACCAGCAGAGAAGAAGCCTACTACAGAAGAAAAGAAGC CAGCTGCA

58 Insert name Sequence hRPL4 ATGGCGTGTGCTCGCCCACTGATATCGGTGTACTCCGAAAAGGG GGAGTCATCTGGCAAAAATGTCACTTTGCCTGCTGTATTCAAGG CTCCTATTCGACCAGATATTGTGAACTTTGTTCACACCAACTTG CGCAAAAACAACAGACAGCCCTATGCTGTCAGTGAATTAGCAG GTCATCAGACTAGTGCTGAGTCTTGGGGTACTGGCAGAGCTGT GGCTCGAATTCCCAGAGTTCGAGGTGGTGGGACTCACCGCTCT GGCCAGGGTGCTTTTGGAAACATGTGTCGTGGAGGCCGAATGT TTGCACCAACCAAAACCTGGCGCCGTTGGCATCGTAGAGTGAA CACAACCCAAAAACGATACGCCATCTGTTCTGCCCTGGCTGCCT CAGCCCTACCAGCACTGGTCATGTCTAAAGGTCATCGTATTGAG GAAGTTCCTGAACTTCCTTTGGTAGTTGAAGATAAAGTTGAAG GCTACAAGAAGACCAAGGAAGCTGTTTTGCTCCTTAAGAAACT TAAAGCCTGGAATGATATCAAAAAGGTCTATGCCTCTCAGCGAA TGAGAGCTGGCAAAGGCAAAATGAGAAACCGTCGCCGTATCCA GCGCAGGGGCCCGTGCATCATCTATAATGAGGATAATGGTATCAT CAAGGCCTTCAGAAACATCCCTGGAATTACTCTGCTTAATGTAA GCAAGCTGAACATTTTGAAGCTTGCTCCTGGTGGGCATGTGGG ACGTTTCTGCATTTGGACTGAAAGTGCTTTCCGGAAGTTAGATG AATTGTACGGCACTTGGCGTAAAGCCGCTTCCCTCAAGAGTAA CTACAATCTTCCCATGCACAAGATGATTAATACAGATCTTAGCAG AATCTTGAAAAGCCCAGAGATCCAAAGAGCCCTTCGAGCACCA CGCAAGAAGATCCATCGCAGAGTCCTAAAGAAGAACCCACTGA AAAACTTGAGAATCATGTTGAAGCTAAACCCATATGCAAAGAC CATGCGCCGGAACACCATTCTTCGCCAGGCCAGGAATCACAAG CTCCGGGTGGATAAGGCAGCTGCTGCAGCAGCGGCACTACAAG CCAAATCAGATGAGAAGGCGGCGGTTGCAGGCAAGAAGCCTG TGGTAGGTAAGAAAGGAAAGAAGGCTGCTGTTGGTGTTAAGAA GCAGAAGAAGCCTCTGGTGGGAAAAAAGGCAGCAGCTACCAA GAAACCAGCCCCTGAAAAGAAGCCTGCAGAGAAGAAACCTAC TACAGAGGAGAAGAAGCCTGCTGCATAA

59 Insert name Sequence eRF1 (mouse/human) ATGGCGGACGACCCCAGTGCTGCCGACAGGAACGTGGAGATCT GGAAGATCAAGAAGCTCATTAAGAGCTTGGAGGCGGCCCGCGG CAATGGCACCAGCATGATATCATTGATCATTCCTCCCAAAGACC AGATTTCACGAGTGGCAAAAATGTTAGCGGATGAGTTTGGAAC TGCATCTAACATTAAGTCACGAGTAAACCGCCTTTCAGTCCTGG GAGCCATTACATCTGTACAACAAAGACTCAAACTTTATAACAAA GTACCTCCAAATGGTCTGGTTGTATACTGTGGAACAATTGTAAC AGAAGAAGGAAAGGAAAAGAAAGTCAACATTGACTTTGAACC TTTCAAACCAATTAATACGTCATTGTATTTGTGTGACAACAAATT CCATACAGAGGCTCTTACAGCACTACTTTCAGATGATAGCAAGT TTGGATTCATTGTAATAGATGGTAGTGGTGCACTTTTTGGCACAC TCCAAGGAAACACAAGAGAAGTCCTGCACAAATTCACTGTGGA TCTCCCAAAGAAACACGGTAGAGGAGGTCAGTCAGCCTTGCGT TTTGCCCGTTTAAGAATGGAAAAGCGACATAACTATGTTCGGAA AGTAGCAGAGACTGCTGTGCAGCTGTTTATTTCTGGGGACAAA GTGAATGTGGCTGGTCTAGTTTTAGCTGGATCCGCTGACTTTAA AACTGAACTAAGTCAATCTGATATGTTTGATCAGAGGTTACAAT CAAAAGTTTTAAAATTAGTTGATATATCCTATGGTGGTGAAAATG GATTCAACCAAGCTATTGAGTTATCTACTGAAGTCCTCTCCAAC GTGAAATTCATTCAAGAGAAGAAATTAATAGGACGATACTTTGA TGAAATCAGCCAGGACACGGGCAAGTACTGTTTTGGCGTTGAA GATACACTAAAGGCTTTGGAAATGGGAGCTGTAGAAATTCTAAT AGTCTATGAAAATCTGGATATAATGAGATATGTTCTTCATTGCCA AGGCACAGAAGAGGAGAAAATTCTCTATCTAACTCCAGAGCAA GAAAAGGATAAATCTCATTTCACAGACAAAGAGACCGGACAGG AACATGAGCTTATCGAGAGCATGCCCCTGTTGGAATGGTTTGCT AACAACTATAAAAAATTTGGAGCTACGTTGGAAATTGTCACAG ATAAATCACAAGAAGGGTCTCAGTTTGTGAAAGGATTTGGTGG AATTGGAGGTATCTTGCGGTACCGAGTAGATTTCCAGGGAATGG AATACCAAGGAGGAGACGATGAATTTTTTGACCTTGATGACTAC TAG

60 Insert name Sequence eRF3 (mouse) ATGGCCGCGGTGGCCGAGGCCCAGCGTGAGAACCTCAGCGCG GCTTTCAGCCGGCAACTCAACGTCAACGCCAAGCCTTTCGTCC CCAACGTCCACGCCGCTGAATTCGTGCCGTCCTTCCTGCGGGG TCCGGCCCAGCCGCCGCTATCCCCAGCTGGCGCAGCCGGCGGC GACCACGGAGCAGGCAGCGGCGCGGGAGGCCCTTCGGAACCT GTAGAGTCCTCTCAAGACCAGTCGTGTGAAGGTTCAAATTCTA CTGTTAGCATGGAACTTTCAGAACCTGTTGTGGAAAATGGAGA GACAGAAATGTCCCCAGAAGAATCATGGGAGCACAAAGAAGA AATAAGTGAAGCGGAGCCAGGGGGTGGTTCCTCAGGAGATGG AAGGCCACCAGAGGAAAGCACCCAAGAAATGATGGAGGAGGA GGAAGAAATACCGAAACCTAAGTCAGCGGTGGCACCACCAGG TGCTCCCAAGAAGGAACATGTAAATGTAGTGTTCATCGGACATG TTGATGCAGGCAAGTCAACCATTGGAGGACAAATAATGTATTTG ACTGGAATGGTCGACAAAAGGACACTTGAGAAGTATGAACGA GAAGCTAAAGAAAAAAACAGAGAAACTTGGTACTTGTCTTGG GCCTTAGACACGAATCAGGAAGAACGAGACAAGGGCAAAACC GTGGAGGTGGGTCGTGCCTATTTTGAAACGGAAAAGAAGCATT TTACAATTCTAGATGCTCCTGGCCATAAGAGTTTTGTCCCAAATA TGATTGGTGGTGCCTCTCAAGCTGACTTGGCTGTACTGGTAATC TCAGCCAGGAAAGGAGAGTTTGAAACTGGATTTGAAAAAGGA GGACAGACAAGAGAACATGCAATGTTGGCAAAGACAGCAGGT GTCAAACACTTAATTGTGCTTATTAATAAGATGGATGATCCAACG GTAAACTGGAGCAACGAGAGATATGAAGAATGCAAAGAGAAA CTAGTACCATTTTTGAAAAAAGTTGGCTTCAATCCCAAAAAGG ACATTCATTTTATGCCGTGCTCAGGACTGACAGGAGCGAATCTT AAAGAACAATCAGATTTCTGTCCTTGGTACATTGGCTTACCATT TATTCCATATCTGGATAATTTGCCAAACTTCAATAGATCAGTTGA TGGACCAATCAGGCTGCCAATTGTCGATAAGTACAAGGATATGG GGACTGTGGTCCTGGGAAAGCTGGAATCTGGATCTATTTGTAAA GGCCAGCAGCTTGTAATGATGCCAAATAAGCACAACGTGGAAG TTCTTGGAATACTTTCTGATGATGTAGAAACGGACTCTGTAGCT CCAGGTGAAAACCTCAAAATCAGACTGAAAGGAATTGAAGAG GAAGAGATTCTTCCAGGATTCATCCTTTGTGATCTTAATAATCTT TGTCATTCTGGACGCACATTTGATGCTCAGATAGTGATTATAGAG CACAAGTCCATCATCTGCCCAGGGTATAATGCGGTGCTGCATATT CATACCTGTATCGAAGAAGTCGAAATAACAGCCTTAATCTGCTT GGTAGACAAGAAATCAGGAGAGAAAAGTAAGACTCGACCCCG TTTTGTAAAACAAGATCAAGTATGCATTGCGCGTTTAAGGACAG CAGGAACCATCTGCCTGGAGACCTTTAAGGACTTCCCTCAGAT GGGACGTTTTACCTTAAGAGATGAGGGTAAGACCATTGCAATTG GAAAAGTTCTGAAACTGGTTCCAGAGAAAGACTAA

61 Insert name Sequence

PABP ATGAACCCCAGCGCCCCCAGCTACCCCATGGCCTCTCTGTACGT GGGGGACCTGCACCCCGACGTGACCGAGGCGATGCTCTACGAG AAGTTCAGCCCGGCCGGGCCCATCCTCTCCATCCGGGTCTGCA GGGACATGATCACCCGCCGCTCCTTGGGCTACGCGTACGTGAA CTTCCAGCAGCCGGCGGACGCGGAACGTGCTTTGGACACCATG AATTTTGATGTTATAAAGGGCAAGCCAGTACGCATCATGTGGTC TCAGCGTGATCCATCACTTCGCAAAAGTGGAGTAGGCAACATAT TCATTAAAAATTTGGACAAATCCATCGACAATAAAGCACTATAT GATACGTTTTCTGCGTTTGGTAACATCCTTTCATGTAAGGTGGTT TGTGATGAAAATGGCTCCAAGGGCTATGGATTTGTACACTTTGA AACACAGGAAGCAGCTGAAAGAGCTATTGAAAAAATGAATGG GATGCTTCTAAATGATCGTAAAGTGTTTGTTGGACGATTTAAATC TCGGAAGGAACGAGAAGCAGAGCTTGGAGCCAGGGCAAAGGA GTTCACCAATGTTTACATCAAGAACTTTGGAGAAGACATGGATG ATGAGCGCCTTAAGGAACTCTTTGGCAAGTTTGGGCCTGCCTTA AGTGTGAAAGTAATGACAGATGAAAGTGGAAAATCCAAAGGAT TTGGATTTGTAAGCTTTGAAAGGCATGAAGATGCGCAGAAAGC TGTGGATGAGATGAATGGGAAGGAGCTCAATGGAAAACAGATT TATGTTGGACGAGCTCAGAAAAAAGTGGAACGGCAGACGGAA CTTAAGCGCAAATTTGAGCAGATGAAGCAAGATAGGATCACCA GATATCAGGGTGTGAACCTTTATGTGAAAAATCTTGATGACGGG ATTGATGATGAGCGTCTCCGGAAGGAGTTTTCTCCGTTTGGTAC AATCACCAGTGCAAAAGTAATGATGGAGGGTGGGCGCAGCAAA GGGTTTGGTTTTGTATGTTTCTCATCCCCTGAAGAAGCCACTAA AGCAGTTACAGAGATGAATGGTAGAATTGTGGCCACGAAGCCA CTGTATGTAGCTTTAGCTCAGCGCAAAGAAGAGCGCCAGGCTC ACCTCACTAACCAGTATATGCAGAGGATGGCAAGTGTACGAGCT GTGCCCAACCCCGTGATCAACCCCTACCAGCCAGCACCTCCTTC AGGTTACTTCATGGCAGCTATCCCACAGACTCAGAACCGTGCTG CATACTATCCTCCTAGCCAAATTGCTCAACTAAGACCAAGTCCT CGCTGGACTGCTCAGGGTGCCAGACCTCATCCATTCCAGAATAT GCCCGGTGCTATCCGCCCAGCTGCTCCTAGACCACCATTTAGTA CGATGAGACCAGCTTCCTCACAGGTTCCACGAGTCATGTCAAC ACAGCGTGTTGCTAACACATCAACACAGACAATGGGTCCACGT CCTGCAGCTGCTGCTGCTGCAGCCACTCCTGCTGTCCGCACCG TTCCCCAGTATAAATATGCTGCGGGAGTCCGCAATCCCCAGCAA CATCTTAATGCACAGCCACAAGTTACCATGCAACAGCCTGCTGT TCATGTGCAAGGTCAAGAACCTTTAACTGCTTCCATGTTGGCAT CTGCGCCCCCGCAAGAGCAGAAGCAAATGTTGGGTGAACGGC TGTTTCCTCTTATCCAAGCCATGCACCCTTCTCTTGCTGGTAAAA TCACTGGCATGCTGTTGGAGATTGATAACTCAGAATTACTTCAC ATGCTCGAGTCTCCAGAGTCTCTCCGCTCAAAGGTTGATGAAG CTGTAGCTGTACTACAAGCCCACCAAGCGAAAGAGGCTGCCCA GAAAGCAGTGAACAGTGCCACTGGTGTTCCAACTGTCTAA

62 Insert name Sequence

RPS3a* ATGGCGGTCGGGAAGAACAAGCGCCTGACGAAAGGTGGCAAG AAGGGAGCTAAGAAGAAAGTGGTCGATCCATTTTCTAAGAAAG ACTGGTACGATGTGAAAGCTCCAGCCATGTTCAATATTAGGAAC ATCGGGAAGACACTAGTCACGAGGACTCAAGGAACCAAAATTG CCTCTGATGGCCTCAAGGGTCGTGTGTTTGAAGTGAGCCTTGCT GATCTACAGAATGATGAAGTTGCGTTTAGAAAATTCAAGCTAAT CACTGAGGACGTTCAGGGCAAAAACTGTCTGACTAACTTCCAT GGTATGGATCTTACCCGGGATAAAATGTGCTCCATGGTCAAGAA GTGGCAGACCATGATCGAAGCTCACGTTGATGTCAAGACGACC GATGGGTATTTGCTCCGACTTTTCTGTGTTGGCTTCACTAAGAA ACGCAACAACCAGATAAGAAAGACATCCTATGCGCAGCACCAG CAAGTCCGCCAGATCCGGAAGAAGATGATGGAAATCATGACCC GAGAGGTGCAGACCAATGACTTGAAGGAAGTAGTTAATAAACT GATTCCAGACAGCATTGGGAAAGACATAGAAAAGGCTTGCCAG TCCATTTATCCTCTCCATGACGTCTTTGTTAGAAAAGTAAAAATG CTGAAGAAACCCAAGTTTGAATTGGGCAAGCTCATGGAGCTGC ATGGGGAAGGCGGCAGCTCTGGCAAAGCAGCTGGAGATGAGA CGGGTGCTAAGGTTGAGCGAGCTGACGGATATGAACCGCCAGT CCAAGAATCCGTTTAA

RPS4* ATGGCTCGTGGTCCCAAGAAACACCTGAAGCGTGTAGCTGCTC CAAAACATTGGATGCTGGATAAGTTGACTGGCGTGTTTGCTCCT CGTCCATCCACTGGTCCTCACAAGCTGAGGGAATGCCTGCCTCT CATCATTTTCCTAAGGAACAGACTTAAGTATGCCCTGACTGGAG ATGAAGTAAAAAAGATTTGCATGCAGCGATTCATTAAGATTGAT GGGAAAGTCAGGACCGATATAACCTACCCTGCTGGGTTTATGGA TGTCATCAGCATTGACAAGACCGGAGAGAACTTCCGTCTGATCT ATGACACCAAGGGTCGCTTTGCTGTTCATCGTATTACACCGGAG GAGGCCAAGTACAAGTTGTGCAAAGTGAGAAAGATCTTTGTAG GCACAAAAGGAATCCCGCACCTGGTGACCCATGATGCTCGTAC TATTCGGTACCCTGATCCCCTCATCAAGGTGAACGACACCATTC AGATTGATTTGGAGACAGGCAAAATAACTGACTTCATCAAGTTT GACACTGGGAACCTGTGTATGGTGACTGGAGGTGCTAACTTGG GAAGAATTGGTGTAATCACCAACAGAGAGAGACATCCCGGCTC TTTTGATGTGGTTCATGTGAAAGATGCCAATGGCAACAGCTTTG CCACTCGGCTGTCCAACATTTTTGTTATTGGCAAGGGTAACAAA CCATGGATCTCTCTTCCCAGAGGAAAAGGAATCCGCCTCACCAT TGCTGAAGAGAGAGACAAGAGGCTTGCGGCCAAACAGAGCAG TGGG

RPS10* ATGTTGATGCCTAAGAAGAATCGGATTGCCATCTACGAACTCCT TTTTAAGGAGGGCGTGATGGTCGCCAAAAAGGATGTCCACATG CCTAAGCACCCGGAGCTGGCAGACAAAAATGTGCCCAACCTTC ATGTAATGAAGGCCATGCAGTCTCTCAAGTCTCGAGGCTACGTG AAGGAACAGTTTGCTTGGAGACATTTCTACTGGTACCTTACGAA CGAGGGCATCCAGTATCTCCGAGACTACCTGCACCTACCCCCGG AGATCGTGCCCGCCACCCTGCGTCGCAGCCGTCCCGAGACCGG CAGGCCTCGGCCCAAAGGTCCAGAGGGTGAGCGACCTGCAAG ATTCACAAGAGGGGAGGCTGACAGAGACACCTACAGAAGGAG CGCTGTGCCCCCTGGAGCTGACAAGAAAGCTGAGGCTGGGGCT GGCTCAGCCACTGAGTTCCAGTTTAGAGGCGGCTTTGGTCGTG GACGTGGTCAGCCACCTCAGTGA

63 Insert name Sequence

RPS14* ATGGCGCCACGCAAGGGGAAGGAAAAGAAGGAAGAGCAGGTC ATCAGCCTGGGACCTCAGGTGGCTGAGGGAGAGAATGTGTTTG GTGTCTGCCACATCTTTGCATCCTTCAATGACACCTTTGTCCATG TTACCGATCTTTCTGGCAAGGAAACCATCTGCCGAGTGACTGGT GGGATGAAGGTGAAGGCTGACCGAGATGAGTCCTCTCCATATG CAGCCATGTTGGCTGCCCAGGATGTGGCCCAGAGGTGCAAGGA GTTGGGCATCACTGCCCTGCACATCAAACTCCGGGCCACAGGA GGAAACAGGACCAAGACCCCTGGACCTGGAGCCCAGTCAGCC CTCAGAGCTCTTGCTCGCTCTGGGATGAAGATTGGGCGGATTGA GGATGTCACCCCCATTCCCTCTGATAGCACTCGAAGAAAAGGG GGTCGTCGTGGTCGCCGTCTGTGA

RPS20* ATGGCATTTAAAGATACCGGAAAGACGCCCGTGGAGCCCGAAG TGGCGATTCACCGAATTCGAATCACGCTCACCAGCCGCAACGT GAAGTCGCTGGAGAAGGTTTGTGCGGACTTGATCAGAGGCGCC AAGGAAAAGAATCTGAAAGTGAAAGGACCGGTGCGCATGCCT ACCAAGACTTTGAGAATCACTACCAGAAAAACACCTTGTGGTG AAGGTTCCAAGACCTGGGATCGATTCCAGATGAGGATCCACAA GCGACTCATTGATTTACATAGTCCTTCTGAGATTGTTAAGCAGAT TACTTCCATCAGTATTGAGCCGGGAGTTGAGGTTGAAGTCACCA TTGCAGATGCCTAA

RPL5* ATGGGGTTTGTGAAAGTTGTCAAGAATAAGGCCTACTTTAAAA GATACCAAGTGAGATTTCGAAGGCGGCGAGAGGGTAAAACTGA CTACTATGCTCGAAAACGATTGGTGATCCAGGACAAGAATAAGT ACAACACACCCAAATATAGGATGATAGTTCGTGTAACTAACAGA GATATCATCTGCCAGATTGCATATGCCCGTATAGAAGGGGATATG ATCGTCTGTGCAGCATATGCACATGAACTTCCAAAATATGGTGT GAAAGTTGGCCTGACAAATTATGCTGCAGCCTATTGCACTGGCC TGCTGCTGGCCCGCAGGCTTCTGAATAGGTTTGGCATGGACAA GATCTATGAAGGCCAAGTGGAGGTGAATGGAGGTGAATACAAT GTGGAAAGCATTGACGGTCAGCCTGGTGCCTTCACTTGCTATCT GGATGCAGGTCTTGCCCGAACTACAACTGGCAATAAAGTTTTTG GGGCCCTGAAGGGAGCTGTGGATGGAGGCTTGTCTATCCCTCAT AGTACCAAACGATTCCCTGGTTATGACTCTGAAAGCAAGGAGT TCAATGCAGAGGTACATCGGAAGCACATCATGGGTCAGAATGT GGCAGACTACATGCGCTACCTAATGGAGGAAGATGAAGATGCG TATAAGAAACAGTTCTCTCAGTACATCAAGAACAACGTAACTCC AGACATGGAGGAGATGTATAAGAAAGCTCATGCTGCTATCCGAG AGAATCCAGTCTATGAGAAGAAGCCCAAGAGAGAAGTGAAGA AGAAGAGGTGGAATCGTCCCAAAATGTCTCTTGCCCAGAAGAA AGATCGGGTTGCTCAAAAGAAGGCAAGCTTCCTCAGAGCTCAG GAAAGGGCTGCTGAAAGCTAA

64 Insert name Sequence

RPL7* ATGGAGGCTGTTCCAGAGAAGAAAAAGAAGGTTGCCACTGTG CCAGGAACCCTTAAGAAAAAGGTTCCTGCTGGGCCAAAAACTC TCAAGAAAAAGGTTCCCGCTGTGCCAGAAACCCTCAAGAAAA AGCGAAGGAATTTCGCAGAGTTGAAGGTGAAGCGCCTGAGGA AGAAGTTTGCCCTGAAGACACTTCGAAAGGCAAGGAGGAAGC TCATCTATGAGAAGGCAAAGCACTATCACAAGGAGTACGGGCA GATGTACCGCACTGAGATTCGGATGGCCAGGATGGCAAGGAAA GCTGGCAACTTCTATGTGCCCGCAGAACCAAAGCTGGCCTTTG TCATCAGAATTCGAGGTATCAATGGAGTAAGCCCAAAGGTTCGT AAGGTTCTGCAGCTCCTTCGTCTTCGACAGATCTTCAATGGCAC CTTTGTTAAGCTCAACAAGGCTTCAATTAACATGCTGCGGATTG TGGAGCCATACATTGCATGGGGGTACCCCAACCTGAAGTCAGTA AACGAGCTCATCTACAAGCGAGGCTACGGCAAAATCAACAAGA AGCGGATTGCCTTGACAGATAATTCCTTGATTGCTCGGTCTCTT GGTAAGTTTGGCATCATCTGCATGGAGGATCTAATTCATGAGAT CTATACAGTCGGGAAACGCTTCAAGGAAGCAAATAACTTCCTG TGGCCCTTCAAGTTATCTTCCCCACGAGGTGGGATGAAGAAAA AGACAACTCACTTTGTAGAAGGTGGAGATGCTGGCAACAGGGA AGACCAGATAAACAGGCTTATTAGACGGATGAACTAA

RPL11* ATGGCGCAAGATCAAGGGGAAAAGGAGAACCCCATGCGGGAA CTGCGCATCCGCAAGCTCTGCCTCAATATCTGCGTCGGGGAGAG CGGAGACAGACTGACCCGGGCAGCCAAGGTGTTGGAGCAGCT CACAGGCCAGACCCCGGTGTTCTCCAAAGCTAGATACACTGTC AGGTCCTTTGGCATCCGGAGAAATGAGAAGATTGCTGTTCACT GCACAGTCCGCGGAGCCAAGGCAGAGGAAATTCTGGAGAAAG GCCTGAAGGTGCGGGAGTATGAGTTGCGGAAAAATAACTTCTC GGATACTGGAAACTTTGGTTTTGGAATTCAAGAACACATTGACC TGGGCATCAAATACGACCCAAGCATTGGGATCTACGGCCTGGA CTTCTATGTGGTGCTGGGTAGGCCAGGGTTCAGCATCGCAGAC AAGAAGCGCAGAACAGGCTGCATTGGGGCCAAACACAGAATC AGCAAGGAGGAGGCCATGCGCTGGTTCCAGCAGAAGTACGATG GAATCATCCTTCCTGGAAAACTCGAGTCTAG

RPL22* ATGGCGCCTGTGAAAAAGCTTGTGGCGAAGGGGGGCAAAAAA AAGAAGCAGGTTTTGAAGTTCACCCTGGACTGCACTCACCCTG TAGAAGATGGAATCATGGATGCTGCCAATTTTGAGCAGTTCCTC CAGGAGAGAATCAAGGTGAATGGGAAAGCTGGCAACCTCGGC GGAGGAGTCGTGACCATCGAACGCAGCAAGAGCAAGATCACT GTCACTTCAGAGGTGCCTTTCTCCAAAAGGTATTTGAAATATCT CACCAAAAAATATTTGAAGAAGAACAACCTCCGAGACTGGCTG CGTGTTGTCGCCAACAGCAAAGAGAGTTACGAGCTGCGTTACT TCCAGATTAACCAGGATGAAGAGGAGGAGGAAGACGAGGATT AG

65 Insert name Sequence

RPL4 C trunc 359 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGA GCACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATC CACTGAAGAACCTGAGAATCATGTTGAAGCTGAACCCTTACGC CAAGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGAAATC ACAAACTTCGTGTGAAAAAGCTGTAG

RPL4 C trunc 388 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGA GCACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATC CACTGAAGAACCTGAGAATCATGTTGAAGCTGAACCCTTACGC CAAGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGAAATC ACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACTGCACT GGCAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGGACTGC AGACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAAGTGA

66 Insert name Sequence

RPL4 C trunc 402 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGA GCACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATC CACTGAAGAACCTGAGAATCATGTTGAAGCTGAACCCTTACGC CAAGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGAAATC ACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACTGCACT GGCAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGGACTGC AGACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAAGGTGG ATGCCAAGAAGCAGAAGCCTGCAGGGAAAAAGGTGGTTGCCT GA

RPL4 HBD 264 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATACTATGCGCAGGAATACCATTCTTCGCCAGG CCAGAAATCACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAG CTACTGCACTGGCAACCAAATCCGAGAAGGTTGTTCCAGAGAA GGGGACTGCAGACAAGAAGCCAGCGGTAGGCAAGAAAGGAA AGAAGGTGGATGCCAAGAAGCAGAAGCCTGCAGGGAAAAAGG TGGTTGCCAAGAAACCAGCAGAGAAGAAGCCTACTACAGAAG AAAAGAAGCCAGCTGCATAA

67 Insert name Sequence

RPL4 HBD 265 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCAGAATCTTGAAAAGCCCAGAAATCCAA AGAGCCCTCCGAGCACCACGCAAGAAGATTCATCGCAGAGTCT TGAAGAAGAATCCACTGAAGAACCTGAGAATCATGTTGAAGCT GAACCCTTACGCCAAGACTATGCGCAGGAATACCATTCTTCGCC AGGCCAGAAATCACAAACTTCGTGTGAAAAAGCTGGAAGCAG CAGCTACTGCACTGGCAACCAAATCCGAGAAGGTTGTTCCAGA GAAGGGGACTGCAGACAAGAAGCCAGCGGTAGGCAAGAAAG GAAAGAAGGTGGATGCCAAGAAGCAGAAGCCTGCAGGGAAAA AGGTGGTTGCCAAGAAACCAGCAGAGAAGAAGCCTACTACAG AAGAAAAGAAGCCAGCTGCATAA

68 Insert name Sequence

RPL4 HBD 290 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGA AATCACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACTG CACTGGCAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGGA CTGCAGACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAAG GTGGATGCCAAGAAGCAGAAGCCTGCAGGGAAAAAGGTGGTT GCCAAGAAACCAGCAGAGAAGAAGCCTACTACAGAAGAAAAG AAGCCAGCTGCATAA

69 Insert name Sequence

RPL4 HBD 320 ATGGCTTGTGCCCGTCCCCTCATATCGGTGTACTCCGAAAAGGG AGAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAA GCTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTT GAGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCA GGTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTG TGGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATC TGGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATG TTTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGA ATACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCC TCGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGA GGAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAA GGTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAA CTCAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGA GAATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGA TCCAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGT ATCATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAAT GTAAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATG TGGGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTT GGATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAG AGTAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCT TAGCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGA GCACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATC CACTGAAGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAG AAATCACAAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACT GCACTGGCAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGG ACTGCAGACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAA GGTGGATGCCAAGAAGCAGAAGCCTGCAGGGAAAAAGGTGGT TGCCAAGAAACCAGCAGAGAAGAAGCCTACTACAGAAGAAAA GAAGCCAGCTGCATAA

MoMLV 2FP GACCCTAGATGACTAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGATAACCCTCAAAGTCGGGGGGCAACCCGTC

CEMPK3 CCAAGCTGGCTAGTTAAGCTTACCATGAAAAAGATGAAGAAAA AGAAGATGAAGATGAAAAAGAAAAAGAAAAAGTAGGGAGGTC AGGGTCAGGAGCCCCCCCCTGAACCCAGGATAACCCTCAAAGT CGGGGGGAAAAAGAAAAAGAAAAAGAAAAAGTAATAACTGA ATGATTCAACAAGAATTAAGGAGTTTATATGGAAACCCTTATAA ACTCGA

Flag-CEMPK-noQ ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACAT CGATTACAAGGATGACGATGACAAGTTGCTTACCATGGCCTACT GCAAAGAAAAGGGGCACTGGGCTAAAGATTGTCCCAAGAAAC CACGAGGACCTCGGGGACCAAGACCCGAGACCTCCCTCCTGA CCCTAGATGACTAGGGAGGTGAGGGTCAGCAGCCCCCCCCTGA ACCCAGGATAACCCTCAAAGTCGGGGGGCAACCCGTCACCTTC CTGGTAGATACTGGGGCCTAATAACTGAATGATTCAACAAGAAT TAAGGAGTTTATATGGAAACCCTTATAAACTCGAGGGAGCTTGG TACCGAGCTCGGATCCACTAGTCCAGTGTG

70 Insert name Sequence

L4-FS ATGGCTTGTGCCGTCCCCTCATATCGGTGTACTCCGAAAAGGGA GAGTCATCTGGCAAGAATGTCACTTTGCCAGCTGTGTTCAAAG CTCCCATTCGACCAGATATTGTGAACTTCGTTCACACCAACTTG AGGAAAAACAACAGACAGCCCTATGCCGTCAGTGAATTGGCAG GTCATCAGACCAGTGCTGAGTCTTGGGGTACTGGCAGAGCTGT GGCTCGAATTCCAAGAGTTCGAGGTGGTGGGACCCACCGATCT GGCCAGGGTGCCTTTGGAAATATGTGTCGTGGGGGACGCATGT TTGCACCAACCAAAACCTGGCGTCGTTGGCACCGCAGAGTGAA TACAACCCAAAAACGATATGCCATCTGTTCTGCCCTGGCTGCCT CGGCCTTACCAGCTTTGGTGATGTCTAAAGGTCATCGTATTGAG GAGGTTCCTGAGCTGCCGCTGGTGGTTGAAGATAAGGTTGAAG GTTACAAGAAGACCAAGGAGGCTGTTCAGCTGCTCAAGAAACT CAAAGCTTGGAATGACATCAAAAAGGTCTATGCCTCTCAGAGA ATGAGAGCTGGCAAGGGCAAAATGAGAAACCGACGGCGGATC CAGCGCAGGGGGCCCTGCATCATCTATAATGAAGATAATGGTAT CATCAAGGCCTTCCGCAACATCCCTGGTATTACTCTGCTTAATGT AAGCAAGCTGAACATTTTGAAACTGGCTCCTGGTGGGCATGTG GGCCGTTTCTGCATCTGGACGGAGAGTGCTTTCCGGAAGTTGG ATGAGCTGTATGGCACTTGGCGGAAGGCTGCTTCCCTCAAGAG TAACTATAACCTGCCCATGCACAAGATGATGAACACCGACCTTA GCAGAATCTTGAAAAGCCCAGAAATCCAAAGAGCCCTCCGAGC ACCACGCAAGAAGATTCATCGCAGAGTCTTGAAGAAGAATCCA CTGAAGAACCTGAGAATCATGTTGAAGCTGAACCCTTACGCCA AGACTATGCGCAGGAATACCATTCTTCGCCAGGCCAGAAATCAC AAACTTCGTGTGAAAAAGCTGGAAGCAGCAGCTACTGCACTGG CAACCAAATCCGAGAAGGTTGTTCCAGAGAAGGGGACTGCAG ACAAGAAGCCAGCGGTAGGCAAGAAAGGAAAGAAGGTGGATG CCAAGAAGCAGAAGCCTGCAGGGAAAAAGGTGGTTGCCAAGA AACCAGCAGAGAAGAAGCCTACTACAGAAGAAAAGAAGCCAG CTGCATAA

13PK6 U38A GACCCTAGATGACTAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGAAAACCCTCAAAGTCGGGGGGCAACCCGTC

13PK6 U38A-C GACCCTAGATGACCAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGAAAACCCTCAAAGTCGGGGGGCAACCCGTC

13PK6 U38C GACCCTAGATGACTAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGACAACCCTCAAAGTCGGGGGGCAACCCGTC

13PK6 U38C-C GACCCTAGATGACCAGGGAGGTCAGGGTCAGGAGCCCCCCCCT GAACCCAGGACAACCCTCAAAGTCGGGGGGCAACCCGTC

Nucleotides in bold denote stop and corresponding sense codons in experimental and control dual luciferase reporters.

* Native protein coding sequence. C-terminal myc-tagged inserts eliminate the stop codon to allow translation of the myc epitope.

71 Chapter 3: RPL4 effects on retroviral recoding

In an attempt to understand the cellular role in the phenomenon of readthrough, our lab conducted a yeast 3-hybrid screen (Bernstein, Buter et al. 2002) to find host factors that interact with the MoMLV pseudoknot mRNA. Briefly, the bait was designed as the

MoMLV PK RNA transcriptionally fused to the MS-2 stem loop. The MS-2 RNA motif interacts with a hybrid protein of the MS-2 RNA-binding coat protein and Lex-A DNA binding protein, tethering the bait upstream of His3 and LacZ reporters on the yeast chromosome. The prey consisted of a hybrid protein containing the activation domain of the Gal4 transcription factor fused to coding sequences from a pooled plasmid mouse cDNA library. An interaction between RNA bait and protein prey results in translation of the reporter genes that can be detected by growth on his(-) media and verified by positive

X-gal staining. From over 200 positive hits, we focused on two that showed high levels of reporter activity. Large ribosomal protein 4 (RPL4) was recovered for its ability to interact with the MoMLV pseudoknot sequence; in particular the C-terminus of the protein was found to bind RNA. RPL4 was also able to bind a no-pseudoknot control, as verified in gel shift assays, though the strength or frequency of the interaction has not been tested (Houck-Loomis 2009). Small ribosomal protein 3a (RPS3a) was also recovered.

RPL4 is highly conserved among all kingdoms (Lecompte, Ripp et al. 2002). In bacteria,

RPL4 lines the exit tunnel of the ribosome, and has extensions near the peptidyl transferase center (Zengel 2003), and eukaryotic RPL4 appears to occupy a similar

72 position in the 80S ribosome (Taylor, Devkota et al. 2009; Ben-Shem, Garreau de

Loubresse et al. 2011; Klinge, Voigts-Hoffmann et al. 2011) (Figure 3-1). The eukaryotic version of RPL4 has a large extension that is not present in the prokaryotic protein and is located in close proximity to the peptidyl transferase center and is interconnected to eukaryotic specific extensions of other large subunits proteins which ring the peptide exit tunnel. Single point mutations in bacterial RPL4 can confer antibiotic resistance in E. coli although there appears to be no direct contact between RPL4 and the decoding center where aminoglycosides interact with the ribosome (O'Connor 2004) . Mouse RPL4 has been shown to interact with helicase Gu alpha in the nucleolus and to have a role in the processing of ribosomal RNA (Yang, Henning et al. 2005). It also has a nuclear function, increasing the transcription of myb-regulated genes (Egoh, Nosuke Kanesashi et al.

2010).

To measure changes RPL4 might have on readthrough efficiency, we employed a dual luciferase assay with a two-plasmid reporter system (Grentzmann, Ingram et al. 1998).

The MoMLV gag stop codon and pseudoknot sequence was cloned into a plasmid between Renilla and firefly luciferase coding sequences, thus allowing expression of firefly luciferase only when the readthrough event has occurred (Figure 3-2), paralleling the expression of gag and gag-pol. To control for effects on overall translation, and to allow normalization of basal readthrough levels independently of background or transfection efficiency, we also constructed a control plasmid containing the same

MoMLV sequence with a glutamine codon replacing the wild type stop codon; the control

73 A Type to

B

Figure 3-1. Architecture of 60S Ribosomal subunit and detail of RPL4. (A)View of the solvent exposed side of the 60S subunit., RPL4 circled in green (B) Evolutionary representation of RPL4; protein core found in all kingdoms are depicted in light blue, protein extensions unique to eukaryotes are in red. Positions of the N and C terminus are indicated. From (Klinge, Voigts-Hoffmann et al. 2011). Reprinted with permission from AAAS.

74 A Readthrough/Pseudoknot: MoMLV (13PK6)

Rluc UAG PK Fluc Rluc CAG PK Fluc

Wildtype XMRV/MoMLV PK Control XMRV/MoMLV PK

Rluc UAG PK Rluc CAG PK Fluc

Wildtype XMRV/MoMLV PK Control XMRV/MoMLV PK

B

% recoding = (Fluc wildtype/Rluc wildtype) x 100 (Fluc control/Rluc control)

Figure 3-2. Schematic of 13PK6 luciferase assay reporter constructs and calculation of readthrough. (A) The dual luciferase reporters contain 83 base pairs of of the MoMLV gag:pol junction, with 13 base pairs before the UAG stop codon and 6 base pairs after the end of stem 2 of the pseudoknot structure. The gag:pol junction sequence is between the renilla luciferase and firefly luciferase genes, allowing translation of firefly luciferase only when a readthrough event occurs. The experimental/wildtype construct contains the UAG stop codon, and the control construct substitutes a glutamine CAG to allow maximum translation of firefly luciferase to allow for normalization. (B) Readthrough efficiency is calculated as the ratio of firefly to renilla luciferase signals from the experimental construct divided by ratio of firefly to renilla luciferase signals from the control construct, multiplied by 100 to give percent readthrough.

75 plasmid was transfected in parallel into separate cells. Percent readthrough was calculated as the ratio of firefly:renilla signal from wild-type sequence divided by the ratio of firefly:renilla signal from control sequence multiplied by 100. A series of plasmids containing various lengths of the sequences upstream of the gag stop codon and after the pseudoknot structure were initially tested. Flanking sequences affect the amount of readthrough, and the 13PK6 reporter pair (13 amino acids before the stop codon and 6 amino acids after) was chosen as readthrough levels were most consistent and were equivalent with those obtained with longer sequences (Houck-Loomis 2009).

mRPL4 enhances readthrough at the MoMLV gag:gag-pol junction.

To investigate whether the interaction between RPL4 and the pseudoknot had a functional effect, an expression vector for mouse RPL4 was transfected into 293A cells along with the 13PK6 and 13PK6-C reporter plasmids. Results of the dual luciferase assay (DLA) showed a dose dependent increase in the amount of readthrough that correlated with the amount of RPL4 transfected (Figure 3-3). Readthrough increased from a basal level of about 4% to over 9% in cells transfected with the maximum amount of RPL4 cDNA.

Small ribosomal protein 3a (S3a), which was also a hit in the yeast 3-hybrid screen, was transfected in parallel in separate cells as a control. S3a transfection did not result in an increase in readthrough. Raw values of firefly and Renilla luciferase indicate that the transfection of ribosomal cDNAs did not significantly affect the translation levels of reporter constructs.

76 A

Type to enter text

B

Type to enter text

Figure 3-3. mRPL4 effects on readthrough of MoMLV gag-pol junction. (A) Dual luciferase analysis of readthrough of MoMLV pseudoknot and control reporter constructs 13PK6 and 13PK6-C transfected into HEK 293A cells, transfected in 96-well format. Error bars correspond to standard deviation of averages derived from 3 wells transfected with either wild-type or control reporter. (B) Raw values of luciferase from transfection in figure A. Fluc and Rluc refer respectively to firefly and renilla values of the wild-type reporter; Fluc control and Rluc control correspond to firefly and renilla values of the control reporter.

77 RPL4 effects on readthrough are cell line dependent

To investigate whether enhancement of readthrough was a universal effect we tested

RPL4 and RPS3a in a number of cell lines from different species and tissues (Table 3-1).

RPL4 increased readthrough in many, but not all, cell lines tested (Figure 3-4).

Differences were not attributable to species or lineage of cell types; both mRPL4- responding and non-responding groups contained human, rodent, fibroblasts, and tumor- derived or transformed cells. Mouse and human cells responded to mRPL4 transfection with increased readthrough of 13PK6, indicating that the effect seen in 293A cells was not attributable solely to a mouse protein being transfected into a human line.

Additionally, RPL4 did not increase readthrough in several human cell lines tested

(HeLa, Te671, A549). Although these non-responding cells were all tumor derived, RPL4 did not increase readthrough in Rat2 fibroblast cells, nor in other mouse lines tested (data not shown). Variations in absolute values of luciferase signals indicated that transfection efficiencies or other conditions may have affected basal levels of readthrough; however, there was no correlation between response to RPL4 and basal levels of readthrough. All signals were well above background and within the linear range of the assay.

There was a surprisingly wide range in the basal level of readthrough among the cell lines surveyed. Among the non-responding lines, basal readthrough ranged from 5% to greater than 10%. In responding cell lines the range was lower, from 3% to 7%. All cell lines were transfected with S3a as a negative control and showed no increase in readthrough

(data not shown). The variety in response suggests that readthrough may be modulated by

78 Cell line Species Tissue type

293A human embryonic kidney

293T human embryonic kidney transformed with T antigen

A549 human lung carcinoma

Balb mouse embryonic fibroblast

BHK hamster kidney

HeLa human cervical carcinoma

La-4 mouse lung adenoma

NH3T3 mouse embryonic fibroblast

Sirc rabbit corneal epitheluem

Te671 human medullablastoma

Rat2 rat embryonic fibroblast

Table 3-1: Cell lines used in dual luciferase assay for survey of RPL4 effects.

79 20

16

12

8 % Readthrough

4

0 Sirc Balb La­T BHK Rat2 293T A549 293A HeLa Te671

VC!"#$%&"%' NIH3T3 L4 60 ng()'*+',- L4 120 ng()'./+',- A L4 240 ng()'/)+',-

109 108 107 106 105 104 103 Luciferase Units 102 101 100 Sirc Balb Rat2 BHK La­T 293T A549 HeLa 293A Te671 NIH3T3 C­ Fluc!"#$%& B C­ Rluc!"'(%&

!"#$%&'()'*+,'&--&./0'12'313+4'#5#6718'%&59/:%1$#:;'.&88'8"2&'0$%<&=)Figure 3-4. RPL4 effects on MoMLV gag-pol readthrough: cell line survey. >)'?$58'8$."-&%50&'5258=0"0'1-'%&59/:%1$#:'1-'313+4'70&$91@21/'529'.12/%18'%&71%/&%'.120/%$./0'ABCDE'529'(A) Dual luciferase analysis of readthrough of MoMLV pseudoknot and control ABCDE6F'/%520-&./&9'"2/1'5'752&8'1-'%19&2/G'%5HH"/'529':$*52'.&88'8"2&0'I"/:'"2.%&50"2#'5*1$2/0'1-'*+,'.?J>)'reporter constructs 13PK6 and 13PK6-C transfected into a panel of rodent, rabbit and K%57:'"0'5<&%5#&'1-'(6,'&L7&%"*&2/0'-1%'&5.:'.&88'8"2&G'/%520-&./&9'"2'ME6I&88'-1%*5/)'N)'O5I'<58$&0'1-'8$."-human cell lines with increasing amounts of mRPL4 cDNA. Graph is average of 2-4 6 &%50&'-%1*'%&71%/&%'128='<&./1%'.12/%18'P4FQ'-1%'&5.:'&L7&%"*&2/0'$0&9'-1%'!"#$%&'A)'!68$.'.12/%18'529'O68$.'experiments for each cell line, transfected in 96-well format. (B) Raw values of !"#$%"&'!"%%()*"#+'$"',%(-.'/#+'%(#0&&/'1/&2()'"3'$4('!"#$%"&'%(*"%$(%luciferase from reporter only vector control (VC) for each experiments5' used in (A). Fluc control and Rluc control correspond to firefly and renilla values of the control reporter.

80 additional cellular factors, and is not purely a cis-acting function of the pseudoknot- containing viral mRNA.

At the time of this work, xenonotropic murine leukemia-like virus (XMRV) had been recently discovered and its role in prostate cancer (Urisman, Molinaro et al. 2006) and chronic fatigue syndrome (Lombardi, Ruscetti et al. 2009) was being evaluated. XMRV and MoMLV share the same sequence at the gag-pol junction; XMRV also utilizes readthrough for expression of gag-pol. Retroviruses known to cause pathology in humans employ frameshift; XMRV offered an opportunity to study readthrough in a human system. Thus we choose the human cell line 293A for the majority of our experiments in anticipation that our work might be directly applicable to human disease.

Mouse and human RPL4 both enhance MoMLV/XMRV gag-pol readthrough

RPL4 is considered a conserved protein among kingdoms, and the overall sequence is very similar among mammals. The N-terminal region from amino acids 1-350 are essentially identical between mouse and human (residue 170 is glutamate and 208 is methionine in mouse, leucine and isoleucine respectively in human) and the human protein is slightly longer overall. The C-terminal 17 amino acids, as well several other lysine-containing regions close to the C-terminus, are identical. We cloned the human version (hRPL4) and transfected it into 293A cells with the DLA reporter constructs; readthrough was also increased, although to a lesser degree than with mouse RPL4

(Figure 3-5).

81 A !"#

2.0

1.5

1.0

0.5 Fold change in readthroguh

0 VC 60 ng 120 ng 240 ng

!"#mouse L4!"# Type $"#human L4$"# A to B 108 C 107 106 105 104 103

2

Luciferase units 10 101 100 VC 60 ng 120 ng 240 ng F­luc!"#$%&'( B R­luc!"#$)&'( F­luc control!"*#$%+'(,- R­luc control!"*#$)+'(,- Type108 to10 7 106 105 104

3 Figure 3-5.10 mRPL4 and hRPL4 induced increase of MoMLV gag-pol readthrough. Luciferase units 2 (A) Fold change10 in readthrough of 13PK6 and 13PK6-C transfected into 293A with 101 increasing amounts of mouse RPL4 or human RPL4. Data shown from single 100 representative experiments.VC 60 ng Fold120 ng change240 ng calculated from readthrough level in +m/ hRPL4 conditionsC divided by basal readthrough. (B) Raw values of luciferase for mouse RPL4 transfection used for (A). (C) Raw values of luciferase for human RPL4 !"#$%&'()'*+,'-./'0+,'"./$1&/'".1%&-2&'34'535+6'#-#7839'%&-/:0%3$#0)transfection used for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enhancement of readthrough is distinctive among ribosomal proteins

To address whether the increase in readthrough with mRPL4 was a general consequence of over-expression of a ribosomal protein we repeated the dual-luciferase assay with additional ribosomal proteins. The proteins chosen included several with known extra- ribosomal functions, and also some with native positions deep within the ribosome.

Transfection of 293A cells with cDNAs encoding these proteins resulted in no significant increase in readthrough, in contrast to the level of enhancement seen with RPL4 (Figure

3-6). With the exception of the acidic stalk protein RPLP0, transfection of cDNAs for all proteins gave similar levels in raw reporter luciferase values .

To verify that the cDNAs were being expressed, c-terminal myc-tagged versions of all proteins were constructed, and tested in the DLA system. However, addition of the C- terminal myc tag to RPL4 abolished the enhancement of readthrough, and reduced the slight increase seen with S20 and L7 to the basal level; effects of all other tagged ribosomal proteins corresponded with the non-tagged version (Figure 3-7). Expression of the myc-tagged proteins, with the exception of L22, was virtually undetectable by western blot (Figure 3-8). This suggests that expression of many of these ribosomal proteins is tightly regulated; it may not be possible to accumulate a detectable above- normal amount in vivo. The addition of the tag may inhibit proper folding of the proteins and promote degradation; this could also account of the absence of proteins seen in the blot.

83 A 22

1.-0

!"#$!%"!&$'() 0.0 *+&',&-. /0+&',&-. 01+&',&-. !"#$%&'()*+,( -./0

7 )486 )456 )48/ )480 )45-7 )4822 )4527 )48-- )4897 )4526

!"#$%&"%' ()'*+',- Type to ()'./+',- enter3 text ()'/)+',- B

271

27/ 27<

270

276

27; 8+=>?$)%@$"AB>'@ 27-

272

277 )486 )456 )48/ )480 )45-7 )4822 )4527 )48-- )4897 )4526

CDE+=!"#$%&'( #DE+=!"#$)&'(

CDE+="=*B')*E!"*#$%+'(,- Type :to #DE+="=*B')*E!"*#$)+'(,- enter text !"#$%&'()'*$%+&,'-.'&..&/0'-.'122"0"-314'%"5-6-714'8%-0&"36'-3'9-9:;'#1#<8-4'%&120=%-$#=) >)'?&120=%-$#='8&%/&301#&6'516&2'-3'2$14'4$/".&%16&'1661,'%&6$406'-.'9-9:;'86&$2-@3-0'132'/-30%-4'%&8-%0&%' /-360%$/06'ABCDE'132'ABCDE'"3'JKB>'Figure 3-6. Survey of effect of additional ribosomal proteins on MoMLV gag-pol /&446'"3'KE'G&44'.-%710)'L%18='6=-G6'%&8%&6&3010"+&'&M8&%"7&306'-.'10'4&160'B'1661,6'G"0='&1/='8%-0&"3)'N%%-%'readthrough. (A) Readthrough percentages based on dual luciferase assay results of 51%6'/-%%&68-32'0-'601321%2'2&+"10"-3'-.'1+&%1#&6'2&%"+&2'.%-7'B'G&446'0%136.&/0&2'G0='&"0=&%'G"42<0,8&'-%'/-3MoMLV pseudoknot and control reporter constructs 13PK6 and 13PK6-C co-transfected < 0%-4'%&8-%0&%)'O)'?1G'+14$&6'-.'4$/".&%16&'+14$&6'.-%'%&8-%0&%'-34,'P;FQ'/-30%-46'.-%'&M8&%"7&306'$6&2'"3'!"#$%&'with increasing amounts of ribosomal protein cDNA in 293A cells in 96 well format. !"#$$%&'()$*+,$-&'()$./0/.$./12/)345/'6$37$8./96$*+,$./+4''*$5*'(/1$70$3:/$;4',&362/$./27.3/.<$%&'()$)7+3.7'$*+,$Graph shows representative experiments of at least 3 assays with each protein. Error bars -&'()$)7+3.7'$)7../127+,$37$8./96$*+,$./+4''*$5*'(/1$70$3:/$)7+3.7'$./27.3/.correspond to standard deviation of averages derived from triplicate# readings of each condition. (B) Raw values of luciferase values for reporter only (VC) controls for experiments used in A.

84 A 10

8

6 5.4 5.2 5.5 5.5 4.6 4.8 4.7 4.7 4.3 4.3 4.1 4 % readthrough 2

0 VC RPL4 RPL5 RPL7 RPS4 RPL11 RPL22 RPS10 RSP14 RPS20 RPS3a Ribosomal protein

B 1E+08 1E+07 1E+06 1E+05 Av. Fluc Av. Rluc 1E+04 Ave. FLuc-C Ave. RLuc-C 1E+03

Luciferase Units 1E+02 1E+01 1E+00 VC RPL4 RPL5 RPL7 RPS4 RPL11 RPL22 RPS10 RSP14 RPS20 RPS3a Ribosomal protein Figure 3-7. Effect of myc-tagged ribosomal proteins on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of MoMLV pseudoknot and control reporter constructs 13PK6 and 13PK6-C co-transfected with increasing amounts of myc-tagged ribosomal protein cDNA in 293A cells in 96 well format. Graph shows representative experiment, error bars denote standard deviation of averages from 3 wells transfected with either wild-type or control reporter. (B) Raw values of luciferase values for reporter only (VC) controls for experiments used in (A).

85 Vector only

Endogenous

Figure 3-8. Expression of myc-tagged ribosomal proteins: western blot of myc- tagged ribosomal proteins. 293A cells were co-transfected with 6 ug of each myc- tagged ribosomal protein and 1 ug 13PK6 or 13PK6-C in 6 well format. 40 mg of total protein was loaded into each lane, and blots were probed with anti-myc antibody. Boxes highlight proteins detected at the expected size for each tagged ribosomal protein.

86 We next prepared an N-terminal Flag-tagged version of RPL4. We transfected 293A cells with the Flag-tagged RPL4 and the 13PK6 reporter pair, and performed the DLA. In multiple retrials we saw no increase in readthrough with an N-terminal tagged mRPL4, and we were only able to see able to see faint expression of this construct via western blot. We subsequently re-cloned RPL4 into a new 3X N-terminal flag vector. We could see robust expression of this construct via western blot. However, as we observed before, the 3X N-terminal flag tagged RPL4 did not increase readthrough in the DLA (Figure

3-9).

In an attempt to avoid use of tags, we obtained several commercial antibodies against human/mouse RPL4, and examined lysates via western blot. Although we could visualize

RPL4, we could not detect a significant increase in protein levels corresponding to increased transfection of either mouse or human RPL4 cDNA (Figure 3-10). As an alternate method, we performed q-PCR on RNA obtained from 293A cells transfected with mouse RPL4. To distinguish between endogenous and induced expression of RPL4 we designed primers to a region unique to the mouse protein. Transfection of increasing levels of mouse RPL4 cDNA resulted in proportional increases in levels of mouse RPL4 mRNA accumulating in the cells (Figure 3-11). Therefore, we can be confident that the

RPL4 transcript is produced by the expression plasmid. However, we cannot detect any increase in protein levels.

Since we observed an increase in mRNA, and because of the difficulty in visualizing

87 A B 10

8 IP for Flag-tagged L4, !-hL4

5.43 M Flag IP Flag- IP Flag IP Flag-IP hL4 hL4 Endogenous 6 Lysate E lysate C C E C E 4 3.19 3.21 2 % readthrough 0 VC mL4 Flag-tagged mL4

C D Exp Control 10 Flag Flag 8 6.6 VC mRPL4 mRPL4 VC mRPL4 mRPL4 6 3.4 3.7 4 2 % readthrough 0 VC mL4 3XFlag mL4 Figure 3-9. Expression of N-terminal Flag-tagged RPL4. (A) Readthrough percentages based on dual luciferase assay results of MoMLV pseudoknot and control reporter constructs 13PK6 and 13PK6-C co-transfected with 6 ug of p2tag flag-tagged or untagged mRPL4 in 293A cells in 6 well format. (B) Western blot of lysates transfected with tagged mRPL4 or untagged hRPL4 and 13PK6 reporter pair. Flag tagged lysates were immunoprecipitated with anti-Flag beads and lysates were blotted with poly-clonal anti RPL4. (C) Readthrough percentages based on dual luciferase assay results of MoMLV pseudoknot and control reporter constructs 13PK6 and 13PK6-C co-transfected with 6 ug of 3X-flag-tagged or untagged mRPL4 in 293A cells in 6 well format. (D) Western blot of lysates from (C) probed with anti-flag antibody.

88 6 ug 6 ug A VC mRPL4 hRPL4 BG RPL4 actin

B 6

5

3

% readthrough 2

0 VC mL4 hL4

C 0.20 0.15 0.10 L4/actin 0.05 0 VC mL4 hL4 endogenous

Figure 3-10. Expression of mRPL4 and hRLP4. (A)Western blot of lysates from 293A cells transfected with 6 ug of mRPL4 or hRPL4 and the 13PK6 reporter pair. (B) Readthrough from cells in (A) analyzed by DLA. (C) Quantification of signals from western blot in (A), ratio of RPL4 to actin.

89 0.020

0.015

0.010

0.005 Mean Qty L4/actin

0 1ug 3ug 6ug 9ug endogenous vector only RPL4 cDNA

Figure 3-11. qPCR analysis of RPL4 mRNA levels in 293 cells. Cells were transfected mRPL4 expression vector and harvested 24 hours later for qPCR analysis. mRPL4 mRNA signals were normalized to actin mRNA expression.

90 over-expression of protein, we considered that the effect on readthrough could be induced by the RNA itself. RPL4 regulates it own transcription in bacteria (Zengel and Lindahl

1992; Zengel, Vorozheikina et al. 1995); we hypothesized that the increased levels of transcribed mRNA may not be translated, but that the RNA itself could be interacting with the translation machinery to enhance readthrough. We designed an RPL4 frameshift

(L4-FS) mutant by deleting the 11th nucleotide from the coding sequence; the sequence downstream of the deletion has an artificially +1 shifted reading frame that contains multiple stop codons (Figure 3-12). This produces an mRNA with essentially the same sequence, retaining the overall secondary structure, but the altered coding sequence results in a different, shorter protein. Transfection of the L4-FS into 293A cells did not significantly increase readthrough of the 13PK6 reporter pair over basal levels (Figure

3-13) . Thus, increased readthrough is likely not caused by the RPL4 mRNA.

RPL4 mutants do not increase readthrough

Concurrent with our efforts to visualize L4 overexpression, we cloned two panels of L4 mutants to try to define regions essential for the increase in readthrough. Initial work in our lab showed a requirement for the N terminus of RPL4 (Houck-Loomis 2009); versions of RPL4 with deletion of the N-terminus failed to stimulate an increase in readthrough. This work was done before the availability of RPL4 antibodies, so expression of the mutants was not verified. However, it is likely that this region is necessary since the 3X terminal flag tagged versions of full-length RPL4 were produced in vivo, but had no effect on readthrough.

91 A RPL4 cDNA

ATG GCT TGT GCC CGT CCC CTC ATA TCG GTG TAC TCC GAA AAG GGA GAG TCA TCT GGC AAG AAT GTC ACT TTG CCA GCT GTG TTC AAA GCT CCC ATT CGA CCA GAT ATT GTG AAC TTC GTT CAC ACC AAC TTG AGG AAA AAC AAC AGA CAG CCC TAT GCC GTC AGT . . .

B RPL4

MACARPLISVYSEKGESSGKNVTLPAVFKAPIRPDIVNFVHTNLRKNNRQPYAVSELA......

C RPL4-FS

MACAVPSYRCTPKRESHLARMSLCQLCSKLPFDQIL*TSFTPT*GKTTDSPMPSVNW ......

Figure 3-12. RPL4-FS mutant. (A). The beginning portion of the mRPL4 coding sequence. The RPL4-FS mutant was constructed by eliminating the 11th base from the coding sequence. The secondary structure of the mRNA should remain relatively unchanged from the single deletion (B) The N-terminal region of the normal RPL4 protein. (C) The N-terminal region of RPL4-FS. The RPL4-FS has a different peptide sequence after the fourth amino acid and multiple stop codons.

92 8 6 5.31

3.09 4 2.66

% readthrough 2 0 VC L4-FS mRPL4 RPL4 expression construct

Figure 3-13. Effect of L4-FS on readthrough of the MoMLV gag-pol junction. Readthrough percentages based on dual luciferase assay results of MoMLV 13PK6 reporter pair co-transfected with 6 ug of mRPL4 or mRPL4-FS n 293A cells in 6 well format. Graph shows representative experiment.

93 Similarly, we designed a series of RPL4 mutants that were truncated at the C terminus.

We designed these mutants to end the protein at the start of regions of non-homology between the mouse and the human version (Figure 3-14). None of the C-terminal truncation mutants supported a significant increase in readthrough (Figure 3-15). This result correlates with the C-terminal myc-tagged RPL4 result, indicating a requirement for the C terminus of the protein. We were not able to see expression of these mutants via

Western blot.

RPL4 interacts directly with helicase Guα to facilitate rRNA processing in the nucleolus

(Yang, Henning et al. 2005) and therefore we thought perhaps RPL4 could also interact with the ribosomal helicase or another helicase which may be involved in unwinding the pseudoknot structure. Yang et. al. mapped the region of RPL4 that binds to Guα to amino acids 264-428. We designed 4 mutants in which portions of or the entire of this region were deleted (Figure 3-16). We tested these mutants in the dual luciferase system. No increase in readthrough was seen with the mutants (Figure 3-17). We were not able to directly visualize expression of the altered proteins; the deletions may have been extreme enough to prevent the proteins from proper folding or to induce another measure of quality control. Therefore, the importance of the helicase binding domain can not be evaluated from these experiments. Additionally, the deletions substantially decreased the length of the protein, which may be a defining property in the promotion of readthrough.

94 Figure 3-14: C-terminal differences between mouse and human RPL4 and schematic of RPL4 c-terminal truncation mutants. (A) Alignment of C-terminal regions of mouse and human RPL4. The first 340 base pairs are essentially identical among species. (B) Schematics of mRPL4 c-terminal truncation mutants.

95 8 6.9

6 4.6 3.9 4 3.6 3.5

% Readthrough 2

0 Vector Full length trunc 402 trunc 388 trunc 354 RPL4 expression construct

Figure 3-15. RPL4 C-terminal truncation mutants effect on readthrough. Readthrough percentages based on dual luciferase assay results of MoMLV 13PK6 reporter pair co-transfected with 6 ug of mRPL4 or C-terminal truncation mutants in 293A cells in 6 well format. Graph represents summary of 3+ independent experiments. Error bars denote standard deviation.

96 Figure 3-16. Schematic of RPL4 helicase-binding domain mutants. The helicase- binding domain has been mapped to amino acids 264 to 333. Straight lines denote segments deleted in each construct.

97 8

6.1 6

4 3.4 3.4 3.6 3.5 3.0 % readthrough 2

0 VC del 264 del 265 del 290 del 320 full length RPL4 expression vector

Figure 3-17. Effect of RPL4 helicase-binding domain mutants on MoMLV gag-pol readthrough. Readthrough percentages based on dual luciferase assay results of MoMLV 13PK6 reporter pair co-transfected with 6 ug of mRPL4 or helicase-binding domain mutants in 293A cells in 6 well format. Graph shows representative experiment.

98 RPL4 Time Course

It was observed previously in our lab that increased expression of RPL4 is likely down- regulated over time. Attempts by a former lab member to create a stably over-expressing cell line revealed that the increase in readthrough is limited to approximately 48 hours.

In addition, the obstacles to visualizing RPL4 levels complicated the verification of over- expression. We normally harvest the lysates from our transfections at 24 hours, so we wondered if an early spike in RPL4 over-expression was responsible for starting a series of events that resulted in increased readthrough. We theorized that perhaps by the time we harvested samples for Western blot the expression of RPL4 returned to normal levels, but downstream effects were still present. We thus conducted a time course to observe levels of readthrough and corresponding levels of RPL4.

We transfected 293A cells with RPL4, and took samples for DLA and WB analysis at various time points. Readthrough reached the highest level at 24 hours with co- transfection of RPL4 (Figure 3-18). To measure RPL4 expression, we quantified the intensity of the RPL4 signal and compared it to the intensity of the actin signal in the western blots. Quantification of RPL4:actin showed the highest expression at 24 hours compared to other time points during the trial. This ratio was much lower than the level of RPL4:actin in untransfected cells, as seen previously (Figure 3-10) which may be an artifact of transfection. However, we were able to verify that timing of our DLA and WB assays were correct: we were not missing the peak of the effect or an early spike in RPL4 expression as detectable by our methods.

99 A 5.00

4.00

3.00 VC L4 2.00 % readthrough

1.00

0 6 hours 10 hours 24 hours 28 hours Time

B 6 hr 10 hr 24 hr 28 hr endo E C E C hRPL4 actin

C 1.00 0.75 0.50 +OE RPL4 endogenous L4/actin 0.25 0 6h 10h 24h 28h Time

Figure 3-18: Timecourse of RPL4 enhancement of readthrough and corresponding RPL4 expression. (A) Readthrough of 13PK6 reporter pair in 293A cells co-transfected with 6 ug hRPL4. (B) Western blot analysis of human RPL4 in 293A cells; lysates were blotted with poly-clonal anti-RPL4 and anti-actin. (C) Quantification of signals in (B), endogenous sample taken from untransfected cells.

100 RPL4 enhances translation of other functional viral recoding sequences

Recoding also occurs in other contexts in the translation of viral proteins, i.e the -1 frameshifting of the HIV-1 transcript; the HIV-1 sequence does not contain a pseudoknot, but forms a stem-loop downstream of the recoding site. The genome of the alphavirus

Sindbis utilizes a leaky termination sequence, a UGA stop codon followed by a cytidine, to control the ratio of its structural proteins. It has been believed that this stop codon and downstream base, termed the tetranucleotide code, was solely responsible as the viral recoding sequence, without any contribution of a secondary structure (Li and Rice 1989;

Li and Rice 1993). Recently it has been shown that in the context of the virus additional mRNA sequences promote readthrough to the levels necessary for viral replication (Firth,

Wills et al. 2011). However, for this work, we included only the leaky stop codon sequence UGA C; the additional sequence contributions were not known at the time of the experiments.

We tested RPL4 for effects on HIV-1 frameshift and the Sindbis readthrough sequences in our dual luciferase assay. The reporter plasmids for HIV-1 contain the slippery sequence and downstream 62 nucleotides containing the proposed extended stem loop mRNA structure for the wild-type construct; the control disables the native slippery sequence to prevent frameshifting and adds an additional nucleotide to allow translation of the functional fluc reporter. The HIV-1 construct showed about 6% frameshift efficiency. To test Sindbis readthrough we cloned a small fragment containing the UGA C

101 tetranucleotide code in the cloning site between the reading frames of the reporter; no other viral sequences were included, eliminating the possibility of viral specific secondary mRNA structure. The control plasmid replaced the UGA stop with CGA to allow maximum translation of the reporter. The UAG C tetranucleotide sequence promoted suppression of termination at a significant rate, about 3% of the time using our

DLA reporter, which is comparable to the low end of MoMLV readthrough.

Transfection of the RPL4 expression construct increased HIV-1 frameshift in a dose dependent manner similar to MoMLV readthrough, indicating that the effects of RPL4 are not limited to suppression of termination, or dependent on a specific pseudoknot structure (Figure 3-19). Surprisingly, RPL4 also increased readthrough of the Sindbis recoding sequence when transfected at high levels. Since Sindbis recoding is not dependent on a secondary structure, the RPL4 effect may be due in part to an interaction that affects decoding or interferes with termination.

In an attempt to verify whether the RPL4 enhancement of readthrough was related to the presence of a recoding sequence, rather than a general effect on termination, we tested mRPL4 in the DLA with a construct containing a scrambled pseudoknot (ScrPK) which retains the nucleotide composition of the native sequence but eliminates the predicted secondary structure. However, raw values of this reporter construct were below the linear range of the DLA suggesting that the mRNA was not being translated at a significant level. We considered that this reporter construct was being targeted by the nonsense

102 15 15

12 12

15 15 9 VC!"#$%&"%' 9 L4 60 ng()'*+',- 12 12 L4 120 ng()'./+',- 6 L4 240 ng()'/)+',- 6 9 VC!"#$%&"%' 9 L4 60 ng()'*+',- Recoding

L4 120 ng()'./+',- % Readthrough

% Readthrough 3 6 3 L4 240 ng()'/)+',- 6 % Readthrough

% Readthrough 3 3 0 0 VC 0 0 VC VC VC HIV­1 ScrPK HIV­1 MoMLV ScrPK Sindbis 3 ug mL4 3 ug mL4 MoMLV

A Sindbis 6 ug mL4 3 ug mL4 6 ug mL4 A 3 ug mL4 F 6 ug mL4 F 6 ug mL4 U38C U38A U38C U38A

108 108 108 8 8 8 107 10 107 10 107 10 106 107 106 107 106 107 5 5 5 10 6 10 6 10 6 10 10 4 10 104 104 10 5 103 105 103 105 103 10 102 4 102 4 102 4

Luciferase units 10 Luciferase units 10 Luciferase units 10 1 1 1 10 103 10 103 10 103 100 100 100 2 2 2 VC VC 10 VC 10 VC 10 Luciferase units Luciferase units Luciferase units 60 ng 1 60 ng 1 1

120 ng 10 240 ng 10 10 120 ng B 240 ng C !

3 ug mL4 0 0 0 3 ug mL4 10 10 6 ug mL4 10 6 ug mL4 108 108 U38C U38A VC 7 7 VC VC 10 VC 10 106 106 60 ng 60 ng

120 ng 5 240 ng 105 10 120 ng B 240 ng C F­luc!"#$%&'( ! 4 4

10 10 3 ug mL4 R­luc!"#$)&'( 3 ug mL4 3 3 6 ug mL4 10 10 F­luc control!"*#$%+'(,- 6 ug mL4 8 108 102 10 102 R­luc control!"*#$)+'(,- U38C U38A Luciferase units Luciferase units 7 7 101 10 101 10 100 106 100 106 VC 105 VC 105

60 ng F­luc!"#$%&'( 60 ng 240 ng 4 120 ng 240 ng 4 120 ng D 10 E 10 R­luc!"#$)&'( 103 103 F­luc control!"*#$%+'(,- 102 102 R­luc control!"*#$)+'(,- Luciferase units

Figure 3-19.Luciferase units Effect of RPL4 on additional viral recoding sequences. !"#$%&'()'*++&,-'.+'/0'.1'.-2&%'3"%45'416'7$-41-'8.8/9':;&$6.<1.-';&=$&1,&;> 101 101 (A) Readthrough?)'@.7:4%";".1'.+'%&46-2%.$#2'.+'8.8/9'%&46-2%.$#2A''BC9DE'+%47&;2"+-A'F"16G";'%&46-2%.$#2A'416'F,%HI' percentages of 293A cells co-transfected with dual luciferase 0 100 reporters %&46-2%.$#2'"1'JKL?',&55;',.D-%41;+&,-&6'M"-2'6$45'5$,"+&%4;&',.1;-%$,-':4"%;'416'"1,%&4;"1#'47.$1-;'.+'7/0A'for 10HIV-1 frameshift, Sindbis readthrough, and ScrPK readthrough and VC increasing-%41;+&,-&6'"1'K('M&55'+.%74-)'N&46-2%.$#2'5&3&5;'+%.7'%&:%&;&1-4-"3&'&O47:5&'.+'L'-%41;+&,-".1;':&%',.1;-%$,-' amounts of mRPL4, transfected in 96 well format. ReadthroughVC levels from 60 ng representative:4"%)'*%%.%'G4%;',.%%&;:.16'-.';-4164%6'&%%.%'.+'43&%4#&;'.+'L'M&55;'-%41;+&,-&6'M"-2'&"-2&%'M"56D-P:&'.%',.1-%.5' examples per reporter60 ng pair. Error bars correspond to standard error of 240 ng 120 ng 240 ng D 120 ng averages %&:.%-&%',.1;-%$,-)'Q)'N4M'345$&;'.+'5$,"+&%4;&'+.%'8.8/9'64-4'"1'!"#$%&'R?)'@)'N4M'345$&;'.+'5$,"+&%4;&'+.%'of 3 wells. (B) Raw values of luciferase for MoMLVE data in (A). (C) Raw BC9DE'64-4'"1'!"#$%&'(?)'S)'N4M'345$&;'.+'5$,"+&%4;&'+.%'F"16G";'64-4'"1'!"#$%&'(?)'*)'N4M'345$&;'.+'5$,"+&%4;&' values of +.%'F,%HI'64-4'"1'!"#$%&'(?)''!)'N&46-2%.$#2'5&3&5;'G4;&6'.1'6$45'5$,"+&%4;&'4;;4P'.+'TLU@'2P:.'D'416'TLU?'luciferase for HIV-1 data in (A). (D) Raw values of luciferase for Sindbis data in (A). (E)2P:&%D4,-"3&'8.8/9':;&$6.<1.-'7$-41-;',.D-%41;+&,-&6'M"-2'"1,%&4;"1#'5&3&5;'.+'7/0'"1'('M&55'+.%74-)'*%%.%' Raw values of luciferase for ScrPK data in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mediated decay (NMD) pathway. This idea is supported by recent work from our lab which correlates readthrough translation with increased mRNA stability (Hogg and Goff

2010). Therefore the lack of the readthrough stimulating pseudoknot may predispose the

ScrPK reporter to nonsense-mediated decay at a rate that does not allow accumulation of substantial levels of luciferase. Even so, addition of mRPL4 to reactions containing

ScrPK did not rescue readthrough to levels within linear range of the assay.

RPL4 effect on readthrough of non-recoding premature stop codons is undetermined

The ScrPK construct we used as a no-pseudoknot control lacked the flanking sequences

— the 13 base pairs before the stop codon, and the 6 nucleotides after the proposed end of the structure. Since it has been previously shown that the presence of the flanking sequences do have an effect on readthrough, we felt it was important to also test these sequences in our study of the RPL4 enhancement on readthrough, in case the leader peptide was playing a role. More importantly, in scrambling of the pseudoknot sequence, the nucleotide immediately following the stop codon was changed from a G to an A. This tetranucleotide sequence is known to have a profound effect on the incidence of readthrough; UAG A naturally does not readthrough as frequently as UAG G (Fan-

Minogue and Bedwell 2007). Therefore, the decrease in readthrough we observed on the

ScrPK sequence could be affected by differences other than the lack of the secondary structure. We thus designed a new construct, 13ScrPK6 which retained the tetranucleotide stop codon sequence of the wildtype gag-pol junction, contained the 13 nucleotides

104 before the stop codon and 6 base paris after the putative 2nd stem of the pseudoknot, and had the scrambled nucleotide sequence of ScrPK. We transfected the new 13ScrPK sequence into 293A cells. However, the overall levels of the reporters were still too low to be interpretable (Figure 3-20). The signal from firefly luciferase is inherently lower than Renilla, and an analysis of serial dilutions of Renilla and firefly luciferase shows that below total counts of 600 the assay is not linear (Figure 3-21). Thus the assay can fail to register accurate levels of very low readthrough, and an increase of a very low level remains unverifiable with these methods.

We observed that the renilla levels with the stop codon containing ScrPK and 13ScrPK constructs were noticeably lower than the rluc with the corresponding 13PK6 construct.

Low rluc levels, the denominator in our readthrough equation, result in the appearance of very high readthrough. Since rluc is placed before the pseudoknot in our reporters, renilla values give information on the general translation of the construct, indicating global cellular effects or specific attributes of the reporter. As with the ScrPK reporter, we wondered whether the 13ScrPK6 construct was more susceptible to NMD and if so, how we could assay for the effect of RPL4 on a simple premature termination codon. We thus attempted to reduce the effects of NMD by manipulating the cellular environment and also the reporter construct.

UPF1 is an RNA binding protein that is involved in mRNA decay (Isken and Maquat

2007) Although the specific mechanism of its effects are still unknown, UPF1

105 A

100

80 72.3

60

40

% Readthrough 20 3.4 0 13PK6 + C 13ScrPK6 + C Reporter construct

B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 13PK6 13ScrPK6 Reporter construct

Figure 3-20. Readthrough frequency of the 13ScrPK6 reporter. (A) Readthrough levels as determined by DLA of 13ScrPK6 reporter pair transfected into 293A cells in 6 well format. (B) Raw values of luciferase for data in A.

106 6827449 1E+07 4271383 2182898 1248698 1E+062382721 654946 1251858 348825 167996 466121 274443 84242 1E+05 197451 48102

5103152517 14013 30060 7606 1E+04 4004 11963 1942 1168 3120 2561 1E+03 611 487 535 366 1288 252

Raw Luciferase Units 469 402 380 288 289 294 1E+02 184

1E+01

1E+00 Untitled 1 Untitled 4 Untitled 7 Untitled 10 Untitled 13 Untitled 16 Untitled 19 IVT lysate dilution

Firefly Renilla

Figure 3-21. Linear range of the dual luciferase assay. Serial two-fold dilution of in-vitro translation reaction with HIV-1 control construct. This construct has very high expression of firefly and Renilla luciferase. Lysates were diluted in passive lysis buffer.

107 accumulation on mRNAs with a premature stop codon (PTC) seems, in many cases, to promote nonsense mediated decay. This is thought to be a sequence non-specific binding which is undisrupted by translation on mRNA’s with long 3’ UTR after the stop codon

(Hogg and Goff 2010). We attempted to circumvent NMD effects on our no-pseudoknot containing reporter, which is a transcript containing a premature stop codon and should appear as an aberrant mRNA, by using dominant negative UPF1 mutants.

We tested two UPf1 mutants, DD636AA and K498A (Kashima, Yamashita et al. 2006) with 13ScrPK6. We continued to see what looked like high levels of readthrough according to our calculation of ratios. However the raw values of the reporters again pointed to an anomaly; over-all levels of both were still low, and renilla in particular was lower (Figure 3-22). UPF1 also interacts with the termination machinery, and the dominant negative forms may be preventing termination. This would result in a delay in translation as ribosomes would be unable to be efficiently recycled, and a resultant decrease in total products. Therefore, we determined that interfering with the functions of

UPF1 may have effects on multiple aspects of translations which would be impossible to interpret in our system. Additionally, our two reporter system is designed so the maximum readthrough is 100%, as normalized to normal translation; readthrough percentages about 100% should not be possible, as the fluc signal from the control reporter should signify maximum translation.

We then utilized another retroviral motif, the Rous sarcoma virus stability element (RSE).

108 A 600 554.9 480 415.0 380.9 382.3 363.3 360 240 120 % Readthrough 4.5 0 13PK6 13ScrPK6 + UPF1 K498A .5 ug + UPF1 K498A .1 ug + UPF1 DD636AA 1 ug + UPF1 DD636AA .5 ug Reporter and UPF1 mutant constructs B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 13PK6 13ScrPK6 + UPF1 K498A .5 ug + UPF1 K498A .1 ug + UPF1 DD636AA 1 ug + UPF1 DD636AA .5 ug Reporter and UPF1 mutant constructs Figure 3-22. UPF1 dominant negative mutant effects on readthrough of 13ScrPK6. (A) Readthrough levels as determined by DLA of 1 ug 13ScrPK6 reporter pair co- transfected with dominant negative UPF1 mutants in 293A cells in 6 well format. The 13PK6 reporter pair was used as a positive transfection control. (B) Average raw values of luciferase for data in (A).

109 The RSE is an RNA sequence downstream of the gag stop codon, and is necessary for the stability of the mRNA (Weil and Beemon 2006; Weil, Hadjithomas et al. 2009). Deletion or movement of this element more than 500 bp away from the gag stop codon results in decreased amounts of gag-pol mRNA. RSV utilizes a -1 framesift for the translation of gag-pol, much like HIV-1. However, the RSE is not implicated in frameshift. The full- length RSE is approximately 400 bps long, but a fragment of about 250 bp containing the

3’ end (“C fragment”) has been shown to confer most of the protection against NMD. We cloned the C fragment into our 13ScrPK6 construct, after the scrambled pseudoknot and before the start of the fluc coding sequence. We transfected the 13ScrPK6_RSE constructs into 293A cells and performed the DLA. We again saw levels of rluc remained comparable with 13ScrPK6 (Figure 3-23), indicating the same amount of translation and/ or mRNA stability. However, the expression of fluc was markedly lower in the control reporter; fluc in the experimental (stop codon containing) reporter was still below the linear range of the assay. The straight readthrough calculated from the raw numbers showed a higher level than 13PK6 or 13ScrPK6, but the raw values suggested that additional factors could be altering expression of the constructs. We surmised that the

13ScrPK6 and RSE sequences could be translating a peptide moiety that was interfering with the functionality of the luciferase reporters, and thus not an appropriate control.

We thus decided to disregard the ScrPK containing constructs as proper controls for the presence of a recoding element. We designed a new dual luciferase reporter pair that utilized a segment of the collagen open reading frame which began with a glutamine

110 A 30 28.4

24

18

12

% readthrough 6 2.8 0 13PK6 13ScrPK6_RSE Reporter construct B 1E+08 1E+07 1E+06 Av. Fluc 1E+05 Av. Rluc 1E+04 Ave. FLuc-C Ave. RLuc-C 1E+03 1E+02

Luciferase Units 1E+01 1E+00 13PK6 13ScrPK6_RSE Reporter construct

Figure 3-23. Readthrough efficiency of 13ScrPK6_RSE reporter. (A) Readthrough levels as determined by DLA of 1 ug 13PK 6 or 13ScrPK6_RSE reporter pair transfected in 293A cells in 6 well format. (B) Average raw values of luciferase for data in (A).

111 CGA and extended the same length as the pseudoknot sequence. We replaced the CGA with a UAG to make an artificial PTC and designated this CollStop. The control plasmid contained the native sequence. We tested this construct for expression of rluc and fluc.

Levels of rluc were comparable to those of 13PK6 and 13PK6C, indicating a similar stability and/or overall expression of the mRNA. Fluc levels were also comparable for the control construct (Figure 3-24). The level of fluc in the experimental construct had a readthrough frequency of about 0.5%. Although fluc levels were in the 100s, still lower than the reliable limit of detection for the assay, the high rluc signal confirmed that this construct represented a context where NMD was not significant, and the lack of readthrough was a true event.

We transfected the CollStop reporter pair in to 293A cells along with increasing amounts of mRPL4. In some trials, we observed slight increases in the levels of fluc (Figure 3-25).

The levels were still below the linear range of the assay, but calculation of readthrough showed an increase in the presence of RPL4. Because this increase was not seen in all trials, and when present it was still below the range of the assay, it is hard to interpret the actual effect of RPL4 on any PTC or normal termination. However, the presence of a recoding stimulus appears to enhance the effect of RPL4.

PseudoKnot mutants and RPL4

Our lab has previously tested RPL4 on various pseudoknot mutants with impaired readthrough; RPL4 was not able to rescue readthrough of these hypoactive mutants

112 A 100 80 60 52.8 40

% readthrough 20 2.9 0.2 0 13PK6 CollStop 13ScrPK6 Reporter construct

B 1E+08 Av. Fluc 1E+07 1E+06 Av. Rluc 1E+05 Ave. FLuc-C 1E+04 Ave. RLuc-C 1E+03 1E+02

Luciferase Units 1E+01 1E+00 13PK6 CollStop 13ScrPK6 Reporter construct

Figure 3-24. Comparison of readthrough efficiency of reporter constructs. (A) Readthrough levels as determined by DLA of 1 ug 13PK6, 13ScrPK6_RSE, or CollStop reporter pairs transfected in 293A cells in 6 well format. Graph shows representative experiment. Error bars correspond to standard deviation of averages derived from triplicate readings of cells lysates. (B) Average raw values of luciferase for data in (A).

113 A 10

8

6 4.6 5.0 3.8 4 2.7

% Readthrough 2 0.2 0.3 0.5 0.4 0 13PK6 VC CollStop VC 13PK6 + L4 1 ug 13PK6 + L4 3 ug 13PK6 + L4 6 ug Collstop + L4 3 ug CollStop + L4 1 ug CollStop + L4 6 ug Reporter construct and RPL4 B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C 1E+02 Ave. RLuc-C Luciferase Units 1E+01 1E+00 13PK6 VC CollStop VC 13PK6 + L4 1 ug 13PK6 + L4 3 ug 13PK6 + L4 6 ug Collstop + L4 3 ug CollStop + L4 1 ug CollStop + L4 6 ug Reporter construct and RPL4

Figure 3-25. RPL4 effects on Collstop readthrough. (A) Readthrough based on dual luciferase assay results of 13PK6 or CollStop reporter pair co-transfected with 1, 3, or 6 ug of mRPL4 in 293A cells in 6 well format. Graph shows representative experiment. Error bars correspond to standard deviation of averages derived from triplicate readings of cell lysates. (B) Average raw values of luciferase for data in (A).

114 (Figure 3-26). These constructs contain point mutations within pseudoknot sequence which have been shown to alter the normal frequency of readthrough, presumably by changing the stability of the pseudoknot structure or the ability of ribosomal or other host factors to interact with the viral mRNA. We also tested a pair of pseudoknot mutants.

U38C has been shown to be hypoactive: this point mutation results in reduced readthrough; changing the same base to an A gives a hyperactive mutant (Alam, Wills et al. 1999). Again, mRPL4 was not able to increase readthrough for the hypoactive mutant

(Figure 3-27), in agreement with earlier experiments, although the same considerations of mRNA stability as the ScrPK are applicable given the raw luciferase levels. RPL4 enhanced the readthrough of the hyperactive pseudoknot mutant U38A about 2 fold, which is comparable to its effects on the wildtype sequence (Figure 3-27). Therefore,

RPL4 does not rescue readthrough of a non-functional recoding signal, but is able to enhance the readthrough frequency of a more efficient pseudoknot.

RPL4 increases readthrough at all three stop codons

Previous studies on the MoMLV gag-pol junction showed that readthrough could occur with any of the three stop codons (Feng, Levin et al. 1989). We tested the RPL4 effect on pseudoknot-containing sequences containing the UGA and UAA stop codons. We transfected 293A cells with dual luciferase constructs containing the alternate stop codons in front of the MoMLV pseudoknot sequence, with and without RPL4. We observed that

RPL4 increased the incidence of readthrough in all three cases (Figure 3-28).

115 5

4.5

4

) 3.5 % ( 3 h g u

o 2.5 r h t

d 2 a e

R 1.5

1

0.5

0 K G U A A C A 1C A 6 6 4 4 U C 7 P 6 1 20 2 2 2 2 39 40 5 V U G C & :C & & A A G L 5 A 0 1 M 2 5 2 2 o :C 1 C C M C G 16 5& 1 G Pseudoknot Mutation

Control (5µg) Rps3a (5µg) Rpl4 (5µg)

Figure 4­6. The effects of Rpl4 expression on the readthrough­stimulating activity of mutant pseudoknots. Only the G15&16C:C25&26G compensatory mutation, which retains near wild type readthrough activity, shows an Rpl4­dependent enhancement of Figurereadthrough. Error bars indicate standard error. 3-26. Effects of RPL4 on MoMLV pseudoknot mutants. Only the double compensatory mutant G15&16C:C25&26G compensatory mutant, which retains near wild-type readthrough activity, shows an RPL4 dependent enhancement of readthrough. Error bars indicate standard error. Figure and legend used with permission from (Houck-Loomis 2009).

116 15 15

12 12 15 15 A 12 9 12 VC!"#$%&"%' 9 L4 60 ng()'*+',- 9 L4 120 ng()'./+',- VC!"#$%&"%' 9 L4 240 ng()'/)+',- 6L4 60 ng()'*+',- 6 L4 120 ng()'./+',- 6 L4 240 ng()'/)+',- 6 % Readthrough % Readthrough % Readthrough 3 3

% Readthrough 3 3

0 0 0 0 VC VC VC VC HIV­1 ScrPK MoMLV Sindbis Type to 3 ug mL4 A 3 ug mL4 HIV­1 6 ug mL4 F 6 ug mL4 ScrPK

MoMLV enter text U38C U38A Sindbis 3 ug mL4 A 3 ug mL4 6 ug mL4 F 6 ug mL4 108 108 U38CPseudoknot108 mutantU38A and RPL4 107 107 107 106 106 B 106 8 8 8 10 105 10 105 10 105 107 104 107 104 107 104 3 3 3 106 10 106 10 106 10 102 102 102 Luciferase units Luciferase units 5 5 Luciferase units 105 10 101 10 101 101 4 104 100 104 100 10 100 3 3 3 VC VC VC 10 VC 10 10 60 ng 2 60 ng 2 2 120 ng 240 ng 120 ng 10 10 B 10 240 ng C Luciferase units Luciferase units Luciferase units ! 3 ug mL4 3 ug mL4 1 1 1 6 ug mL4 10 10 10 6 ug mL4 108 108 U38C U38A 0 0 0 10 107 10 107 10 106 106 VC VC VC VC 5 105 10 F­luc!"#$%&'( 60 ng 60 ng 4 4 10 10 Type to !"#$)&'(

120 ng R­luc 240 ng 120 ng B 10240 ng 3 C 103 ! F­luc control!"*#$%+'(,- 3 ug mL4

enter text 3 ug mL4 102 102 6 ug mL4 R­luc control!"*#$)+'(,- Luciferase units 6 ug mL4 Luciferase units 108 101 108 101 U38C U38A 7 100 107 100 10 Pseudoknot mutant and RPL4 VC 106 106 VC 60 ng 60 ng 240 ng 5 120 ng 240 ng 5 D 10 120 ng 10 E F­luc!"#$%&'( 4 4 10 10 R­luc!"#$)&'( 103 103 F­luc control!"*#$%+'(,- 102 !"#$%&'()'*++&,-'.+'/0'.1'.-2&%'3"%45'416'7$-41-'8.8/9':;&$6.<1.-';&=$&1,&;>102 R­luc control!"*#$)+'(,- Luciferase units Luciferase units ?)'@.7:4%";".1'.+'%&46-2%.$#2'.+'8.8/9'%&46-2%.$#2A''BC9DE'+%47&;2"+-A'F"16G";'%&46-2%.$#2A'416'F,%HI' 101 101 %&46-2%.$#2'"1'JKL?',&55;',.D-%41;+&,-&6'M"-2'6$45'5$,"+&%4;&',.1;-%$,-':4"%;'416'"1,%&4;"1#'47.$1-;'.+'7/0A' 100 100 -%41;+&,-&6'"1'K('M&55'+.%74-)'N&46-2%.$#2'5&3&5;'+%.7'%&:%&;&1-4-"3&'&O47:5&'.+'L'-%41;+&,-".1;':&%',.1;-%$,-' VC :4"%)'*%%.%'G4%;',.%%&;:.16'-.';-4164%6'&%%.%'.+'43&%4#&;'.+'L'M&55;'-%41;+&,-&6'M"-2'&"-2&%'M"56D-P:&'.%',.1-%.5'VC Figure 3-27. Effects of RPL4 on hyperactive pseudoknot mutant. (A) Readthrough 60 ng 60 ng %&:.%-&%',.1;-%$,-)'Q)'N4M'345$&;'.+'5$,"+&%4;&'+.%'8.8/9'64-4'"1'!"#$%&'R?)'@)'N4M'345$&;'.+'5$,"+&%4;&'+.%'levels based on dual luciferase assay of U38C hypo - and U38A hyper-active MoMLV 240 ng 120 ng 240 ng D 120 ng BC9DE'64-4'"1'!"#$%&'(?)'S)'N4M'345$&;'.+'5$,"+&%4;&'+.%'F"16G";'64-4'"1'!"#$%&'(?)'*)'N4M'345$&;'.+'5$,"+&%4;&'E pseudoknot mutants co-transfected with increasing levels of mRPL4 in in 293A cells +.%'F,%HI'64-4'"1'!"#$%&'(?)''!)'N&46-2%.$#2'5&3&5;'G4;&6'.1'6$45'5$,"+&%4;&'4;;4P'.+'TLU@'2P:.'D'416'TLU?'in 6 well format. Graph is from representation experiment. Error bars correspond to 2P:&%D4,-"3&'8.8/9':;&$6.<1.-'7$-41-;',.D-%41;+&,-&6'M"-2'"1,%&4;"1#'5&3&5;'.+'7/0'"1'('M&55'+.%74-)'*%%.%'standard deviation of averages derived from triplicate readings of cells lysates. (B) G4%;',.%%&;:.16'-.';-4164%6'6&3"4-".1'.+'43&%4#&;'6&%"3&6'+%.7'-%":5",4-&'%&46"1#;'.+',&55;'5P;4-&;)'V)'N4M'345D !"#$%&'()'*++&,-'.+'/0'.1'.-2&%'3"%45'416'7$-41-'8.8/9':;&$6.<1.-';&=$&1,&;>Average raw values of luciferase for data in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

8

5.89 6 4.53 4 3.18 3.05 % readthrough 1.93 2 0.96

0 UGA UGA MLVPK UAA MLVPK UAG MLVPK CGA MLVPK UAA +L4 MLVPK UAG + L4 MLVPK CGA + L4 Pseudoknot mutant/RPL4

B 3

2.3

2 1.5 1.5

1

Fold Increase in Readthrough 0 MLVPK UAG + L4 MLVPK CGA + L4 MLVPK UAA + L4 Pseudoknot mutant

Figure 3-28. RPL4 enhancement of readthrough of alternate stop codons. (A) Readthrough from dual luciferase assay of 13PK6 reporter pair with UAG, UGA or UAA stop codon co-transfected with 6 ug of mRPL4 in 293A cells in 6 well format. (B) Fold change of readthrough (RPL4 overexpression levels/reporter alone) for data in (A). Error bars denote standard deviation of average of triplicate fold changes for each condition.

118 The UAA stop codon containing construct showed the least amount of basal readthrough.

This result corresponds with the findings of PTC in other transcripts. UAA is known to be most resistant to readthrough in general, even with the addition of readthrough promoting drugs (Fan-Minogue and Bedwell 2007). Interestingly, although the level of RPL4 induced readthrough was only 2% percent with UAA, as opposed to 1% percent without

RPL4, the fold increase was actually higher than the RPL4 enhancement of UGA- and

UAG-containing transcripts in these trials. RPL4 may affect readthrough by a different mechanism than the one that allows differential suppression of termination of the three stop codons.

RPL4 does not enhance readthrough in in-vitro translation reactions

In an attempt to further characterize the RPL4 effect, and to clarify the role of the RPL4 protein (as opposed to the RNA or some other effect) we attempted to co-express RPL4 and the dual luciferase reporters in a rabbit reticulocyte lysate in-vitro translation (IVT) system. We initially employed an in-vitro translation system only using mRNAs for the dual luciferase reporters, mRPL4 and S3a that were produced in separate transcription reactions using T7 polymerase. These mRNAs were then added to the IVT lysates for translation. No increase in readthrough was evident with the addition of RPL4 mRNA.

Because some mRNAs are highly labile, we thought perhaps there was degradation of

RPL4 mRNA, which would diminish its translation and subsequently diminish the RPL4 effect. We switched to a transcription and translation (TnT) system; in this system transcription of mRNA by T7 polymerase occurs in the same reaction tube as translation.

119 cDNA is added directly to the reaction, and the issues of mRNA stability and purity are bypassed. We observed a small increase in readthrough with the addition of any cDNA; increase in readthrough was not unique to RPL4 (Figure 3-29). We concluded that there must be an active competition for transcription and translation machinery in these reactions, and that we were not getting adequate levels of RPL4 to have an effect. We then pre-translated RPL4, by adding the RPL4 cDNA alone for 30 minutes before the addition of the dual luciferase reporter constructs. We still did not observe a significant, specific increase in readthrough attributable to RPL4 (figure 3-30).

We next attempted to add the RPL4 protein directly to the IVT reaction. We expressed

GST-tagged RPL4 and S3a in bacteria, purified the proteins on a glutathione column, and added the tagged protein directly to the IVT lysate. We did not observe an increase in readthrough with either RPL4 or S3a (Figure 3-31). Since our N-terminal flag tagged

RPL4 and N-terminal truncation mutants of RPL4 did not enhance readthrough we concluded that the tag might be interfering with the function of RPL4 in the IVT system.

We attempted to cleave the GST tag with thrombin. RPL4 could not be recovered from this procedure, although we were able to recover S3a, as viewed by Coomassie staining of the recovered recombinant protein. Therefore, we were unable to produce a stable, soluble untagged version of RPL4 to test in the IVT system. These difficulties prevented further use of the IVT system, as there appears to be a block to the RPL4 effect in the reconstituted translation mix. This could include a saturation of a readthrough-prohibiting or promoting factors, or a role for transcriptional up-regulation of some other component.

120 A 20

16 11.86 12 9.44 9.28 8 6.77 % readthrough 4

0 L4 VC L22 L4-FS Expression construct

B 1E+08 1E+07 1E+06 Fluc 1E+05 Rluc 1E+04 C Fluc 1E+03 C Rluc 1E+02 Luciferase Units 1E+01 1E+00 L4 VC L22 L4-FS Expression construct

Figure 3-29. RPL4 effect on readthrough in TnT IVT reaction. (A) Readthrough based from dual luciferase assay of 0.25 ug of 13PK6 reporter pair transcribed and translated with 1 ug RPL4, RPL4-FS, or RPL22 in in-vitro transcription and translation reaction. Results shown from representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each IVT reaction. (B) Average raw values of luciferase for data in (A).

121 A 15 12 10.68 9.91 8.38 9 6 3 % readthrough 0 RPL4 RPS3a reporter only Expression construct

1E+08 1E+07 1E+06 1E+05 Fluc 1E+04 Rluc 1E+03 C Fluc C Rluc 1E+02

Luciferase Units 1E+01 1E+00 RPL4 RPS3a reporter only Expression construct

Figure 3-30. Effect of pre-translated RPL4 on MoMLV readthrough in TnT IVT reaction. (A) Readthrough based on dual luciferase assay results of 1 0.25 ug of 13PK6 reporter pair transcribed and translated with RPL4 or RPS3a in in-vitro transcription and translation reaction. 1 ug cDNA for RPL4 or RPS3a was added to TnT IVT reactions 30 minutes before reporter DNA. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each IVT reaction. (B) Average raw values of luciferase for data in (A).

122 A 9.68 10

8 6.24 6.12 5.68 4.97 5.23 5 3.99 2.89 3 % readthrough 0 VC L4 1.0 ug L4 2.0 ug L4 0.5 ug S3a 0.5 ug S3a 1.0 ug S3a 2.0 ug Gluthathione GST-tagged recombinant protein

B 1E+08 1E+07 1E+06 1E+05 Fluc 1E+04 Rluc 1E+03 C Fluc C Rluc 1E+02 Luciferase Units 1E+01 1E+00 VC L4 1.0 ug L4 2.0 ug L4 0.5 ug S3a 0.5 ug S3a 1.0 ug S3a 1.0 ug Gluthathione GST-tagged recombinant protein

Figure 3-31. Effect of RPL4-GST tagged protein on readthrough in TnT IVT reaction. (A) Readthrough based on dual luciferase assay results of 1 0.25 ug of 13PK6 reporter pair transcribed and translated with 0.5 ug, 1.0 ug, or 2.0 ug GST-tagged RPL4 or RPS3a protein in in-vitro transcription and translation reaction. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each IVT reaction. (B) Average raw values of luciferase for data in (A).

123 Release factor over-expression negates RPL4 enhanced readthrough

We next examined the effect of over-expression of termination factors on readthrough.

Our lab has previously discovered an interaction between reverse-transcriptase and eRF1

(Orlova, Yueh et al. 2003). Reverse transcriptase can bind eRF1, perhaps to sequester it and allow readthrough to occur. Additionally, overexpression of eRF1 reversed the increase in readthrough which resulted from overexpression of reverse transcriptase.

Concurrent with our RPL4 work, we immuno-depleted eRF1 from IVT lysates as a step in customizing translation mixes for a cryo-EM visualization of the pseudoknot on the ribosome, described in the appendix (Figure A-10). We observed an increase in readthrough, and in frameshift, in lysates with reduced eRF1.

We transfected 293A cells with cDNAs for eRF1 and eRF3 and the 13PK6 reporter pair.

We performed western blot analysis to verify over-expression and saw only slight increases in levels. Since eRF1 and eRF3 are well conserved among mammals, a high affinity specific antibody is likely hard to produce. There also may be regulatory elements that control the levels of termination factors in cells, similar to what we hypothesize for RPL4. Surprisingly, we saw no decrease in readthrough in the dual luciferase assay, with transfection of eRF1 or eRF3 separately or together (Figure 3-32).

However, when we performed a series of assays with RPL4 and eRF1 and eRF3, we saw that expression of the termination factors could abrogate the RPL4 increase in readthrough (Figure 3-33). As a control we tested a combination of RPL4 with RPL7, another ribosomal protein that induces slight readthrough. Expression of RPL7 with

124 A 8

6

4 2.94 3.20 2.68 2.76 2.82 2.49 2 % readthrough

0 VC erf1 6 ug eRF1 3 ug eRF1 6 ug eRF3 3 ug erf1 + erf3, 3 ug Release factor B 1E+08 1E+07 1E+06

1E+05 Fluc 1E+04 Rluc 1E+03 C Fluc 1E+02 C Rluc Luciferase Units 1E+01 1E+00 VC erf1 6 ug erf1 6 ug eRF1 3 ug eRF3 3 ug erf1 + erf3, 3 ug Release factor

Figure 3-32. Release factor effect on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pairs co- transfected with 1, 3, or 6 ug of eRF1 and/or eRF3 in 293A cells in 6 well format. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

125 A 10

8 5.41 5 3.44 3.00 3.29 3 % readthrough 0 VC L4 6 ug F1 3 ug, F3 ug L4 6 ug, F1 3 F3 ug Expression constructs B 1E+08 1E+07 1E+06 1E+05 Fluc 1E+04 Rluc 1E+03 C Fluc 1E+02 C Rluc

Luciferase Units 1E+01 1E+00 VC L4 6 ug F1 3 ug, F3 ug L4 6 ug, F1 3 F3 ug Expression constructs

Figure 3-33. Combined effects of Release Factor and RPL4 on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pairs co-transfected with 6 ug RPL4 and/or 3 ug of eRF1 and eRF3 in 293A cells in 6 well format. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

126 RPL4 did not reduce readthrough; rather readthrough levels remained constant (Figure

3-34).

eRF1 has been shown to be able to cause termination by itself, although at a slower rate than when eRF3 is present (Alkalaeva, Pisarev et al. 2006). We surmised that eRF1 was the key component in the abatement of RPL4’s effect on readthrough. We saw that indeed, by itself, eRF1 had the ability to reduce the RPL4 enhancement of readthrough.

Increasing the ratio of RPL4 to eRF1 lessened the eRF1 effect; when equal amounts of the constructs are transfected together, the reduction of readthrough is most pronounced

(Figure 3-35).

RPL4 and G418 act additively to increase MoMLV readthrough

Concurrent work, described in the next chapter, investigates the effects of readthrough promoting drugs on the gag-pol ratio. G418 is a widely used aminoglycoside which induces miscoding and suppression of termination in bacteria (reviewed in (Houghton,

Green et al. 2010). It has been shown that many aminoglycosides bind to rRNA in the decoding center, and force a conformation which allows non-cognate interactions to be tolerated. Derivatives of G418 are used to treat human diseases that result from premature stop codons; readthrough events allow production of otherwise truncated proteins.

Treatment of cells with G418 increased MoMLV readthrough in a dose dependent manner (Figures 3-36 and 4-4, discussed in detail in Chapter 4). We were curious whether

G418 would eclipse or augment RPL4’s effect on readthrough, suggesting a role for

127 A 20.0 15.2 16.0 14.9 15.0 11.9 12.0 9.8 9.5 7.9 8.0

% readthrough 4.0 0 VC L4 60 ng L7 60 ng L4 120 ng L7 120 ng L4 & L7 60 ng each L4 & L7, 120 ng each Expression construct

B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 VC L4 60 ng L7 60 ng L4 120 ng L7 120 ng L4 & L7 60 ng each L4 & L7, 120 ng each Expression constructs

Figure 3-34. Combined effects of RPL4 and RPL7 on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pairs co-transfected with increasing amounts of RPL4 and RPL7 alone and together in 293A cells in 96 well format. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

128 A 12.0 10.0 8.9 8.2 7.3 7.8 7.7 8.0 6.2 6.0 4.7 3.7 4.0 3.2 2.9 3.2 2.0 0 VC L4 120ng eRF1 60ng eRF1 120ng eRF1 240ng F1 60ng, L4 60ng L4 120ng, F1 30ng L4 120ng, F1 60ng F1 60ng, L4 120ng F1 60gn, L4 180ng L4 120ng, F1 120ng eRF1 and RPL4 transfection mix B 1E+08 1E+07 1E+06 Av. Fluc 1E+05 Av. Rluc 1E+04 Ave. FLuc-C 1E+03 Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 VC L4 120ng eRF1 60ng eRF1 120ng eRF1 240ng F1 60ng, L4 60ng L4 120ng, F1 30ng L4 120ng, F1 60ng F1 60ng, L4 120ng F1 60gn, L4 180ng L4 120ng, F1 120ng eRF1 and RPL4 transfection mix

Figure 3-35. eRF1 effects on RPL4 enhancement of readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pairs co-transfected with increasing amounts of RPL4 and eRF1 alone and together in 293A cells in 96 well format. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each condition. (B) Average raw values of luciferase for data in (A).

129 RPL4 in manipulation of the decoding center similar to that of an aminoglycoside.

We transfected RPL4 and the 13PK6 reporter pair into 293A cells, and added G418. We saw that G418 and RPL4 worked in an additive manner. The readthrough was higher in cells with RPL4 and exposed to G418 at over 4 fold (Figure 3-36). The result was slightly higher than the combination of both RPL4 and G418 alone, but not an exponential increase that would suggest a synergistic effect. It appears that RPL4 and G418 do not function on the same site to alter decoding. If RPL4 simply perturbs the decoding site out of the restrictive conformation, it would be expected that G418 would have no additional effect. Therefore, RPL4 may function at another point in the proofreading process.

RPL4 enhancement of readthrough is not due to ribosome biogenesis

In an effort to further elucidate the nature of RPL4 enhancement of readthrough we attempted to analyze whether transfection of RPL4 cDNA was stimulating ribosome biogenesis. RPL4 has a role in the processing of rRNA, and is perhaps a trigger for ribosome biogenesis (Egoh, Nosuke Kanesashi et al. 2010), and therefore it is plausible that in our system we were triggering an up-regulation of ribosome biogenesis.

Depending on the stoichiometry of ribosomes to termination machinery, an increase in ribosome number could deplete the available pool of termination factors, thus allowing more opportunity for readthrough to occur.

130 4.8 5.00 A 3.75 2.4 2.50 1.6 1.0 1.2 1.25 Fold increase 0 VC G418 1 mg/ml G418 2 mg/ml G418 .5 mg/ml G418 .25 mg/ml drug concentration

B C

20 1E+08 16 12.0 1E+06 12 1E+04 8 4.1 4.6 4 2.7 1E+02 % readthrough Luciferase Units 0 1E+00 VC VC L4 9 ug L4 9 ug

Av. Fluc G418 1mg/ml Av. Rluc G418 1mg/ml Ave. FLuc-C Ave. RLuc-C L4 9 ug + G418 1 mg/ml L4 9 ug + G418 1 mg/ml Drug and/or expression construct Drug and/or expression construct

Figure 3-36. Effects of G418 and combined G418/RPL4 on readthrough (A) Fold increase of readthrough percentages from dual luciferase assay of the 13PK6 reporter pairs transfected in 293A cells treated with G418, in 6 well format. (B) Readthrough percentages from dual luciferase assay of the 13PK6 reporter pairs transfected with 9 ug of RPL4 and/or 1 mg/ml G418 in 293A cells in 6 well format. Graphs are of representative experiments, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (C) Average raw values of luciferase for data in (A).

131 We also theorized that the cell line dependence of the RPL4 effect could be used to determine what was causing the increase in readthrough in some cells. If ribosome biogenesis was being triggered by transfection of RPL4 cDNA, perhaps responding cells had a different basal level of ribosomes than non-responding cells. We first attempted to quantify the level of endogenous RPL4 in a sampling of responding and non-responding cell lines. We picked 293A and 293T for responding cells, and A549 and TE671 as non- responders. Our intention was to expand our test to include all the cell lines surveyed if we found anything of interest in a smaller pool.

We verified that the RPL4 monoclonal antibody A43 would detect human and rodent

RPL4 (Figure 3-37) by testing its ability to detect GST-tagged RPL4 we had made for in- vitro translation and endogenous RPL4 in 293A and NIH3T3 cells. We observed an increase in signal with increase in total protein loaded, but differences of two fold were hard to distinguish. We lysed 500,000 cells (80% confluency) of each cell type in RIPA buffer, quantitated protein concentration using the Bradford assay, and loaded equal amounts of total protein per sample. We analyzed the lysates via Western blot with the

RPL4 monoclonal antibody A43. We could see no distinguishable variation in the amount of RPL4 in responding and non-responding cells (Figure 3-38). Therefore we could not conclude that differences in endogenous levels of RPL4 were responsible for the cell line dependence of the RPL4 effect, as a catalyst for ribosomal biogenesis. We also transfected the same panel of cells with 6 ug of mRPL4 cDNA and performed WB analysis of the lysates collected 24 post transfection. We observed no difference in RPL4

132 GST GST-mL4 GST-mL4 A B

NIH3T3 C Neat NIH3T3 NIH3T3 (~ 50 ug) 1:2 1:4

RPL4

293A Neat 293A 293A D (~ 80 ug) 1:2 1:4

RPL4

Figure 3-37. Monoclonal antibody detection of endogenous mouse and human RPL4. (A) Commassie stained gel of GST-tagged RPL4 protein. (B) Western blot of GST-tagged RPL4 protein, probed with monoclonal antibody to RPL4. (C) Western blot of endogenous mouse RPL4 in NIH3T3 cells. Lysates were loaded in decreasing concentrations to determine if differences in RPL4 levels could be detected. (D) Western blot of endogenous human RPL4 in 293A cells. Lysates were loaded in decreasing concentrations to determine if differences in RPL4 levels could be detected.

133 293A 293T TE671 A549

tubulin RPL4

RPL4 responsive RPL4 non-responsive

Figure 3-38: Levels of endogenous RPL4 in responsive and non-responsive cells. Western blot of endogenous RPL4 in 293A, 293T, Te671 and A549 cells, probed with monoclonal anti-RPL4 and and anti-tubulin as a loading control.

134 expression between endogenous and transfected cells (Figure 3-39).

We examined the expression of the ribosomal protein RPL10a as an additional indicator of ribosome number. L10a is component of the large subunit with no known extra- ribosomal or cytoplasmic function, and therefore increase in the amount of RPL10a would be attributable to an increase in ribosome number. We again transfected the 6 cell lines with 6 ug of RPL4, and performed WB analysis on endogenous and transfected samples. We saw no increase in L10a among any of the cell lines (Figure 3-40).

We then attempted to use qPCR methods to quantitate ribosomes. Translation could only be affected by an increase in 80S ribosomes, which could be measured by 28S rRNA via qPCR. We transfected the same cell lines with 6 ug of mRPL4 cDNA and extracted RNA

24 h post transfection. We made cDNA pools from each cell type and performed qPCR using primers against 28S rRNA, with actin as a control. We saw slight increases in 28S rRNA amounts in cells transfected with RPL4, which could indicate an increase in ribosomes. However, this result was consistent among all the cell types; thus ribosome biogenesis was not the singular factor in dictating the response to RPL4 or the RPL4 enhancement of readthrough (Figure 3-41).

RPL4 increase in gag-pol readthrough has deleterious effects on viral replication

We investigated the RPL4 enhancement of readthrough on production of wild-type

135 293A 293T TE671 A549 Endo OE Endo OE Endo OE Endo OE

Tubulin RPL4

2.0 RPL4 responsive 1.6 RPL4 non-responsive 1.5 1.1 1.0 0.8

OE/endo 0.5 0 Figure 3-39: RPL4 overexpression0 in responsive and non-responsive cell lines. Western blot of RPL4 in 293A,293A 293T, Te671293T and A549TE671 cells, probedA549 with monoclonal anti-RPL4 and and anti-tubulin as a loading control. O/E lysates were transfected with 6 ug mRPL4 in 6 well format.

Tuesday, October 25, 2011

136 293A 293T TE671 A549 Endo OE Endo OE Endo OE Endo OE tubulin RPL4

RPL10a

2.0 1.6 RPL4 non-responsive RPL41.5 responsive 1.1 1.0 0.8

OE/endo 0.5 Figure 3-40. Figure 2-40. RPL4 overexpression effects on RPL10a0 expression. 0 Western blot of RPL10a in 293A,293A 293T, Te671293T and A549TE671 cells, A549probed with monoclonal anti-RPL10a and anti-RPL4, and anti-tubulin as a loading control.

Tuesday, October 25, 2011

137 1E+06

1E+05

1E+04

1E+03 Endogenous

CT L4/actin mL4 O/E Δ 1E+02

1E+01

1E+00 293a 293T TE671 TE671 Cell line

Figure 3-41. Effects of RPL4 overexpression on 28S rRNA in responsive and non- responsive cells. Results of qPCR of 28S rRNA levels in cells transfected with 6 ug RPL4 in 6 well format. Endogenous levels refer to untransfected cells. 28S rRNA levels were normalized to actin mRNA levels. Graph is from representative experiment.

138 MoMLV in vivo. We transfected 293T cells with the pNCS MoMLV provirus, and increasing amounts of mRPL4 or S3a. Cells were lysed 48 hours post transfection and blotted using the anti-capsid antibody R187. In lysates from all cells transfected, the gag precursor Pr65gag could be detected at consistent levels, whereas the gag-pol fusion product Pro200gag-pol was undetectable. However, lysates from cells transfected with

RPL4 exhibited a dose-dependent decrease in processed capsid, even as Pr65gag precursor levels appear unchanged (Figure 3-42). The reduced defect in processing is likely a result of alterations to viral assembly; increased readthrough alters the gag:gag-pol ratio and this is thought to affect the stoichiometry and formation of the virus. Cells transfected with increasing levels S3a show no change in processed capsid levels compared to basal levels in the virus only control.

To monitor viral release, we harvested virus released from producer cells 48 hours after transfection. We pelleted virus from the filtered culture media of cells transfected with

RPL4 and RPS3a and analyzed the collected virus for capsid protein via western blot. In supernatants from cells co-transfected with RPL4 there is a strong dose dependent reduction in capsid levels, indicating inhibition of viral formation and/or release. There was no change in the amount of capsid detected in virus produced with RPS3a overexpression. Therefore, the RPL4 enhancement of readthrough has a negative effect on viral production through alteration of the gag:gag-pol ratio, affecting both assembly and virus release.

139 A B

Gag Gag

Capsid Capsid

Actin Actin Marker A Marker B 4 ug L4 1 ug L4 C 2 ug L4 C 4 ug S3a 1 ug S3a 2 ug S3a No Virus 0.5 ug L4 No Virus Virus only Virus only 0.5 ug S3a

C D Marker Marker 4 ug L4 1 ug L4 2 ug L4 4 ug S3a 1 ug S3a 2 ug S3a No Virus No Virus 0.5 ug L4 Virus only C Virus only D

C C 0.5 ug S3a

!"#$%&'()'*++&,-'.+'/0'.1'2"%34'%&54",3-".1) 6)'7&8-&%1'94.-'3134:8"8'.+';<=>',&448',.?-%318+&,-&@'A"-B'C'$#'5DEF'GH.H/I'5%.2"%$8J'31@'"1,%&38"1#' 4&2&48'.+'K/0L'31@'5%.M&@'A"-B'%3-'31-"?,358"@'5.4:,4.134'31-"M.@:)'9)'7&8-&%1'94.-'3134:8"8'.+';<=>',&448',.? -%318+&,-&@'A"-B'C'$#'5DEF'GH.H/I'5%.2"%$8J'31@'"1,%&38"1#'4&2&48'.+'KF=3L'31@'5%.M&@'A"-B'%3-'31-"?,358"@' 5.4:,4.134'31-"M.@:)'E)'7&8-&%1'M4.-'3134:8"8'.+'5&44&-'2"%$8'+%.K';<=>',&448',.?-%318+&,-&@'A"-B'C'$#'5DEF'Figure 3-42: Effects of RPL4 on viral replication. (A) Western blot of lysates of GH.H/I'5%.2"%$8J'31@'"1,%&38"1#'4&2&48'.+'K/0L'31@'5%.M&@'A"-B'%3-'31-"?,358"@'5.4:,4.134'31-"M.@:)''E)'7&8-? &%1'M4.-'3134:8"8'.+'5&44&-'2"%$8'+%.K';<=>',&448',.?-%318+&,-&@'A"-B'C'$#'5DEF'GH.H/I'5%.2"%$8J'31@'"1,%&38293T cells transfected with 1 ug pNCS and 1ug, 3 ug or 5 ug RPL4, probed with anti- ? "1#'4&2&48'.+KF=3L'31@'5%.M&@'A"-B'%3-'31-"?,358"@'5.4:,4.134'31-"M.@:)'capsid antibody and anti-actin as a loading control. (B) Western blot of lysates of 293T cells transfected with 1 ug pNCS and 1ug, 3 ug or 5 ug RPS3a, probed with anti-capsid antibody and anti-actin as a loading control. (C) Western blot of viral pellets collected from culture media of 293T cells transfected with 1 ug pNCS and 1ug, 3 ug or 5 ug RPL4, probed with anti-capsid antibody. (D) Western blot of viral pellets collected from culture media of 293T cells transfected with 1 ug pNCS and 1ug, 3 ug or 5 ug RPS3a, probed with anti-capsid antibody.

140 Chapter 4: Readthrough Promoting Drugs and Retroviral Recoding

Aminogylcoside antibiotics have been traditionally used to treat bacterial infections due to their effects on prokaryotic translation. Aminoglycosides are highly positively charged molecules that are naturally produced by certain microorganisms. They are medically classified as amino-modified sugars (MeSH), and share a basic structure of a 2- deoxystreptamine (ring II) linked to a glucopyranosyl ring (ring I) (Figure 4-1).

Aminoglycosides are poorly taken up by eukaryotic cells as they do not easily diffuse across the cell membrane. Bacterial cells possess transporters that actively take up aminoglycosides, and this may be a partial cause for the increased toxicity and effect on miscoding in prokaryotic cells (Houghton, Green et al. 2010).

Aminoglycosides promote miscoding by interacting with the rRNA of the decoding center and causing changes in the rRNA conformations that allow for incorporation of near and non-cognate amino acids. This alteration to the proofreading machinery of the ribosome also permits readthrough of stop codons. The family of aminoglycosides is large, but subtle variations in structure determine efficacy and toxicity. These variations consist of oxygen or nitrogen at the 6’ position of ring 1 and additional sugars attached to ring II (Figure 4-2) (Fan-Minogue and Bedwell 2007). Most aminoglycosides have a greater affinity for the prokaryotic decoding center and this difference allows for their use as microbicides in eukaryotic organisms (Xie, Dix et al. 2010).

141 Figure 4‐1: Aminoglycoside structures (Houghton, Green et al. 2010), http:// onlinelibrary.wiley.com/doi/10.1002/cbic.200900779/full.

142 A B C

Figure 4-2. Substitutions and variations in aminoglycoside structure. Structures of (A) G418, (B) paromomycin and (C) neomycin. Relevant differences between G418 and paromomycin at the 6’ position of Ring 1 circled in green. Neomycin shares the same linkage to Ring II and additional structure with paromomycin (circled in orange), but is more toxic. (Drug structures from the National Library of Medicine: G418 http:// pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=123865&loc=ec_rcs, paromomycin http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi? cid=165580&loc=ec_rcs, neomycin http://pubchem.ncbi.nlm.nih.gov/summary/ summary.cgi?cid=8378&loc=ec_rcs

143 Crystal structures of bacterial 30S subunits and aminoglycosides demonstrate the interactions that result in miscoding. Paromomycin has been modeled in complex with the bacterial rRNA; the drug binds the 16S rRNA at helix 44. The region of rRNA contains the proofreading nucleotides A1492 and 1493 and the bound drug displaces their native positions (Figure 4-3). This change in orientation allows acceptance of near and non-cognate tRNAs and results in incorporation of alternate amino acids. The parmomoycin-induced change in conformation is not identical to the shift that occurs upon cognate interactions, suggesting that the default inhibitory conformation is the defining factor in selection.

In addition to their use in combating bacterial infections, aminoglycosides are used to treat human diseases caused by premature termination codon mutations. Duchennes muscular dystrophy and cystic fibrosis are caused by nonsense mutations in coding sequences that cause inappropriate termination at premature stop codons and produce truncated dysfunctional proteins (reviewed in (Hermann 2007)). Treatment with aminoglycosides provides readthrough at a frequency which allows enough translation of the full-length protein to restore normal function. However, at high enough doses, aminoglycosides cause defects in eukaryotic translation and this dosage effect allows for their use in selection in tissue culture. We examined the effects of aminoglycosides on eukaryotic recoding in the context of retroviral readthrough.

144 Figure 4‐3: Paromomycin bound to 16S RNA. (A) The key decoding center nucleotides (green) in an A-site-vacant 30S ribosome subunit. (B) The key decoding nucleotides undergo conformational changes when the 30S subunit binds a cognate anticodon stem loop (ASL, yellow). Adenosine 1492 (A1492) and A1493 are displaced from helix 44 of the 16S rRNA to a position where they can engage the minor groove of the codon-anticodon helix in the A-site. (C) Paromomycin (orange) –binding of the 30S subunit induces similar conformational changes in the key residues. (D) The RF1 (yellow) -bound 70S ribosome structure shows a different conformational change where only A1492 unstacks from helix 44 and A1493 remains stacked within the helix. This positioning of A1493 is stabilized by the movement of A1913 of the 23S rRNA (brick red) into the region to provide a stacking interaction. Reprinted from Cell, Vol. 136, Issue 4, Zaher and Green, Fidelity at the Molecular Level: Lessons from Protein Synthesis, pp. 746-762, 2009,with permission from Elsevier.

145 Aminoglycosides increase MoMLV readthrough in Vivo

G418 is a drug widely used in cell selection and we tested its effects on readthrough of

MoMLV gag-pol. We transfected 293A cells with the 13PK6 reporter pair. Two hours later we replaced the media with media containing increasing doses of G418. We harvested the cells 24 hours after adding the drug, and analyzed readthrough with the dual luciferase assay. G418 treatment results in dose dependent increase in readthrough

(Figure 4-4). Readthrough was increased from the normal basal level of 3-5% to greater than 11% with the highest dose of 2 mg/ml. This concentration is often used for cell selections, and the levels of raw luciferase show that overall translation is inhibited.

Lower levels of drug also result in an increase in readthrough. Thus, G418 does increase

MoMLV pseudoknot controlled readthrough.

We next tested G418 in a transducing virus assay to determine whether the increase in readthrough we saw with the DLA reporters would affect viral replication. We transfected 293T cells with plasmids encoding components for recombinant MoMLV. We used a 3 plasmid system consisting of expression constructs for MoMLV gag-pol, VSV-G envelope and an MoMLV-based vector with the MoMLV packaging signal and LTRs expressing the firefly luciferase gene. This mix produces a recombinant virus that is assembled with MoMLV gag and pol products and an envelope that will allow infection of many cell types. The recombinant virus genome carries only the firefly luciferase

(fluc) gene that will be integrated into cellular DNA, allowing firefly luciferase to

146 A 20

16 11.47 12 8.35 8 5.82 3.54 4.35

% readthrough 4

0 VC G418 1 mg/ml G418 2 mg/ml G418 .5 mg/ml G418 .25 mg/ml drug concentration B 1E+08 1E+07 1E+06 Fluc 1E+05 Rluc 1E+04 C Fluc 1E+03 C Rluc 1E+02 Luciferase Units 1E+01 1E+00 VC G418 1 mg/ml G418 2 mg/ml G418 .5 mg/ml G418 .25 mg/ml drug concentration

Figure 4-4. G418 effects on MoMLV readthrough in 293A cells. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293A cells in 6 well format. G418 was added in increasing amounts. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

147 function as a reporter of infectivity. We also transfected a separate plasmid encoding

Renilla luciferase (rluc) as a control for general translational health in the producer cells.

Two hours after transfection, we replaced the transfection media with media containing

G418 at increasing concentrations. Twenty-four hours post drug treatment we collected virus from the supernatant, and infected naïve 293A cells. Forty-eight hours later we lysed the infected cells and performed a luciferase assay to measure expression of fluc as an indicator of viral production and infectivity. We also measured the rluc in the producer cells to determine if the drugs were having an impact on general translation at the levels used. Cells infected with virus from G418 treated producer cells had lower fluc signals, indicating that less virus was produced, or that there were defects in packaging of the fluc gene and assembly of virus due to improper ratios of gag-pol (Figure 4-5). The rluc signals from producer cells also decreased with increases in G418 concentration, indicating an overall defect in translation from the toxicity of the drug.

We performed a viability assay with G418 using PrestoBlue live cell imaging reagent. We transfected 293A cells with 13PK6 and control. Two hours post transfection we replaced the transfection media with G418 containing media in increasing concentrations. We also treated a separate plate of 293A cells with same G418 mixes to test cell viability. Twenty- four hours post drug addition we assayed for dual luciferase and cell viability. G418 greatly reduced cell viability at concentrations that resulted in highest readthrough

(Figure 4-6). Therefore, it is difficult to determine whether it was a reduction in the number of producer cells or a direct affect on G418 on readthrough that resulted in lower

148 A 6E+06

5E+06

3E+06 Fluc: RLU 2E+06

0E+00 no G418 2 mg G418 1 mg G418 .5 mg G418 .25 mg G418 B drug concentration 3E+07

2E+07

2E+07 Rluc: RLU 8E+06

0E+00 no G418 2 mg G418 1 mg G418 .5 mg G418 .25 mg G418 drug concentration

Figure 4-5. G418 effects on viral production: transducing virus assay. (A) Fluc levels in naive 293A cells infected with recombinant virus produced in cells treated with G418. (B) Rluc levels in 293A producer cells treated with G418.

149 11 100

8 75

6 50

3 25 Average %Viabililty Average % readthrough

0 0 No drug G418 1 mg/ml G418 0.5 mg/ml G418 1.5 mg/ml G418 0.25 mg/ml G418 0.75 mg/ml G418 0.125 mg/ml drug concentration

Readthrough Viability

Figure 4-6. G418 effects on cell viability and readthrough. Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293A cells in 6 well format. G418 was added in increasing amounts 2 hours after transfection. Cell viability was measured with PrestoBlue reagent added to non-transfected cells exposed to the same G418 media.

150 expression of the fluc reporter measured in the transducing virus assay.

We tested additional aminoglycosides, and the small molecule PTC124 (Hirawat, Welch et al. 2007; Welch, Barton et al. 2007; Du, Liu et al. 2008) in similar experiments. We transfected 293A cells with the 13PK6 reporter pair and 2 hours post transfection, replaced the transfection media with media containing drugs. G418, PTC124, paromomycin, amikacin and gentamicin all increased readthrough (Figure 4-7). The average fold increase from at least three trials of each concentration is shown in (Figure

4-8.) Paromomycin has an ~ 2.5 fold effect on readthrough at high concentrations.

PTC124 treatment results in a twofold increase in readthrough. Gentamicin, a derivative of G418, also modestly increases readthrough at doses suggested for cell selection.

Amikacin, also used in eukaryotic cells, did not increase readthrough. Paromomycin and amikacin are used to treat infections in mammals and are well tolerated by eukaryotic cells, as the rluc levels of the reporter demonstrate. PTC124 and gentamicin are also used therapeutically, and do not cause the defects in translation that G418 does. Thus, a moderate amount of readthrough increase can be induced by aminoglycosides in vivo without significant cell death.

We tested Hygromycin, which works by inhibiting racheting of the ribosome and tRNA movement instead of interacting with the decoding center. Hygromycin was extremely toxic to cells; cells detached from the plate by the time of the DLA. Neomycin proved too

151 A 8

6

4

5.0 4.5 4.9 4.8 4.8 4.3 4.6 % readtrhough 2 3.7 3.6 3.4 2.7 0 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Amikacin 1.5 mg/ml Amikacin 3.0 mg/ml Gentamicin 25 ug/gml Gentamicin 50 ug/gml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml B drug concentration 1E+08 1E+07 1E+06 Av. Fluc 1E+05 Av. Rluc 1E+04 Ave. FLuc-C 1E+03 Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Amikacin 1.5 mg/ml Amikacin 3.0 mg/ml Gentamicin 25 ug/gml Gentamicin 50 ug/gml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration

Figure 4-7. Readthrough promoting drug effects on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293A cells in 6 well format. Drugs were added in indicated amounts two hours after transfection. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

152 5

4

3.15 3 2.61 2.51 2.50 2.22 2 1.74 1.57 1.59 1.40 1.22 1.00 1 Fold Increase in readthrough

0 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Amikacin 1.5 mg/ml Amikacin 3.0 mg/ml Gentamicin 25 ug/gml Gentamicin 50 ug/gml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration

Figure 4-8. Readthrough promoting drug effects on MoMLV readthrough. Fold changes in readthrough of the 13PK6 reporter in 293A cells treated with aminoglycosides or PTC124. Graph shows results of 2-4 trials per condition, error bars correspond to standard deviation of the mean of average fold changes per condition.

153 toxic to use as well. Kanamycin did not give any increase in readthrough. Since we routinely use the aminoglycoside streptomycin in our cell culture media, we also tested its effect on readthrough. We saw no increase in readthrough at doses of streptomycin at and above standard tissue culture concentrations (Figure 4-9).

Readthrough promoting drugs can affect retroviral replication

G418 affects retroviral replication

We looked at the effect of these drugs on viral production with a transduction assay

(Figure 4-10), following the protocol described previously. G418 had the most profound effect on viral production, as measured by firefly luciferase levels. At the highest concentrations (2 mg/ml and 1mg/ml), G418 reduced signals of firefly luciferase by up to

3 logs in the infected cells, compared to the no drug control (Figure 4-11). However, renilla luciferase levels in the producer cells were reduced correspondingly (Figure 4-12), indicating that the high levels of G418 were impeding normal translation, as we observed in previous experiments. At lower levels, G418 had a more modest effect on the levels of fluc in infected cells, and had a correspondingly milder effect on translation as measured by rluc. Therefore, cell death is likely a contributing factor to the reduction in viral production. However the DLA results of the same concentrations show increased levels of readthrough, providing evidence that the disruption of the gag-pol ratio is also a plausible factor in the decrease in infectivity.

154 A 10 8 6 4.4 3.7 4 2 % readthrough 0 no drug pen/strep media condition

B 15 1E+08 12 1E+06 8.6 9.5 9 7.9 7.1 1E+04 6 4.7 3 1E+02 % readthrough Luciferase Units 0 1E+00 no drug no drug Streptomycin 1 mg/ml Streptomycin 1 mg/ml Streptomycin .5 mg/ml Streptomycin .5 mg/ml Streptomycin .25 mg/ml Streptomycin .25 mg/ml Streptomycin .125 mg/ml Streptomycin .125 mg/ml drug concentration drug concentration

Figure 4-9. Streptomycin effects on MoMLV readthrough. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293A cells in 6 well format in media +/- penicillin/streptomycin. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293A cells in 6 well format. Drugs were added in indicated amounts two hours after transfection. (C) Average raw values of luciferase for data in (B).

155 2E+07

1E+07

8E+06 RLU: Fluc

4E+06

0E+00 “1:2” “1:4” “1:8” “1:16” Virus dilution Virus only

G418 1mg/ml G418 0.5 mg/ml G418 0.25 mg/ml PTC124 5 mg/ml PTC124 2.5 mg/ml Amikacin 3 mg/ml Amikacin 1.5 mg/ml Parmomycin 2 mg/ml Parmomycin 1 mg/ml

Figure 4-10. Drug effects on viral replication: transducing virus assay. Fluc levels in naive 293A cells infected with virus produced in cells treated with aminoglycosides or G418. Ratios on x-axis refer to dilutions of virus based on neat concentration of media collected from producer cells.

156 1E+07

1E+06

1E+05

1E+04

1E+03 RLU: fluc

1E+02

1E+01

1E+00 Aminoglycoside added

Virus only G418 0.25 mg/ml G418 0.5 mg/ml G418 1mg/ml PTC124 2.5 mg/ml PTC124 5 mg/ml Amikacin 1.5 mg/ml Amikacin 3 mg/ml Parmomycin 1 mg/ml Parmomycin 2 mg/ml

Figure 4-11: Drug effects on viral replication: transducing virus assay. Detail of firefly luciferase levels in infected cells from virus produced in cells treated with drugs, 1:4 dilution of virus.

157 1E+08

1E+07

1E+06

1E+05

1E+04

RLU: Rluc 1E+03

1E+02

1E+01

1E+00 Aminoglycoside added

Virus only (no drug) G418 0.25 mg/ml G418 0.5 mg/ml G418 1 mg/ml PTC124 2.5 mg/ml PTC124 5 mg/ml Amikacin 1.5 mg/ml Amikacin 3 mg/ml Parmomycin 1 mg/ml Parmomycin 2 mg/ml

Figure 4-12: Drug effects on viral replication: transducing virus assay. Renilla luciferase levels in 293A producer cells treated with drugs; an rluc expressing plasmid was co-transfected with the recombinant virus mix to measure drug effects on overall translation.

158 PTC124 decreases retroviral replication

PTC124 treatment of virus-producing cells reduced the amount of firefly luciferase in infected cells by more than half, suggesting that the increase in readthrough of gag-pol had a deleterious effect on viral production. In the DLA PTC124 caused a higher increase in readthrough than G418; therefore, we expected that the effect on viral production would be greater than G418. PTC124 also slightly reduced rluc expression in producer cells, despite its relative non-toxicity in cells. There is a controversy concerning PTC124 and its interaction with firefly luciferase; it has been reported that PTC124 can bind to firefly luciferase and increase its stability, which in turns skews the readthrough ratio

(Auld, Thorne et al. 2009; Peltz, Welch et al. 2009; Auld, Lovell et al. 2010) Therefore, an increase in firefly signal could be due to drug-firefly luciferase interactions rather than a reduction of the amount of firefly luciferase produced. Although PTC 124 treated cells may show an artificially elevated fluc signal due to the drug:luciferase interaction, in this context, fluc measures infectivity of naive cells that should not be affected by PTC124.

Amikacin does not significantly affect retroviral replication

Cells infected with virus produced in the presence of amikacin had slightly lower levels of firefly expression. Rluc expression was only slightly reduced in producer cells, indicating no gross effects on translation. Amikacin is used therapeutically, and therefore toxicity was not expected to be extreme.

159 Paromomycin decreases retroviral replication

Cells infected with virus produced in paromomycin treated cells had lower firefly luciferase levels, with signals decreasing by almost a log. Producer cells showed the least change in Renilla luciferase expression compared with the no-drug control.

Paromomycin is also used therapeutically, and does not seem to impair overall translation.

Aminoglycosides and PTC124 increase Sindbis/Leaky stop codon readthrough in vivo

We tested the effects of the amino-glycoside dirges on the Sindbis/Leaky stop codon dual luciferase reporter to compare with the MoMLV readthrough results, and to corroborate our findings with previously published data (Fan-Minogue and Bedwell 2007). We repeated our transfection and drug treatment of 293A cells with the Sindbis “UGA C” reporter pair and examined readthrough with the DLA in the presence of the aminoglycosides. We adjusted the levels of G418 to only include the lowest concentrations to minimize the toxic effects. In cells treated with G418, readthrough was higher than the comparable 13PK6 results (Figure 4-13). PTC124, paromomycin and gentamicin treated cells had modestly greater increases in readthrough of the UGA C stop codon than the comparable MoMLV pseudoknot containing 13PK6. These results are in agreement with studies on the variance in effect of aminoglycosides on different stop codons and their context.

160 A 20

16 13.1 12 9.7 8.5 8 6.9 6.6 4.6 4.1 3.2 3.1 % readthrough 4

0 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Gentamicin 25 ug/ml Gentamicin 50ug /ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration B 1E+08 1E+07 1E+06 Av. Fluc 1E+05 Av. Rluc 1E+04 Ave. FLuc-C 1E+03 Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Gentamicin 25 ug/ml Gentamicin 50ug /ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration Figure 4-13: Drugs effects on Sindbis leaky stop codon readthrough. (A) Readthrough percentages based on dual luciferase assay results of the Sindbis reporter pair transfected in 293A cells in 6 well format. Drugs were added in indicated amounts two hours after transfection. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

161 G418 and PTC124 may enhance HIV-1 frameshift in Vivo

To observe whether the putative drug-induced manipulation of the decoding center could have an effect on the frameshift mechanism, we repeated the transfection and drug treatment of 293A cells with the HIV-1 frameshift reporter pair (Figure 4-14). We observed a minor increase in frameshift with 0.5 mg/ml of G418, and no increase with lesser amounts. PTC124 almost doubled frameshift frequency at the highest concentration tested, which may partly be a result of the putative luciferase:drug interaction. Paromomycin treatment resulted in a slight decrease in frameshift efficiency.

Gentamicin had no effect.

Paromomycin has also been implicated in potential drug:RNA actions with the HIV-1 genome: a portion of the dimerization signal in HIV-1 folds to assume a secondary structure that is theoretically modeled with similarity to the decoding region of the 16S rRNA. Paromomycin is thought to bind to this region and to prevent dissolution of the

RNA genome dimer, which in turn prevents reverse transcription and integration

(McPike, Goodisman et al. 2002; Ennifar, Paillart et al. 2003; Kaluzhny, Beniaminov et al. 2008). Our p2luc HIV-1 gag-pol junction containing reporter does not contain the dimerization signal and thus this specific interaction cannot be involved in the slight decrease in readthrough.

162 A 20 17.4 15.6 15 11.4 9.0 9.1 9.7 9.2 10 7.3 8.2 5 % readthrough 0 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Gentamicin 25 ug/ml Gentamicin 50 ug/ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 no drug G418 0.5 mg/ml PTC124 5 ug/ml G418 0.25 mg/ml PTC124 2.5 ug/ml Gentamicin 25 ug/ml Gentamicin 50 ug/ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration

Figure 4-14: Drug effects on HIV-1 frameshift. (A) Readthrough percentages based on dual luciferase assay results of the HIV-1 reporter pair transfected in 293A cells in 6 well format. Drugs were added in indicated amounts two hours after transfection. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

163 Aminoglycosides do not increase readthrough of MoMLV in drug-resistant cells

To test whether the increase in MoMLV readthrough observed in the presence of aminoglycosides and PTC124 is a result of interactions of aminoglycosides with the translation machinery and not the mRNA itself we repeated the transfection of the 13PK6 reporter pair and subsequent drug treatment in 293T cells. 293T cells are neo-resistant; they carry a bacterial resistance gene as a result of being immortalized with T-antigen encoded by a neo-resistance containing plasmid. None of the aminoglycosides increased readthrough of the MoMLV PK containing reporter (Figure 4-15). Therefore inactivation of the drug results in no increase in normal readthrough levels.

Aminoglycosides increase MoMLV/PTC readthrough in Vitro

We tested the aminoglycoside effect on readthrough in in-vitro lysates to determine the maximum affect of the drugs on readthrough without the in-vivo limitations of drug take- up and cell toxicity. We prepared IVT reactions with dual luciferase reporter constructs and increasing concentrations of aminoglycosides. After a 2 hour incubation we tested the reactions for luciferase expression using the DLA. We tested G418 and paromomycin because of the enhancement of readthrough in vivo and effects on viral production. We did not test PTC124 due to the controversy surrounding its interaction with luciferase; a fluorescent 13PK6 reporter (described in the appendix) will be more appropriate for testing the effects of this drug on readthrough.

164 A 5.00

4.00

3.00

2.00 1.2 1.2 1.2 1.0 1.1 1.1 0.9 1.1 1.1 % Readthrough 1.00

0 no drug G418 0.5 mg/ml G418 0.25 mg/ml Amikacin 3 mg/ml Amikacin 1.5 mg/ml Gentamicin .05 ug/ml Gentamicin .025 ug/ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration B 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 no drug G418 0.5 mg/ml G418 0.25 mg/ml Amikacin 1.5 mg/ml Gentamicin .05 ug/ml Gentamicin .025 ug/ml Paromomycin .5 mg/ml Paromomycin 1.0 mg/ml drug concentration

Figure 4-15. Drug effects on MoMLV readthrough in 293T neo-resistant cells. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transfected in 293t cells in 6 well format. Drugs were added in indicated amounts two hours after transfection. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

165 Increasing concentrations of G418 caused increasing enhancement of MoMLV readthrough (Figures 4-16 and 4-17). The highest final concentration of 20 ug/ml induced high levels of readthrough, but the raw values of the control luciferase reporter (FLuc-C and RLuc-C) indicate that overall translation was impaired. G418 had little effect on overall control reporter luciferase levels at concentrations of 5 ng/ml and 20 ng/ml, but also did not increase readthrough levels. At 78 ng/ml G418 caused a modest increase in readthrough and no effect on translation over the no drug control. Readthrough was increased 2 fold at .3125 ug/ml, with a corresponding reduction in control reporter luciferase levels. This trend of increased readthrough with decreased control luciferase expression continued with consecutively higher G418 levels. Therefore, although G418 increases readthrough in a dose dependent manner, it appears to affect translation of all mRNAs.

To determine whether the pseudoknot structure enhanced the drug-induced efficiency of readthrough we tested the no-pseudoknot containing reporter CollStop, described previously, in the IVT system. The experimental CollStop reporter contains the same

UAG G stop codon sequence as 13PK6, and allows comparison of the effects of the pseudoknot structure without variations of aminoglycoside sensitivity to the tetranucleotide code. G418 addition seems to increase readthrough of the CollStop reporter at a higher efficiency (Figure 4-18). Concentrations of 2 ng/ml to .3125 ug/ml of

G418 caused a 2 to 5 fold increase in readthrough efficiency of the CollStop reporter

(Figure 4-19). At levels higher than 0.3125 ug/ml the numbers become unreliable, as the

166 98.6 A 100

80

60 45.3 40

% readthrough 17.2 20 12.5 5.7 5.5 5.5 6.9 0 no drug G418 5 ng/ml G418 5 ug/ml G418 20 ng/ml G418 78 ng/ml G418 20 ug/ml G418 1.25 ug/ml G418 .3125 ug/ml drug concentration

B 1E+08 1E+07 1E+06 Av. Fluc 1E+05 Av. Rluc 1E+04 Ave. FLuc-C 1E+03 Ave. RLuc-C 1E+02 Luciferase Units 1E+01 1E+00 no drug G418 5 ng/ml G418 5 ug/ml G418 20 ng/ml G418 78 ng/ml G418 20 ug/ml G418 1.25 ug/ml G418 .3125 ug/ml drug concentration

Figure 4-16. G418 effects on MoMLV readthrough in vitro. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transcribed and translated in in-vitro reactions with G418. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

167 20

14.8 15

10 7.7

5 4.1 Fold Increasein readthrough 2.9 2.2 1.0 1.3 0.8 0 no drug G418 5 ng/ml G418 5 ug/ml G418 20 ug/ml G418 78 ng/ml G418 20 ug/ml G418 1.25 ug/ml G418 .3125 ug/ml drug concentration

Figure 4-17. G418 effects on MoMLV readthrough in vitro: fold change. (A) Fold changes in readthrough of the 13PK6 reporter in in-vitro reactions with G418. Graph shows results 4 trials, error bars correspond to standard error of the mean of average fold changes per condition.

168 A 100

80

60

40

% readthrough 20 5.6 0.6 1.6 2.1 3.7 0 no drug G418 5 ng/ml G418 20 ng/ml G418 78 ng/ml G418 .3125 ug/ml drug concentration

B 1E+08 1E+07 1E+06

1E+05 Av. Fluc 1E+04 Av. Rluc Ave. Fluc-C 1E+03 Ave. Rluc-C

Luciferase Units 1E+02 1E+01 1E+00 no drug G418 5 ng/ml G418 20 ng/ml G418 78 ng/ml G418 .3125 ug/ml drug concentration Figure 4-18. G418 effects on CollStop readthrough in vitro. (A) Readthrough percentages based on dual luciferase assay results of the Collstop reporter pair transcribed and translated in in-vitro reactions with G418. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

169 12.00

10.00 8.7 8.00

5.8 6.00

4.00 3.3 2.4 2.00 1.0

0 no drug G418 5 ng/ml G418 20 ng/ml G418 78 ng/ml G418 .3125 ug/ml drug concentration

Figure 4-19. G418 effects on Collstop readthrough in vitro: fold change. Fold changes in readthrough of the CollStop reporter in in-vitro reactions with G418. Graph shows fold change for experiment in Figure 3-18, error bars correspond to standard error of the mean of average fold changes per condition.

170 raw values of luciferase decreased below the linear range of the assay. This effect on overall translation was more pronounced with the CollStop reporter than with 13PK6, making consistency of reading at higher G418 concentrations unreliable and not correlative with G418 concentration. In addition, we noticed anomalies on the translation of the CollStop reporter in vitro. In several trials the rate of rluc was drastically reduced, giving a high basal readthrough. This was not a consistent event, nor has the cause been determined, however, it has made an absolute effect of G418 on this reporter difficult to obtain. Therefore although CollStop reporter appears to be more sensitive to G418 than

13PK6 at low levels, overall effects of G418 on the CollStop reporter cannot be determined from these experiments.

We tested 13PK6 and CollStop with paromomycin. Readthrough of 13PK6 increased in a dose-dependent manner, with the highest concentration of paromomycin increasing readthrough to over 40% (~ 6 fold increase over basal levels) (Figure 4-20 and Figure

4-21). Fold changes of CollStop readthrough efficiency were similar to these of 13PK6 for paromomycin concentrations of 5 ng/ml to 1.25 ug/ml, with increases of 1.5X to 2.5X

(Figure 4-22 and 3-23). At the two highest concentration of drugs, 5 ug/ml and 20 ug/ml, the Collstop reporter had double the fold increase in readthrough efficiency compared to

13PK6. The highest readthrough percentage measured was ~ 7% — a 16 fold increase over the average basal level of 0.5% . Levels of both 13PK6 and CollStop reporter constructs were relatively unchanged by the addition of paromomycin with only small decreases occurring at the highest concentration. However, the CollStop expression

171 A 100 80

60 46.2 40 22.6 20 8.0 8.9 13.4 % readthrough 6.1 6.3 6.9 0 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration

B 1E+08 1E+07 1E+06 1E+05 1E+04 Av. Fluc 1E+03 Av. Rluc Ave. FLuc-C 1E+02 Ave. RLuc-C Luciferase Units 1E+01 1E+00 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration

Figure 4-20. Paromomycin effects on MoMLV readthrough in vitro. (A) Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transcribed and translated in in-vitro reactions with paromomycin. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

172 8.00 6.2 6.00

4.00 3.7 2.5

Fold Increase 2.00 1.7 1.4 1.6 1.0 1.1

0 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration

Figure 4-21. Paromomycin effects on MoMLV readthrough in vitro: fold change. Fold changes in readthrough of the 13PK6 reporter in in-vitro reactions with paromomycin. Graph shows results 3-4 trials per condition, error bars correspond to standard error of the mean of average fold changes per condition.

173 A 100 80 60 40 20 6.8 % readthrough 0.4 0.8 0.8 0.8 1.0 1.1 3.0 0 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration

B

1E+08 1E+06 Av. Fluc 1E+04 Av. Rluc Ave. FLuc-C 1E+02 Ave. RLuc-C Luciferase Units 1E+00 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration Figure 4-22. Paromomycin effects on CollStop readthrough in vitro. (A) Readthrough percentages based on dual luciferase assay results of the Collstop reporter pair transcribed and translated in in-vitro reactions with paromomycin. Graph is of representative experiment, error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. (B) Average raw values of luciferase for data in (A).

174 20.00 16.2 15.00

10.00 7.1

Fold increase 5.00 2.0 2.4 2.5 1.0 1.9 1.9 0 no drug Paromomycin 5 ng/ml Paromomycin 5 ug/ml Paromomycin 20 ng/ml Paromomycin 78 ng/ml Paromomycin 20 ug/ml Paromomycin 1.25 ug/ml Paromomycin .3125 ug/ml drug concentration

Figure 4-23. Paromomycin effects on Collstop readthrough in vitro: fold change. Fold changes in readthrough of the CollStop reporter in in-vitro reactions with paromomycin. Graph shows fold change for experiment in Figure 3-22, error bars correspond to standard error of the mean of average fold changes per condition.

175 issues previously described have also made it difficult to establish a consistent trend of the effect of paromomycin on this reporter.

176 Chapter 5: Discussion

In the work described here we have investigated trans-acting factors that enhance readthrough at the MoMLV gag-pol junction. The cell-line dependent increase in readthrough by the cellular protein RPL4 indicates that the cis-acting function of the

MoMLV pseudoknot which promotes readthrough may not the sole modulator of readthrough in the cellular environment. The enhancement of MoMLV readthrough by aminoglycoside drugs (and possibly PTC124) also shows that the recoding at the gag stop codon can be affected by extraneous factors. We provide examples that increased readthrough and alterations to the gag;gag-pol ratio reduce retroviral replication, and that slight increases in gag-pol are enough to cause this effect. We demonstrate that the readthrough rate promoted by the pseudoknot can be pushed higher, and the basal level does not represent an inherent ceiling of readthrough in the cell. In addition to the specific impact on MoMLV replication, we show that the process of proofreading and resultant stop codon suppression can be affected by compound factors in the cell. This implies that there are potentially many levels of control in the eukaryotic proofreading and termination mechanism that remain to be elucidated.

RPL4

Here we demonstrate the phenomenon of recoding enhancement by the trans-acting cellular protein, RPL4. We show that RPL4 is capable of increasing readthrough of the stop codon at the gag-pol junction. Co-transfection of cells with RPL4 cDNA and the

DLA reporter constructs results in a dose-dependent increase in readthrough.

177 Interestingly, this increase is cell line dependent. This suggests that participation of other cellular factors with different expression profiles may also influence readthrough, and are able to block or compensate for the RPL4-induced effect. We tested a panel of other ribosomal proteins to rule out the possibility that addition of extra RPL4 mRNA/protein to the cellular environment was causing a general disruption in the stoichiometry of ribosomal proteins, and that increase in readthrough was a non-specific effect due to a ribosomal protein imbalance. Although proteins L7 and S20 showed a modest increase in

MoMLV readthrough over basal levels, all other proteins tested showed had no effect at all on readthrough, indicating that the RPL4 effect on readthrough is not a result of general non-specific disruption of the translation machinery.

Over-expression of RPL4 has proven difficult to verify via Western blot. The use of tags at either the N- or C-terminus eliminate the readthrough enhancement; the N-terminally tagged versions can be visualized with anti-Flag antibodies, but are not functional in enhancing readthrough. C-terminally tagged constructs can not be seen, do not enhance readthrough and may not be produced in vivo. Use of commercial polyclonal and monoclonal antibodies allowed visualization of RPL4, but the signal did not increase with transfection of RPL4 expression vectors; we concluded that the regulation of RPL4 is tightly controlled, and that the increase may not be detectable by Western blot.

However, we have shown that RPL4 mRNA is transcribed in 293A cells, and that enhancement of readthrough is not due to an RNA effect. We surmise that very small, or transient, increases in the levels of protein may be responsible for the effects.

178 We investigated whether RPL4 only affected the MoMLV pseudoknot or was able to perturb other recoding events. HIV-1 frameshift and Sindbis “leaky stop codon” reporters both showed enhancement of recoding with RPL4. Because HIV-1 utilizes a slippery sequence and a stem-loop structure, and the portion of the Sindbis recoding signal used here is merely a leaky stop codon without a secondary structure, we can conclude that the RPL4 enhancement of MoMLV readthrough is not dependent specifically on the MoMLV gag-pol junction sequence, or even on a general pseudoknot motif.

To clarify whether RPL4 was allowing readthrough of any premature termination codon, we constructed a derivative of the MoMLV pseudoknot reporter, ScrPK, that harbored a scrambled pseudoknot sequence with no predicted secondary structures This reporter showed no enhancement of readthrough with RPL4 addition, but overall levels of luciferase were much lower than with the wild-type reporter pair. Additionally, in the scrambling of the pseudoknot sequence the nucleotide immediately following the UAG stop codon was changed from G to A. The UAG A tetranucleotide sequence has inherently lower readthrough than UAG G. We tested another version of the ScrPK reporter which included the flanking sequences and the native UAG G stop codon sequence, but the levels of rluc expression were also very low, indicating a problem with overall expression of the constructs. The abolishment of readthrough and/or the secondary structure likely decreased the stability of these transcripts due to increased

179 susceptibility to the NMD pathway. Subsequently, the translation of the both luciferase proteins were affected to the extent that the data was below the linear range of our DLA.

Attempts to circumvent NMD with dominant negative UPF1, a major regulatory factor in the decay of premature-stop codon containing mRNAs, were unsuccessful due to other effects on the translation and/or termination cycle. Reporter constructs containing the

RSE from Rous Sarcoma Virus, which provides protection from NMD, also failed to show adequate translation of the rluc reporter. A reporter construct containing a portion of the collagen reading frame with an artificial stop codon had rluc levels comparable to the wild-type MoMLV 13K6 reporter was created. RPL4 did not appear to significantly increase readthrough levels of this construct, indicating that the effect is not general to premature termination codons. In addition, it seems unlikely that RPL4 can increase suppression of normal termination events, given the general health of transfected cells.

Rather, RPL4 seems to enhance the propensity for recoding to occur in the presence of some distinct recoding signal.

We confirmed that RPL4 does not rescue an inactive pseudoknot mutant, in accordance with the apparent requirement for a recoding signal. RPL4 increased readthrough of a hyperactive pseudoknot mutant in a manner consistent with the effect on the wild-type

MoMLV pseudoknot; overall readthrough was higher, but the fold change above baseline was similar to that seen with 13PK6. This shows that the RPL4 enhancement does not act synergistically with nor is eclipsed by the mechanism that allows for more readthrough in the hyperactive mutant. Readthrough may be controlled in several ways during

180 translation. We show also that RPL4 enhances readthrough through suppression of termination of all three stop codons, despite the natural resistance to readthrough that occurs with UAA. We also show that RPL4 works additively, and is not eclipsed by the aminoglycoside G418.

It is important to note that crystal structures of the aminoglycoside paromomycin bound to the 16s rRNA of the bacterial decoding center show displacement of the proof-reading nucleotides A1492 and A1493 from their native position which also occurs upon cognate codon:anticodon interactions. The displaced nucleotides are not in the same orientation with paromomycin binding as they are in a cognate codon:anticodon interaction.

Therefore, it seems it is the displacement from the default position of these nucleotides that is the catalyst of proofreading, not the specific orientation that they assume. If RPL4 were simply disrupting the normal alignment of the molecular caliper in the 18S rRNA, either by binding the pseudoknot and/or ribosome, then addition of an aminoglycoside would not be expected to add to readthrough. Likewise, the ability of aminoglycosides to increase readthrough is extremely sensitive to stop codon identity and the tetranucleotide code. The RPL4 enhancement of readthrough does not share these restrictions. Taken together, these findings show that RPL4 may be working beyond manipulation of the decoding center to increase readthrough.

Overexpression of the termination factors eRF1 and eRF3 with 13PK6 had no effect on readthrough in our DLA. However, transfection of these factors, or eRF1 alone abolished

181 the RPL4 effect. We show also that this is not a general affect of co-transfection of multiple expression constructs since co-transfection of RLP4 and RPL7 did not similarly abolish the enhanced readthrough. Therefore, RPL4 overexpression may be interfering with normal termination, and this effect is overcome by increased expression of release factors. If readthrough is viewed as a competition between termination and non-cognate incorporation of glutamine at a UAG stop codon (prompted by a change in pseudoknot conformation), RPL4 could be sequestering or preventing termination factors from recognizing the stop codon, and allowing a longer temporal window for the pseudoknot to assume the active conformation. Alternatively, acceptance of the tRNA in the A site is also dictated by GTP hydrolysis; RPL4 could be altering the rate of GTP hydrolysis by some mechanism, potentially speeding it up and making proofreading less rigorous. Here, a competition between termination and readthrough could possibly be adjusted by an increase in the availability of release factors to recognize stop codons and catalyze termination.

Paradoxically it has been shown that knock-down of eRF1 reduces readthrough in a one plasmid reporter system (Carnes 2003) (although affects on general termination via a control reporter were not explored, and it could not be verified that the decrease in readthrough was not due to overall suppression of termination.) More recent work has documented a role for eRF1 in HIV-1 frameshift that is not related to the position of the stop codon; depletion of eRF1 increases HIV-1 frameshift in HeLa cells, yeast and in- vitro DLA assays (Kobayashi, Zhuang et al. 2010). These findings correlate with our

182 observations and eRF1 be a common factor in the RPL4 enhancement of readthrough and frameshift.

Knockdown of RPL4 has been shown to decrease rRNA processing (Yang, Henning et al.

2005), and it has been proposed that RPL4 stimulates ribosome biogenesis. We wondered if a simple explanation for the RPL4 enhancement of readthrough was that transfection of

RPL4 and transcription and/or expression acted as a trigger in some cells to produce more ribosomes. In IVT reactions, with set amounts of ribosomes and elongation and termination factors, we do not see an RLP4 specific increase in readthrough. We examined the expression of another ribosomal protein, RPL10a, upon transfection with the RPL4 expression construct. We saw no detectable increase in RPL10a levels in either cell lines that responded to RPL4 or in those that did not. We then used qPCR to measure

28S rRNA and although we saw only a slight increase in levels of 28S rRNA with RPL4 overexpression, and this increase did not correlate with RPL4 responsiveness. Thus,

RPL4 may increase the amount of ribosomes, but this alone seems unlikely to promote the increase in readthrough. The lack of effect in IVT reactions may be due to increase of an additional, unknown factor that could also be the distinguishing element between

RPL4 responding and non-responding cell lines.

Readthrough of the gag stop codon allows the optimal proportion of gag and gag-pol proteins to be translated for virus replication. It has been suggested that slight deviations

183 from the normal gag:gag-pol ratio will affect viral replication. The approximately 2-fold

RPL4 enhancement readthrough, although consistent and unique, is moderate compared to the changes seen in the study of other biological processes, and therefore we investigated whether this relatively small change was relevant to viral replication. We show that RPL4 increased readthrough can indeed profoundly limit the replicative ability of the virus. Consistent production of gag but lower capsid levels in cells transfected with

RPL4 indicates that the gag is produced, but there is a block of normal processing.

Analysis of virus produced in the presence of RPL4 suggests that less virus is released from producer cells. In addition to verifying the effect of RPL4 on MoMLV readthrough, these results further stress the importance of the maintenance of the gag:gag-pol ratio in retroviral replication.

Thus we show that RPL4 is a positive enhancer of recoding, which in turn has negative effects on retroviral replication. Although the precise mechanism of RPL4’s effect is still unclear, our work indicates participation of an additional factor(s) which have differential expression among cell lines. The RPL4 effect appears to be independent of other ways of modulation of readthrough, and may interfere at a different point in the mechanisms of translation fidelity than the point affected by the pseudoknot. Given RPL4’s effect on frameshift as well as readthrough, this modulation of recoding may occur as a more general suppression of termination outside the ribosome, or involve another facet of tRNA acceptance on the ribosome.

184 Roles for several ribosomal proteins in extra-ribosomal translational control have been discovered (reviewed in (Warner and McIntosh 2009). RPL13A is phosphorylated as part of the cellular response to IFNγ; phosphorylated RPL13 disassociates from the ribosome and forms a complex that binds the 3‘ UTR of certain mRNAs and prevents their translation (Kapasi, Chaudhuri et al. 2007). RPL38 regulates the translation of hox genes during embryonic development in a tissue specific manner — it prevents proper assembly of the 80S ribosome, thereby disrupting normal initiation (Kondrashov, Pusic et al. 2011).

Microarray analysis of ribosomal protein expression in various cell types shows differential expression of RPL38 and many ribosomal proteins, indicating that many ribosomal components could have extra-ribosomal functions. This also raises the possibility of heterogeneity in ribosome composition; not all ribosomal proteins may be present in all ribosomes (Kondrashov, Pusic et al. 2011).

There are ribosomal proteins that serve as “ribosome biosynthesis sentinels” (Warner and

McIntosh 2009) and defects in ribosome biogenesis result in the activation of p53 and subsequent apoptosis. RPL11 can interact with H/MDM2 and sequester it, thereby stabilizing p53 (Lohrum, Ludwig et al. 2003); treatment of cells with low levels of actinomycin D to disrupt the transcription of ribosomal components increases the association of RPL11 with H/MDM2 and results in p53 mediated cell cycle arrest. RPL5 interacts with H/MDM2 (Marechal, Elenbaas et al. 1994), both alone and in conjunction with RPL11 (Horn and Vousden 2008), to protect p53. RPL23 also interacts with H/

MDM2 to in a manner similar to RPL5 and RPL11(Dai and Lu 2004). Interestingly,

185 RPL5, RPL23 and RPL11 can interact with c-myc (Dai, Sears et al. 2007) and RPL11 inhibits c-myc response to ribosomal stress (Dai, Arnold et al. 2007) and induces degradation of c-myc mRNA (Challagundla, Sun et al. 2011). Disruption of ribosome biogenesis may allow unassembled ribosomal proteins to perform extraribosomal functions to respond to the deregulation and promote apoptosis (Warner and McIntosh

2009).

The mechanism by which RPL4 alters translation to allow greater readthrough remains unclear. RPL4 has multiple roles in the cell, and may affect readthrough in a variety of ways (Figure 5-1). In eukaryotic cells RPL4 has a necessary role in ribosome biogenesis as it is involved in the processing of rRNA; therefore its expression pattern is expected to be consistent in all cell types. Paradoxically, the cell line dependence of the RPL4 response implies either alternate regulation of RPL4 itself or differential expression of an additional factor that is involved in readthrough. RPL4 expression has been associated with apoptosis in a rat neuronal tumor line treated with 5AzC (Kajikawa, Nakayama et al.

1998), but a later study by the same group did not find the same results in embryonic rat brain tissue. Instead, it appears that there is differential expression of RPL4 during development of the rat brain, with increased RPL4 expression corresponding to differentiation and proliferation, perhaps due to higher level of protein synthesis as development occurs (Ueno, Nakayama et al. 2002). In addition, the role of RPL4 as a stimulator of the transcription of c-myc seems to suggest a pro-growth, rather than regulatory, function. Just as specific ribosomal proteins, but not all, stimulate cell cycle

186 arrest (Sun, Wang et al. 2010), others may upregulate ribosomal biogenesis. RPL4 may stimulate the transcription of one or many other factors that contribute to the enhancement of recoding. It also may be worthwhile to consider the role of RPL4 as part of a cellular anti-viral response, although it is unlikely that it dissociates from the assembled ribosome in the manner of RPL13a.

In many cell lines, RPL4 expression appears constant. RPL4 is used as a standard in qPCR, showing stable expression in many tissue types such as equine embryos (Paris,

Kuijk et al. 2011), chicken embryo fibroblasts (Yue, Lei et al. 2010), human ovarian tissue (Fu, Bian et al. 2010), and murine liver (Takagi, Ohashi et al. 2008). Our attempts to visualize levels of normal and over-expressed RPL4 among various cells types did not reveal any significant detectable differences in protein by Western blot. While this is likely due in part to the antibodies used, the difficulty in visualizing changes in RPL4 may be considered evidence that the expression of RPL4 is tightly regulated under normal circumstances and large differences in levels are not tolerated by the cell.

However it may be possible to visualize a significant transient increase that might occur in our experimental system with alternately tagged versions and we are currently attempting to clone a readthrough enhancing RPL4 construct with an interior Flag tag.

This would also allow visualization of RPL4 in various locations in the cell. RPL4 has nucleolar and nuclear localizations as well as the ribosomal position and perhaps an increase in RPL4 mRNA from the expression construct is a stimulus for reorganization of

RPL4 in the cell. Therefore, although overall levels appear unchanged, a redistribution of

187 additional RPL4 Nucleus: Increased transcription of Myb-regulated genes

Nucleolus: Increased Ribosome Biogenesis

Cytoplasm: protein:protein interaction Ribosomal: mRNA interaction eRF1/3 ?

Monday, November 7, 2011

Figure 5-1. Potential Mechanisms for RPL4 enhancement of Recoding. RPL4 may influence recoding directly or indirectly through many roles in the cell. It may trigger upregulation of ribosomes in the nucleolus; may increase the transcription of another gene associated with an aspect of translation or termination; or may interact directly with a termination or other factor in the cytoplasm. An abundance of RPL4 may also interact directly with mRNA to enhance stability of secondary structures or with the ribosome itself to perturb normal aa-tRNA selection or decoding.

188 RPL4 could lead to readthrough enhancement. A reliable way to visualize exogenous

RPL4 also would allow quantification of RPL4 in different cellular fractions, as well as imaging by immunofluorescence.

To understand how RPL4 is affecting readthrough, it will be necessary to determine whether the enhancement of recoding relies on a predisposition of an mRNA to allow a miscoding event. We presently lack a definitive non-viral non-recoding control for gauging RPL4’s effect on readthrough in the context of simple premature termination.

Our attempts at designing control constructs have not been entirely successful due to limitations of detection, mRNA stability or other expression issues. Exploration of the

RPL4 effect using an alternative reporter system to the DLA may allow proper expression of a non-recoding control and provide a clearer result.

The apparent link between RPL4 and release factors is a phenomenon that can be further explored. The fact that RPL4 affects both readthrough and frameshift argues for its specificity to enhance recoding of mRNAs that contain a stimulus for miscoding. eRF1 has also been implicated in both readthrough and frameshift, and may have a role in mRNA surveillance that is not limited to interactions with the stop codon (Kobayashi,

Zhuang et al. 2010). Thus, RPL4 may interfere with the functions of eRF1. Analysis of physical interactions between RPL4 and release factors is an appropriate approach to understanding the relationship between these proteins and their effect on readthrough. We

189 are currently optimizing experimental conditions for immunoprecipitation of cellular lysates to probe for RPL4:eRF1 interactions.

If RPL4 is preventing termination, i.e. interaction with eRF1, this process may be visualized with a footprinting assay in a reconstituted translation system as per the

Pestova lab (Pestova and Hellen 2003; Pestova and Hellen 2005; Alkalaeva, Pisarev et al.

2006). In these assays, binding of release factors to the ribosome results in a conformational change in ribosome structure which can be visualized as a “two nucleotide forward shift of the toeprint” (Alkalaeva, Pisarev et al. 2006). This would require production of a soluble form of RPL4 protein. We were not successful with our attempts to recover RPL4 produced in bacteria after cleavage of the GST tag and further optimization of the purification and recovery of RPL4 are needed. It may be possible to produce RPL4 in a human in-vitro lysate system if an appropriate purification scheme can be developed. The ability to test RPL4 in a customized reconstituted translation system would also help identify additional factors that may be involved in the readthrough and may even allow study of the point of RPL4 effect on readthrough through analysis of GTP hydrolysis during the proofreading process in a controlled environment.

The cell line dependence of the enhancement of readthrough by RPL4 can also be explored. As RPL4 has been shown to stimulate the transcription of myb-regulated genes, microarray analysis of myb-transcribed genes in responding and non-responding cell

190 lines would be a logical first step. Additionally, mass spectrometry analysis of RPL4- bound proteins in responding and non-responding cells could reveal differences in interactions between lines that cannot be detected by microarray. FACS analysis with fluorescent recoding reporters as described in the appendix is also a potential tool for broadening our understanding of the network of factors involved in recoding.

Identification of factors associated with RPL4 in endogenous and overexpression conditions in responding and non-responding cell would contribute not only to the understanding of the specific RPL4 effect on recoding, but would add to the knowledge of general eukaryotic translation regulation.

Readthrough promoting drugs

We have examined the effect of aminoglycoside antibiotics and the small molecule

PTC124 on the readthrough efficiency of the MoMLV gag-pol junction. We show that exposure of mammalian cells to specific aminoglycosides can result in a dose dependent increase of readthrough at the MoMLV junction as measured by our dual luciferase reporter system. G418 and paromomycin exhibit the greatest effects on readthrough in vivo. Exposure of cells to PTC124 also appeared to increase readthrough of the 13PK6 dual luciferase reporter, but this effect may be exaggerated by drug:luciferase interactions. Amakacin, streptomycin, kanamycin and gentamicin demonstrate little effect on readthrough in vivo. As the aminoglycosides vary only slightly from each other in terms of structure, the specific configurations of the G418 and paromomycin result in

191 their efficacy in promoting readthrough of 13PK6. Both G418 and paromomycin have an

OH at the 6’ position of ring I, which may be responsible for facilitating a reaction with the 18S rRNA that favors readthrough (Figure 4-2). However the structure of an additional sugar (ring III) is different between the two, and is attached at a different position on ring II. The combination of ring III structure and linkage may contribute to toxicity of G418, and/or higher tolerance of paromomycin.

We show that exposure of virus-producing cells to readthrough promoting drugs can affect viral production. Expression of the fluc reporter gene was reduced in all cells infected with recombinant virus produced in the presence of drugs. Virus from G418 reduced cells was reduced from 2 to 4 logs compared with the no-drug control. However, toxicity is an issue with G418, which makes interpretation of its effects in vivo difficult.

Rluc levels in producer cells exposed to G418 were reduced in all trials and a viability assay of G418 treated cells confirmed that viability is reduced as G418 concentration and readthrough efficiency increases. Therefore we are unable to determine if it is the readthrough promoting actions of G418 or its general toxicity to the producer cells that is responsible for decreased infection.

Paromomycin had the greatest effect on viral replication with the least disruption of translation in producer cells (measured by rluc expression), with up to a 10 fold reduction in signal reduction. PTC124 also reduced expression of the viral-conferred firefly gene

192 in infected cells 2- to 5-fold; since the naive cells are not exposed to PTC124, this effect is independent of any drug:luciferase artifacts in the DLA. Amikacin treated cells displayed the least effect on virus produced, but display 10 - 50% reduced signal as compared to the no-drug control. Producer cells treated with PTC124 show some reduction in renilla expression in producer cells, while amikacin does not affect rluc signals. Therefore, exposure of mammalian cells to readthrough promoting drugs may be a method for controlling production of viruses that utilize stop codon suppression.

We show that the aminoglycoside effects on readthrough in the DLA are nonexistent in

293T “neo”-resistant cells. Therefore, inactivation of the drug prevents its ability to enhance readthrough. We verify the flexibility of our experimental system with a test of the leaky stop codon Sindbis reporter, which shows expected greater increases in readthrough due to increased sensitivity to aminoglycosides. We also show that aminoglycosides are specific for increasing readthrough and not frameshift at the doses tested with a survey of drugs and the HIV-1 dual luciferase reporter pair.

We examined the affects of G418 and paromomycin on readthrough in an in-vitro system.

We show that G418 is able to promote high levels of readthrough, but that these increases correspond with overall translational dysfunction. Readthrough of the non-pseudoknot containing reporter CollStop also reaches high levels with G418. At high concentrations of G418, there was a large spike in readthrough frequency of Collstop, with increases of

193 up to 40% readthrough (data not shown). However, the levels of control reporter levels were also affected at this point, making interpretation of the G418 effect unreliable. The mechanism by which G4I8 works may affect translation of all mRNAs, and thus a predisposition of an mRNA to recoding may be eclipsed by a a general effect on translation. Paromomycin stimulates moderate increases in readthrough frequency in a dose dependent manner, with less negative impact on translation of the reporter constructs. This result pertains to both 13PK6 and CollStop reporters. Therefore, aminoglycoside effects in readthrough are not limited to overall disruption of all translational events. The interaction of paromomycin with the eukaryotic ribosome may be intrinsically tied to recoding signals or readthrough of premature termination codon and not to a general miscoding mechanism that functions at any codon. However, verification of this theory is beyond the scope of these experiments.

Our work with aminoglycosides also offers novel insight into eukaryotic translation in the handling of miscoding events. We show that readthrough of the MoMLV pseudoknot can be pushed beyond normal levels upon exposure to certain aminoglycosides; the mechanism by which the MoMLV pseudoknot stimulates readthrough is not exclusively the only way to promote recoding on the eukaryotic ribosome. Likewise, aminoglycoside interactions do not eclipse the readthrough stimulated by the pseudoknot, or the additional enhancement by RPL4. This additive effect of factors on readthrough implies many points of regulation that can be altered to allow miscoding.

194 We also demonstrate that the conventional wisdom that ‘antibiotics won’t do anything for a viral infection’ may not be entirely correct. Our survey of aminoglycoside drug affects on MoMLV readthrough shows that it is possible to alter the rate of miscoding slightly, but enough, to affect viral replication. We tested a limited number of aminoglycosides, and the affects of paromomycin are encouraging. Because aminoglycosides work poorly on eukaryotic ribosomes, further tests may reveal others that work just well enough to promote viral readthrough without great toxicity. As with the RPL4 effect, a properly expressing non-viral non-recoding control is needed to distinguish general effects on translation termination from specific enhancement of viral readthrough.

Final thoughts

We have demonstrated that trans-acting factors can affect the frequency of recoding of mRNAs by the eukaryotic ribosome, indicating that regulation of recoding is not regulated exclusively by mRNA secondary structure. Greater understanding of the phenomenon of recoding can be gained through structural studies, and the visualization of the pseudoknot on the ribosome through cryo-EM will be a powerful source of information that our lab is still working towards. We are also considering single molecule imaging of recoding events utilizing fluorescent-tagged translation components (for review see (Petrov, Kornberg et al. 2011) and will be investigating the feasibility of applying these techniques to our studies in the near future.

195 Thus we have laid important groundwork for understanding the process of miscoding in eukaryotic translation. We have revealed an alternate role for RPL4 as an enhancer of readthrough, adding to the known functions of ribosomal proteins beyond their traditional ribosomal purpose. We also reveal a non-traditional application of aminoglycoside antibiotics to alter translation of retroviral proteins. This work provides many opportunities for further studies on retroviral recoding and general miscoding mechanisms.

196 References

Alam, S. L., N. M. Wills, et al. (1999). "Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus." J Mol Biol 288(5): 837-852.

Alkalaeva, E., A. Pisarev, et al. (2006). "In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3." Cell 125(6): 1125-1136.

Auld, D. S., S. Lovell, et al. (2010). "Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124." Proc Natl Acad Sci U S A 107(11): 4878-4883.

Auld, D. S., N. Thorne, et al. (2009). "Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression." Proc Natl Acad Sci U S A 106(9): 3585-3590.

Balada, E., M. Vilardell-Tarres, et al. (2010). "Implication of human endogenous retroviruses in the development of autoimmune diseases." Int Rev Immunol 29(4): 351-370.

Ben-Neriah, Y., G. Q. Daley, et al. (1986). "The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene." Science 233(4760): 212-214.

Ben-Shem, A., N. Garreau de Loubresse, et al. (2011). "The structure of the eukaryotic ribosome at 3.0 A resolution." Science 334(6062): 1524-1529.

Ben-Shem, A., N. Garreau de Loubresse, et al. (2011). "The Structure of the Eukaryotic Ribosome at 3.0 A Resolution." Science.

Ben-Shem, A., L. Jenner, et al. (2010). "Crystal structure of the eukaryotic ribosome." Science 330(6008): 1203-1209.

Bernstein, D. S., N. Buter, et al. (2002). "Analyzing mRNA-protein complexes using a yeast three-hybrid system." Methods 26(2): 123-141.

Brierley, I. and F. J. Dos Ramos (2006). "Programmed ribosomal frameshifting in HIV-1 and the SARS–CoV." Virus Research 119(1): 29-42.

Cardno, T. S., E. S. Poole, et al. (2009). "A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons." Rna 15(8): 1614-1621.

197 Carnes, J. (2003). "Stop codon suppression via inhibition of eRF1 expression." Rna 9(6): 648-653.

Challagundla, K. B., X. X. Sun, et al. (2011). "Ribosomal protein L11 recruits miR-24/ miRISC to repress c-Myc expression in response to ribosomal stress." Mol Cell Biol 31(19): 4007-4021.

Cheng, Z., K. Saito, et al. (2009). "Structural insights into eRF3 and stop codon recognition by eRF1." Genes & Development 23(9): 1106-1118.

Ciuffi, A. (2008). "Mechanisms governing lentivirus integration site selection." Curr Gene Ther 8(6): 419-429.

Coffin, J. M., Hughes, S. H., Varmus, H. (1997). Retroviruses, Cold Spring Harbor Laboratory Press.

Dai, M. S., H. Arnold, et al. (2007). "Inhibition of c-Myc activity by ribosomal protein L11." EMBO J 26(14): 3332-3345.

Dai, M. S. and H. Lu (2004). "Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5." J Biol Chem 279(43): 44475-44482.

Dai, M. S., R. Sears, et al. (2007). "Feedback regulation of c-Myc by ribosomal protein L11." Cell Cycle 6(22): 2735-2741.

Davis, R. L., J. B. Konopka, et al. (1985). "Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties." Mol Cell Biol 5(1): 204-213.

Donnelly, M. L., G. Luke, et al. (2001). "Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'." J Gen Virol 82(Pt 5): 1013-1025.

Dresios, J., P. Panopoulos, et al. (2006). "Eukaryotic ribosomal proteins lacking a eubacterial counterpart: important players in ribosomal function." Molecular Microbiology 59(6): 1651-1663.

Du, M., X. Liu, et al. (2008). "PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model." Proc Natl Acad Sci U S A 105(6): 2064-2069.

Egoh, A., S. Nosuke Kanesashi, et al. (2010). "Ribosomal protein L4 positively regulates activity of a c-myb proto-oncogene product." Genes Cells 15(8): 829-841.

198 Ennifar, E., J. C. Paillart, et al. (2003). "HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics." J Biol Chem 278(4): 2723-2730.

Fan-Minogue, H. and D. M. Bedwell (2007). "Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity." Rna 14(1): 148-157.

Felsenstein, K. M. and S. P. Goff (1988). "Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing." J Virol 62(6): 2179-2182.

Felsenstein, K. M. and S. P. Goff (1992). "Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein." J Virol 66(11): 6601-6608.

Feng, Y. X., T. D. Copeland, et al. (1990). "Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus." Proc Natl Acad Sci U S A 87(22): 8860-8863.

Feng, Y. X., D. L. Hatfield, et al. (1989). "Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA." J Virol 63(5): 2405-2410.

Feng, Y. X., J. G. Levin, et al. (1989). "Suppression of UAA and UGA termination codons in mutant murine leukemia viruses." J Virol 63(6): 2870-2873.

Feng, Y. X., H. Yuan, et al. (1992). "Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot." J Virol 66(8): 5127-5132.

Firth, A. E., N. M. Wills, et al. (2011). "Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element." Nucleic Acids Res 39(15): 6679-6691.

Frank, J. and R. L. Gonzalez, Jr. (2010). "Structure and dynamics of a processive Brownian motor: the translating ribosome." Annu Rev Biochem 79: 381-412.

Fu, J., L. Bian, et al. (2010). "Identification of genes for normalization of quantitative real-time PCR data in ovarian tissues." Acta Biochim Biophys Sin (Shanghai) 42(8): 568-574.

Goff, S. P., E. Gilboa, et al. (1980). "Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA." Cell 22(3): 777-785.

199 Grentzmann, G., J. A. Ingram, et al. (1998). "A dual-luciferase reporter system for studying recoding signals." Rna 4(4): 479-486.

Haller, C. and O. T. Fackler (2008). "HIV-1 at the immunological and T-lymphocytic virological synapse." Biol Chem 389(10): 1253-1260.

Hellen, C. U. (2007). "Bypassing translation initiation." Structure 15(1): 4-6.

Hellen, C. U. (2009). "IRES-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry." Biochim Biophys Acta 1789(9-10): 558-570.

Hellen, C. U. and P. Sarnow (2001). "Internal ribosome entry sites in eukaryotic mRNA molecules." Genes Dev 15(13): 1593-1612.

Hermann, T. (2007). "Aminoglycoside antibiotics: old drugs and new therapeutic approaches." Cellular and Molecular Life Sciences 64(14): 1841-1852.

Hirawat, S., E. M. Welch, et al. (2007). "Safety, Tolerability, and Pharmacokinetics of PTC124, a Nonaminoglycoside Nonsense Mutation Suppressor, Following Single- and Multiple-Dose Administration to Healthy Male and Female Adult Volunteers." The Journal of Clinical Pharmacology 47(4): 430-444.

Hirokawa, G., R. M. Nijman, et al. (2005). "The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits." RNA 11(8): 1317-1328.

Hogg, J. R. and K. Collins (2007). "RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation." Rna 13(6): 868-880.

Hogg, J. R. and S. P. Goff (2010). "Upf1 senses 3'UTR length to potentiate mRNA decay." Cell 143(3): 379-389.

Horn, H. F. and K. H. Vousden (2008). "Cooperation between the ribosomal proteins L5 and L11 in the p53 pathway." Oncogene 27(44): 5774-5784.

Houck-Loomis, B., M. A. Durney, et al. (2011). "An equilibrium-dependent retroviral mRNA switch regulates translational recoding." Nature.

Houck-Loomis, B. R. (2009). Structural and Functional Studies of the Moloney Murine Leukemia Virus Pseudoknot. Graduate School of Arts and Sciences. New York, Columbia University. PhD.

Houghton, J. L., K. D. Green, et al. (2010). "The future of aminoglycosides: the end or renaissance?" Chembiochem 11(7): 880-902.

200 Isken, O. and L. E. Maquat (2007). "Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function." Genes & Development 21(15): 1833-3856.

Jackson, R. J., S. Napthine, et al. (2001). "Development of a tRNA-dependent in vitro translation system." RNA 7(5): 765-773.

Jern, P. and J. M. Coffin (2008). "Effects of retroviruses on host genome function." Annu Rev Genet 42: 709-732.

Jones, D. S., F. Nemoto, et al. (1989). "The effect of specific mutations at and around the gag-pol gene junction of Moloney murine leukaemia virus." Nucleic Acids Res 17(15): 5933-5945.

Kajikawa, S., H. Nakayama, et al. (1998). "Increased expression of rat ribosomal protein L4 mRNA in 5-azacytidine-treated PC12 cells prior to apoptosis." Biochem Biophys Res Commun 252(1): 220-224.

Kaluzhny, D. N., A. D. Beniaminov, et al. (2008). "2-aminopurine fluorescence: discrimination between specific and unspecific ligand binding to the kissing-loop dimer of the HIV-1 RNA." J Biomol Struct Dyn 25(6): 663-667.

Kapasi, P., S. Chaudhuri, et al. (2007). "L13a blocks 48S assembly: role of a general initiation factor in mRNA-specific translational control." Mol Cell 25(1): 113-126.

Kashima, I., A. Yamashita, et al. (2006). "Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay." Genes Dev 20(3): 355-367.

Kirchhoff, F. (2010). "Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses." Cell Host Microbe 8(1): 55-67.

Klinge, S., F. Voigts-Hoffmann, et al. (2011). "Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6." Science 334(6058): 941-948.

Kobayashi, Y., J. Zhuang, et al. (2010). "Identification of a cellular factor that modulates HIV-1 programmed ribosomal frameshifting." J Biol Chem 285(26): 19776-19784.

Kolupaeva, V. G., S. de Breyne, et al. (2007). "In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry." Methods Enzymol 430: 409-439.

201 Kolupaeva, V. G., T. V. Pestova, et al. (2000). "Ribosomal binding to the internal ribosomal entry site of classical swine fever virus." Rna 6(12): 1791-1807.

Kolupaeva, V. G., T. V. Pestova, et al. (1998). "Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA." J Biol Chem 273(29): 18599-18604.

Kondrashov, N., A. Pusic, et al. (2011). "Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning." Cell 145(3): 383-397.

Kontos, H., S. Napthine, et al. (2001). "Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency." Molecular and Cellular Biology 21(24): 8657-8670.

Kothe, U., A. Paleskava, et al. (2006). "Single-step purification of specific tRNAs by hydrophobic tagging." Analytical Biochemistry 356(1): 148-150.

Kuchino, Y., H. Beier, et al. (1987). "Natural UAG suppressor glutamine tRNA is elevated in mouse cells infected with Moloney murine leukemia virus." Proc Natl Acad Sci U S A 84(9): 2668-2672.

Kuchino, Y., S. Nishimura, et al. (1988). "Selective inhibition of formation of suppressor glutamine tRNA in Moloney murine leukemia virus-infected NIH-3T3 cells by Avarol." Virology 165(2): 518-526.

Lecompte, O., R. Ripp, et al. (2002). "Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale." Nucleic Acids Res 30(24): 5382-5390.

Li, G. and C. M. Rice (1993). "The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon." J Virol 67(8): 5062-5067.

Li, G. P. and C. M. Rice (1989). "Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication." J Virol 63(3): 1326-1337.

Li, M. Z. and S. J. Elledge (2007). "Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC." Nature Methods 4(3): 251-256.

Liao, P. Y., Y. S. Choi, et al. (2011). "The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting." Nucleic Acids Res 39(1): 300-312.

Lohrum, M. A., R. L. Ludwig, et al. (2003). "Regulation of HDM2 activity by the ribosomal protein L11." Cancer Cell 3(6): 577-587.

202 Lombardi, V. C., F. W. Ruscetti, et al. (2009). "Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome." Science 326(5952): 585-589.

Luce, M. C., K. D. Tschanz, et al. (1985). "The accuracy of protein synthesis in reticulocyte and HeLa cell lysates." Biochim Biophys Acta 825(3): 280-288.

Macfarlan, T. S., W. D. Gifford, et al. (2011). "Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A." Genes Dev 25(6): 594-607.

Malhotra, A., P. Penczek, et al. (1998). "Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein." J Mol Biol 280(1): 103-116.

Marechal, V., B. Elenbaas, et al. (1994). "The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes." Mol Cell Biol 14(11): 7414-7420.

Marintchev, A. and G. Wagner (2005). "Translation initiation: structures, mechanisms and evolution." Quarterly Reviews of Biophysics 37(3-4): 197.

McPike, M. P., J. Goodisman, et al. (2002). "Footprinting and circular dichroism studies on paromomycin binding to the packaging region of human immunodeficiency virus type-1." Bioorg Med Chem 10(11): 3663-3672.

Mir, M. A., W. A. Duran, et al. (2008). "Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching." Proc Natl Acad Sci U S A 105(49): 19294-19299.

Miyauchi, K., Y. Kim, et al. (2009). "HIV enters cells via endocytosis and dynamin- dependent fusion with endosomes." Cell 137(3): 433-444.

Miyauchi, K., M. Marin, et al. (2011). "Visualization of retrovirus uptake and delivery into acidic endosomes." Biochem J 434(3): 559-569.

Namy, O., S. J. Moran, et al. (2006). "A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting." Nature 441(7090): 244-247.

O'Connor, M. (2004). "Multiple defects in translation associated with altered ribosomal protein L4." Nucleic Acids Research 32(19): 5750-5756.

Odawara, T., H. Yoshikura, et al. (1991). "Analysis of Moloney murine leukemia virus revertants mutated at the gag-pol junction." J Virol 65(11): 6376-6379.

Ogle, J. M., A. P. Carter, et al. (2003). "Insights into the decoding mechanism from recent ribosome structures." Trends in Biochemical Sciences 28(5): 259-266.

203 Orlova, M., A. Yueh, et al. (2003). "Reverse transcriptase of Moloney murine leukemia virus binds to eukaryotic release factor 1 to modulate suppression of translational termination." Cell 115(3): 319-331.

Paris, D. B., E. W. Kuijk, et al. (2011). "Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos." Reprod Fertil Dev 23(2): 353-363.

Peltz, S. W., E. M. Welch, et al. (2009). "Nonsense suppression activity of PTC124 (ataluren)." Proc Natl Acad Sci U S A 106(25): E64; author reply E65.

Pestova, T. V. and C. U. Hellen (2003). "Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA." Genes Dev 17(2): 181-186.

Pestova, T. V. and C. U. Hellen (2005). "Reconstitution of eukaryotic translation elongation in vitro following initiation by internal ribosomal entry." Methods 36(3): 261-269.

Pestova, T. V. and C. U. T. Hellen (2005). "Reconstitution of eukaryotic translation elongation in vitro following initiation by internal ribosomal entry." Methods 36(3): 261-269.

Petrov, A., G. Kornberg, et al. (2011). "Dynamics of the translational machinery." Curr Opin Struct Biol 21(1): 137-145.

Pisarev, A. V., C. U. T. Hellen, et al. (2007). "Recycling of Eukaryotic Posttermination Ribosomal Complexes." Cell 131(2): 286-299.

Pisarev, A. V., M. A. Skabkin, et al. (2010). "The Role of ABCE1 in Eukaryotic Posttermination Ribosomal Recycling." Molecular Cell 37(2): 196-210.

Racine, T. and R. Duncan (2010). "Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus." Nucleic Acids Res 38(20): 7260-7272.

Ryabova, L. A., M. M. Pooggin, et al. (2002). "Viral strategies of translation initiation: ribosomal shunt and reinitiation." Prog Nucleic Acid Res Mol Biol 72: 1-39.

Seshadri, A. and U. Varshney (2006). "Mechanism of recycling of post-termination ribosomal complexes in eubacteria: a new role of initiation factor 3." J Biosci 31(2): 281-289.

Shtivelman, E., B. Lifshitz, et al. (1985). "Fused transcript of abl and bcr genes in chronic myelogenous leukaemia." Nature 315(6020): 550-554.

204 Singh, N. S., G. Das, et al. (2005). "Evidence for a role of initiation factor 3 in recycling of ribosomal complexes stalled on mRNAs in Escherichia coli." Nucleic Acids Res 33(17): 5591-5601.

Smith, T. F., J. C. Lee, et al. (2008). "The origin and evolution of the ribosome." Biology Direct 3(1): 16.

Sun, X. X., Y. G. Wang, et al. (2010). "Perturbation of 60 S ribosomal biogenesis results in ribosomal protein L5- and L11-dependent p53 activation." J Biol Chem 285(33): 25812-25821.

Takagi, S., K. Ohashi, et al. (2008). "Suitable reference genes for the analysis of direct hyperplasia in mice." Biochem Biophys Res Commun 377(4): 1259-1264.

Taylor, D. J., B. Devkota, et al. (2009). "Comprehensive molecular structure of the eukaryotic ribosome." Structure 17(12): 1591-1604.

Thompson, R. C. and A. M. Karim (1982). "The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]." Proc Natl Acad Sci U S A 79(16): 4922-4926.

Ueno, M., H. Nakayama, et al. (2002). "Expression of ribosomal protein L4 (rpL4) during neurogenesis and 5-azacytidine (5AzC)-induced apoptotic process in the rat." Histol Histopathol 17(3): 789-798.

Urisman, A., R. J. Molinaro, et al. (2006). "Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant." PLoS Pathog 2(3): e25.

Warner, J. R. and K. B. McIntosh (2009). "How common are extraribosomal functions of ribosomal proteins?" Mol Cell 34(1): 3-11.

Weil, J. E. and K. L. Beemon (2006). "A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus." Rna 12(1): 102-110.

Weil, J. E., M. Hadjithomas, et al. (2009). "Structural characterization of the Rous sarcoma virus RNA stability element." J Virol 83(5): 2119-2129.

Welch, E. M., E. R. Barton, et al. (2007). "PTC124 targets genetic disorders caused by nonsense mutations." Nature 447(7140): 87-91.

Wills, N. M., R. F. Gesteland, et al. (1991). "Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon." Proc Natl Acad Sci U S A 88(16): 6991-6995.

205 Xie, Y., A. V. Dix, et al. (2010). "Antibiotic selectivity for prokaryotic vs. eukaryotic decoding sites." Chem Commun (Camb) 46(30): 5542-5544.

Yang, H., D. Henning, et al. (2005). "Functional interaction between RNA helicase II/ Guα and ribosomal protein L4." FEBS Journal 272(15): 3788-3802.

Yoon, S. I. and M. R. Walter (2007). "Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli." Protein Expr Purif 53(1): 132-137.

Youngman, E. M., S. L. He, et al. (2007). "Stop Codon Recognition by Release Factors Induces Structural Rearrangement of the Ribosomal Decoding Center that Is Productive for Peptide Release." Molecular Cell 28(4): 533-543.

Yue, H., X. W. Lei, et al. (2010). "Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV." Virol Sin 25(6): 425-431.

Zaher, H. S. and R. Green (2009). "Fidelity at the Molecular Level: Lessons from Protein Synthesis." Cell 136(4): 746-762.

Zengel, J. M. (2003). "The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control." Rna 9(10): 1188-1197.

Zengel, J. M. and L. Lindahl (1992). "Ribosomal protein L4 and transcription factor NusA have separable roles in mediating terminating of transcription within the leader of the S10 operon of Escherichia coli." Genes Dev 6(12B): 2655-2662.

Zengel, J. M., D. Vorozheikina, et al. (1995). "Regulation of the Escherichia coli S10 ribosomal protein operon by heterologous L4 ribosomal proteins." Biochem Cell Biol 73(11-12): 1105-1112.

206 Appendix

Functional Pseudoknot FACS screen

In effort to further explore the phenomenon of readthrough, we endeavored to use FACS technology to screen for additional cellular factors that could affect readthrough. Goff lab member Brian Houck-Loomis designed a dual fluorescent reporter (2FP) containing the MoMLV PK flanked by DsRed1 and eGFP. This reporter would allow visualization of readthrough in a live cell population. It would be possible to sort cells expressing the fluorescent readthrough reporter with FACS analysis and to create populations of cells with changes in readthrough levels that could be further analyzed. We anticipated that we could use an over-expression library of cellular proteins to find those additional factors that affect readthrough (Figure A-1). The ability to collect candidate cells and produce clonal populations would allow for the identification of these factors by PCR or microarray.

The 2FP reporter cassette was cloned into the lentiviral vector QXCIP, which contains a lentiviral packaging signal and the puromycin resistance gene under the expression of an

IRES for a selection marker (Figure A-1). Puromycin inhibits translation as a chain terminator – the molecule is added to the nascent peptide, but the molecule cannot participate in peptide bond formation with a subsequent amino acid.. It does not effect recoding inthe manner of the aminoglycosides. We created recombinant virus using the

MoMLV 2FP reporter, the pCMV-intron plasmid containing MoMLV gag:pol, and an

207 A

Ψ IRES PURO

B

• Make virus (3 plasmid system) • Infect 293A cells, drug selection (Puromycin) • Establish MLVPK-2FP reporter line

• Select a clone via FACS for infection w/cDNA library. Criteria: consistent level of eGFP, responsive to RPL4 and PABP transfection, with discernible increase/ decrease in readthrough (eGFP/DsRed1)

• Infect with cDNA library • Sort with preparative FACS to collect cells with altered readthrough levels. • Analyze insertions to identify factors that modulate readthrough.

Figure A-1. Schematic of 2FP reporter construct and FACS screen overview. (A) MoMLV 2FP reporter cassette with the wildtype gag-pol junction between DsRed1 and eGFP, in the pQCXIP backbone. (B) Overview of proposed FAC-based functional readthrough screen: establishment of fluorescent reporter line, selection scheme and screen.

208 expression vector for VSV-G envelope. We also created virus containing either DsREd1 or eGFP in PQCXIP for single color controls for the FACS analysis. We transfected

293T cells with the 3-plasmid recombinant virus mix, and harvested virus 48 hours post transfection. We infected naïve 293A and Rat2 cells with either single color control or the

2FP virus. Forty-eight hours later we placed the infected cells under puromycin selection to choose only cells with the integrated 2FP reporter.

Single color control (Figure A-2) and the MoMLV 2FP pool were analyzed via FACS

(Figure A-3). The MoMLV pool was distinguishable as a double positive population.

MoMLV 2FP cells were plated at a dilution to give a cell per well of 96 well plate. Clonal lines were established and were analyzed by FACS for DsRed1 and eGFP expression.

We calculated readthrough as the ratio of eGFP intensity/Dsred1 intensity based on the mean fluorescence per cell using FlowJo FACS analysis software. We were able to estabalish lines from clones #1, #18 and #21. The parent pool and three clones showed similar ratios of eGFP/DsRed1 (Figure A-4). The clones had fewer double positive cells overall, although the cells were maintained under puromycin selection. Clone #18 had a slightly higher and broader range of apparent readthrough values perhaps indicating a difference in cellular environment that could support greater recoding.

To verify that the 2FP reporter system was sensitive enough to register a change in readthrough, we transfected the pool and the clones with PABP and RPL4 and analyzed

209 Readthrough eGFP (FITC) DsRed (PE) (PE vs FITC)

104 1000 1000 0 0

800 800 103

600 600 Unstained 102 PE-A SSC-A FSC-A control 400 400

101 200 200

0 100 0 0 0 10 0 1 2 3 4 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 FITC-A PE-A FITC-A

104 1000 1000 0 0

800 800 103

eGFP 600 600 102 SSC-A SSC-A PE-A control 400 400

84.6 1 0 10 200 200

15.2 84.8 0 0 100 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 FITC-A PE-A FITC-A

104 1000 1000 94.7 0.017

800 800 103

600 600 DsRed1 102 SSC-A SSC-A PE-A control 400 400 8.34e-3 1 95.4 10 200 200

5.33 0 0 0 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FITC-A PE-A FITC-A

4 1000 1000 10 17.2 19.6

800 800 103 MoMLV 600 600 102 2FP SSC-A SSC-A PE-A 400 400

pool 3.94 1 85.8 10 200 200

63.1 0.024 0 0 100 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 FITC-A PE-A FITC-A

Figure A-2. MoMLV 2FP and single color controls. FACS analysis of DsRedI and eGFP expression of normal (“unstained”) 293A cells, control 293A lines stabling expressing either DsRed1 or eGFP, and 293A cells stabling expressing the MoMLV 2FP construct. Threshold for expression of fluorescent reporters in readthrough graphs (PE vs. FITC) based on levels from single positive controls. All cells were initially gated on live and single cells populations.

210 MoMLV 2FP Pool 4 10 1.29 92.7

3 10

10 2 PE-A

10 1

5.94 0.089 10 0 10 0 10 1 102 103 104 FITC-A singles 092508_MLVPK 2FP.fcs Event Count: 27023

4 4 4 10 10 10 0.29 97.1 0.36 97.4 2.57 85.4

3 3 3 10 10 10

2 2 2 10 10 10 PE-A PE-A PE-A

1 1 10 10 101

0 2.57 0.07 0 2.24 0.033 0 12 0.021 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FITC-A FITC-A FITC-A singles singles singles 092508_MLVPK 2FP_#1.fcs 092508_MLVPK 2FP_#2.fcs 092508_MLVPK 2FP_#14.fcs MoMLV Event Count: 8630 Event Count: 8975 Event Count: 4862 2FP Clones #1 #2 #14

4 4 4 10 10 10 0.71 83 2.15 85.9 0.42 97.5

3 3 103 10 10

2 2 2 10 10 10 PE-A PE-A PE-A

1 1 101 10 10

0 14 2.27 0 12 0 0 1.96 0.12 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FITC-A FITC-A FITC-A singles singles singles 092508_MLVPK 2FP_#18.fcs 092508_MLVPK 2FP_#21.fcs 092508_MLVPK 2FP_#22.fcs Event Count: 6609 Event Count: 8101 Event Count: 9087 #18 #21 #22

Figure A-3. Readthrough in MoMLV pool and clones. FACS analysis of readthrough in parent pool of 293A cells stabling expressing the MoMLV 2FP construct and selected clones. Threshold for expression of fluorescent reporters in readthrough graphs (PE vs. FITC) based on levels from single positive controls. All cells were initially gated on live and single cells populations.

211 2000

1500 MoMLV 2FP pool MoMLV 2FP clone 1 MoMLV 2FP clone 18

Cells 1000

# MoMLV 2FP clone 21

500

0 0 0.2 0.4 0.6 0.8 1 eGFP/DsRed1

Figure A-4. Readthrough in MoMLV 2FP pool and clones. Histogram of readthrough in parent pool of 293A cells stably expressing the MoMLV 2FP construct and selected clones. Readthrough is determined by ratio of average mean intensity of eGFP/DsRed1 based on data from Figure A-3.

212 the cells by FACS 48 hours post transfection (Figure A-5). In our work on RPL4 we searched for additional translation/termination factors that may affect recoding. One of the proteins we tested was PABP; it has a role in termination, associating with eRF3, and may also contribute to the surveillance of mRNA by NMD factors (We cloned PABP and tested its affects on readthrough; PABP was able to decrease readthrough by about half in

293A cells as measured by the DLA, but did not affect viral replication in a transducing virus assay.) Neither PABP nor RPL4 caused a change in readthrough in the 2FP pool and clones as detected by FACS in cells analyzed 2 day post transfection.

GFP and its derivatives have an approximate half-life of 24 hours. Therefore we thought perhaps subtle changes in eGFP levels were indiscernible against the background of basal expression at the time we analyzed the cells. We transfected clone #18, which had a broader range of eGFP/DsRed1 expression, with RPL4 and PABP and analyzed the cells by FACS 48 hours post transfection (Figure A-6). In addition we confirmed the ability of clone #18 to respond to RPL4 by DLA with the 13PK6 reporter pair and RPL4 (Figure

A-7); RPL4 increased readthrough at the higher concentrations tested. However in the

FACS analysis we could not detect any increase in readthrough associated with RPL4.

We observed a decrease in readthrough in the PABP transfected cells, demonstrating that the experimental system was sensitive to changes in readthrough. The RPL4 effect has been observed to occur in a short period post transfection; RPL4 could be down-regulated by the time the of FACS analysis.

213 MoMLV 2FP pool

1200

900 Cells

# 600

300

0 0 0.2 0.4 0.6 0.8 1 eGFP/DsRed1

MoMLV 2FP clone 1 MoMLV 2FP clone 18 MoMLV 2FP clone 21

100 100 100

80 80 80

60 60 60 % of Max % of Max % of Max 40 40 40

20 20 20

0 0 0

0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 eGFP/DsRed1 eGFP/DsRed1 eGFP/DsRed1 73.3

untransfected092508_#18.fcs L4 5ug.fcs 72.7

5 ug RPL4092508_#18

70.9 5 ug PABP092508_#18 PABP 5ug.fcs

Figure A-5. Readthrough in MoMLV 2FP pool and clones transiently transfected with RPL4 and PABP. Histogram of readthrough in parent pool of 293A cells stabling expressing the MoMLV 2FP construct and selected clones transfected with mRPL4 or PABP. FACS analysis performed 2 days post transfection.

214 200

150

Cells 100 #

50

0

0 0.2 0.4 0.6 0.8

eGFP/DsRed1

73.3

untransfected092508_#18.fcs

L4 5ug.fcs 72.7

5 ug RPL4092508_#18

70.9 5 ug PABP092508_#18 PABP 5ug.fcs

Figure A-6. Effect of RPL4 and PABP on readthrough in MoMLV 2FP pool and clones. Histogram of readthrough in clone 18 transfected with mRPL4 or PABP. FACS analysis performed 2 days post transfection.

215 A 10

8 6.1 6 5.4 3.9 4 2.8

% Readthrough 2

0 VC RPL4 60 ng B RPL4 120 ng RPL4 240 ng 1E+08 1E+07 1E+06 1E+05 Av. Fluc 1E+04 Av. Rluc 1E+03 Ave. FLuc-C Ave. RLuc-C 1E+02 1E+01 1E+00 VC RPL4 60 ng RPL4 120 ng RPL4 240 ng

Figure A-7. Effects of RPL4 on 13PK6 readthrough in MoMLV 2FP clone 18. A. Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pairs co-transfected with increasing amounts of RPL4 into MoMLV 2FP clone 18 in 96 well format. Error bars correspond to standard deviation of averages derived from triplicate readings of each transfection mix. B. Average raw values of luciferase for data in A.

216 To overcome the background of fluorescent proteins in the stably expressing MoMLV

2FP line we tested the reporter with a transient transfection. We transfected 293A cells with the MoMLV 2FP reporter and RPL4. We could not detect an increase in readthrough as measured by the eGFP/DsRed1 levels in the cells (Figure A-8). However, a higher percentage of cells expressed fluorescent proteins in samples transfected with RPL4.

This indicates differential expression of the reporter plasmid in the presence of RPL4.

RPL4 may enhance the half-life or stability of the MoMLV 2FP construct, either by an

RNA:protein interaction or by protection against NMD. Alternately, RPL4 may be predisposing a portion of the cellular population to allow readthrough; perhaps the increase in readthrough measured in DLA is an aggregate of a population where more readthrough product is produced by more singular cells, not a general increase in all cells.

However, further experiments would be needed to verify this theory.

Concurrent with the transient transfection trial, Cardno et al. published a survey of various combinations of fluorescent proteins and their use in bicistronic reporters

(Cardno, Poole et al. 2009). Our reporter uses DsRed1 as the upstream reporter, and eGPF as the readthrough product. The LSR11 flow cytometer is optimized to read eGFP, but not DsRed1. In addition DsRed1 functions as a tetramer, and more protein needs to be produced in order to detect a signal. Therefore we feared the intensity of the DsRed1 was compromised. A low upstream reporter signal coupled with very intense eGFP might make a twofold increase in eGFP hard to visualize on the log scale. Considering the work of Cardo, we decided to redesign the 2FP reporter, with eGFP as the upstream product.

217 A

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L4 5ug.fcs 72.7

!3#&!;<=>?/@,#(/2092508_#18

6 ug RPL4

70.9 3 ug RPL4092508_#18 PABP 5ug.fcs

Figure A-8. RPL4 effect on readthrough in transient transfection of MoMLV 2FP. (A) FACS analysis of readthrough in 293A cells transfected with RPL4 and the MoMLV 2FP reporter construct. Threshold for expression of fluorescent reporters in readthrough graphs (PE vs. FITC) based on levels from single positive controls. All cells were initially gated on live and single cells populations. (B) Histogram of readthrough in cells in (A).

218 We replaced DsRed1 with mCherry, which is brighter and functions as a dimer, for use as the readthrough reporter. The new 2FPgr cassette has been cloned into pcDNA3.1 to use in transient transections, and our lab has tested it in the study of NMD. However, the signal of mCherry was still dim as detected by our OMEGA plate reader. We have not yet tried it in our recoding assays. We have recently installed a tabletop GUAVA flow cytometer and will be using testing the 2FPgr reporter for further use.

Cryo-EM structure: The pseudoknot on the ribosome

Our lab has obtained the structure of the MoMLV gag-pol junctions through NMR analysis and confirmed it to be a classic H-type pseudoknot. Although this structure has revealed a partial mechanism for the promotion of readthrough based on the equilibrium between an inactive and active conformation, interactions with the ribosome are still not known. Therefore we have endeavored to visualize the MoMLV pseudoknot on the ribosome by way of cryo-EM microscopy. Most cryo-EM structures have been resolved using bacterial ribosomes; eukaryotic translation is more complex and structures are relatively rare. This project has not been completed; the preparation of the sample with mammalian ribosomes is a unique situation and has required the novel application of many experimental techniques. However, we are working toward refining a system that will allow us to freeze translation at the act of readthrough.

219 Cryo-EM requires a large number of homogenous particles in a sample. The 3- dimensional structure is constructed of thousands of images that are manipulated by

Fourier transformation to allow alignment. These are further analyzed by computational algorithms that reference common components to construct a composite image. Although ribosomes have large regions of homology, different populations in various stages of translation have been distinguished, i.e. racheted and non-racheted. However, rarer events approach signal:noise threshold. Because readthrough occurs about 5% of the time, it would be difficult to separate a large enough readthrough population under normal conditions to distinguish from noise in a cryo-EM reconstruction. In addition, the NMR structure suggests that the pseudoknot adopts an alternate confirmation to promote readthrough, but it is unknown when this occurs during translation. The shift in stem 2 may occur often, but may promote readthrough at a particular angle, or only in concert with other events. It is also unknown how long the pseudoknot stays in a readthrough promoting conformation; presumably this occurs in a matter of seconds. Therefore it was necessary to design a protocol that would allow us to prepare a sample with a high proportion of ribosomes ‘caught’ in the act of allowing readthrough (Figure A -9).

The first parameter we attempted to adjust was the amount of readthrough. Since we aimed to explore how readthrough occurs in wildtype MoMLV we opted not to use hyperactive pseudoknot mutants. Instead, assuming that readthrough and termination are exclusive, we attempted to reduce termination. We used commercially prepared rabbit reticulocyte lysate in-vitro translation system (IVT). We immuno-depleted eRF1 from

220 $"#

E P A E !"# P #!$A !"# ! ! Translation of arrest translation at PK induce ‘readthrough’ conformation mRNA up to (no readthrough or termination) and with tRNAgln at stop codon PK isolate occupied ribosomes (near cognate pairing)

• Centrifuge sample to separate • New mRNA that lacks Glu codons up • Return ribosomes to translation ribosomes from translation mix to PK. mix treated with micrococcal nuclease (or tRNA depleted). • Reduce glutamine from reaction mix • Use PP7 hairpin to pull out (dialysis). ribosomes with experimental • Add excess of tRNAglu and construct (termination should GDPNNP; pull out ribosomes with • Reduce eRF1 via IP to reduce release mRNA) hairpin (any terminated transcripts termination if necessary shouldn’t be attached to • RnaseH cleavage to separate ribosomes). • Reduce tRNA from mix (?) ribosomes from beads • Freeze sample for Cryo-EM

Friday, November 4, 2011 Figure A-9. Proposed experimental design for cryo-EM structure of MoMLV pseudoknot promoting readthrough on the ribosome.

221 the lysate by conjugating Sigma and Abcam antibodies to eRF1 to IgA beads and incubating IVT lysates overnight at 4°C. We recovered the lysate and added the 13PK6 reporter pair mRNAs. We saw a drastic increase in readthrough with eRF1 depletion

(Figure A-10). We also probed the immuno-depleted lysates for eRF1 and saw a reduction in eRF1 levels. Thus, it appears that we can increase the population of readthrough ribosomes via eRF1 depletion. We tested the HIV-1 reporter pair in eRF1 depleted IVT lysate as well as a negative control, believing that we would see no change in frameshift. However, we observed that eRF1 depletion also increased frameshift efficiency. This result was corroborated by work from Doughterty et al. (Kobayashi,

Zhuang et al. 2010) as described in Chapter 2, defining a role for eRF1 as a modulator of frameshift.

To freeze ribosomes in the act of readthrough of the UAG stop codon, it is necessary to stall the ribosomes at the UAG stop codon. Ideally, we would visualize the tRNAgln in the

A site in conjunction with the UAG stop codon to examine the mechanism by which the near-cognate amino acid in accepted. Replacement of GTP with a non-hydrolyzable analog, GDPNP allows tRNA to load into the A site, but does not allow translocation, thereby stalling translation. This method is widely used to visualize tRNAs in prokaryotic ribosome structures; yet, it is nonspecific and will affect translation at any codon. We designed an mRNA that did not contain glutamine codons upstream of the stop codon to allow translation of the mRNA to the stop codon in the absence of tRNAgln, thereby eliminating the possibility that readthrough would occur and the elongation would

222 50

39.22 40

30

22.35 18.68 20 16.86 % readthrough 12.48 10.10 10 6.44 7.44

0 Sigma anti-eRF1 Abcam anti-eRF1 beads only lysate

MoMLV HIV-1

Figure A-10. Effects of eRF1 depletion on recoding in IVT lysate. A. Readthrough percentages based on dual luciferase assay results of the 13PK6 and HIV-1 reporter pairs translated in in-vitro lysates depleted of eRF1. Error bars correspond to standard deviation of averages derived from triplicate readings of each reaction.

223 continue past the UAG codon to the downstream sequence. In the absence of termination, we anticipated there would be a stalling of the ribosome at the stop codon. We would then purify the ribosomes with the reporter mRNA by the PP7 hairpin purification method

(Hogg and Collins 2007) and add them to a buffer containing only charged tRNAgln and

GDPNP. Since the incorporation possible in this environment would be that of tRNAgln at the stop codon we hoped to freeze enough ribosomes in the act of incorporating glutamine at the stop codon to visualize by cryo-EM.

To complete the experimental system the tRNA population of the IVT mix must be controlled. We discovered a method published by Jackson, Brierley et al. (Jackson,

Napthine et al. 2001) that effectively depletes IVT lysates of tRNAs using an ethanolomine blocked epoxy-sepharose column. We performed this method on the IVT lysates, and translated the 13PK6-C dual luciferase plasmid. Translation was reduced by

~ 85% in treated lysates and was rescued to normal levels with the addition of exogenous calf-liver tRNA (Figure A-11). Thus we show that we are able to clear the IVT lysates of the majority of existing tRNAs which is the first step in customizing the tRNA population of the IVT lysate.

Separation of tRNAs has been accomplished by column chromatography, but is labor intensive and would require considerable optimization in our lab. De-acylation of tRNAs can be accomplished with high pH (Kothe, Paleskava et al. 2006), and tRNAs can be

224 1.50

1.25 1.12 1.12 1.00 1.00

0.75

0.50

0.25 0.16 Fold Change in Rluc Expression 0 (-)tRNA lysate, (-)met mix lysate, (-)met mix, 50X met (-) tRNA lysate, tRNA, (-)met mix (-) tRNA lysate, tRNA, (-)met mix, 50X met

Figure A-11. Translation in tRNA depleted IVT lysates. A. Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transcribed and translated in in-vitro lysates depleted of tRNAs. Error bars correspond to standard deviation of averages derived from triplicate readings of each reaction.

225 recharged in appropriate buffers (Pestova and Hellen 2005). We initially believed we could construct a short pseudoknot containing mRNA that contained methionine and lysine codons up to the UAG stop codon. Commercially available fluorescent labeled tRNAlys would allow visualization of the mRNA and a readout of termination and readthrough products based on size. Initiation and elongation to the point of readthrough could be accomplished in the commercial IVT depleted of eRF1, and tRNAgln would be added in a reconstituted translation mix after ribosome purification. Glutaminyl synthetase was produced in bacteria and purified to use to charge tRNAgln in an in-vitro reaction. However, we would also need to produce eEF1 as a chaperone. eEF1 exists as a trimer and is extremely labile; it has been purified by Pestova et al. ((Kolupaeva, de

Breyne et al. 2007) ), but it is unclear that producing the subunits in bacteria and combining them would yield a functional protein. In addition, we were unable to see translation of the experimental construct. We theorized that was a result of the high number of lysine residues or the small size of the product.

We redesigned our mRNA CEMPK (Figure A-12) to include 6 kb of MoMLV gag upstream of the stop codon, altering wildtype glutamine codons so no gluatmine would be incorporated before the stop codon. We hypothesized that by customizing the amino acid pool in the IVT lysate we could control charging of de-acylated tRNAs. We dialyzed the transcription and translation (TnT) IVT lysate against Buffer E supplemented with

GTP (Kolupaeva, Pestova et al. 1998; Kolupaeva, Pestova et al. 2000; Kolupaeva, de

Breyne et al. 2007) to reduce the amino acid concentration. We then added 13PK6-C

226 CEMPK3

Leader M K K M K K K K M K M K K K K K K * PK . . . * * RnaseH cleavage site, PP7

Flag_CEMPK_noQ

2X flag MoMLV gag-pol fragment * * RnaseH cleavage site, PP7 No Q * PK

~ 6 kDa

~ 9 kDa

Friday, November 4, 2011

Figure A-12. MoMLV pseudoknot constructs for cryo-EM structure. (A) Schematic of an initial construct for cryo-EM structure of the MoMLV pseudoknot on the ribosome. A short spacer of methionine and lysine residues is placed before the UAG stop codon and pseudoknot sequence. ** indicates a double UAA stop codon. The 3’ end of the construct contains the PP7 phage coat interacting RNA sequence for purification, and and RNase H cleavage site to allow separation from beads after purification. (B) Schematic of revised construct for cryo-EM structure of the MoMLV pseudoknot on the ribosome. The construct contains a 3X N-terminal flag tag and native MoMLV gag sequence before the UAG stop codon and pseudoknot sequence encoding a 6 kD termination product. The CAG codons preceding the were changed to CAA. A section of the native MoMLV pol sequence is present after the pseudoknot, encoding a 9 kD readthrough product. ** indicates a double UAA stop codon. The 3’ end of the construct contains the PP7 phage coat interacting RNA sequence for purification, and and RNase H cleavage site to allow separation from beads after purification.

227 1E+06 478860 473811

1E+05

1E+04 5660 1130 1E+03 337 183

RLU: RLuc 78 1E+02

1E+01

1E+00 a b c d e f g

a: TnT lysate b: TnT lysate (+ buffer E) c: Dialyzed TnT Lysate d: Dialyzed TnT Lysate + amino acid mix e: Dialyzed, tRNA depleted TnT Lysate f: Dialyzed, tRNA depleted TnT Lysate + amino acid g: Dialyzed, tRNA depleted TnT Lysate + amino acid + tRNA

Figure A-13. Figure A-13: Translation in dialyzed TnT IVT lysate. Readthrough percentages based on dual luciferase assay results of the 13PK6 reporter pair transcribed and translated in in-vitro lysates dialyzed to deplete amino acids and/or depleted of tRNAs. Error bars correspond to standard deviation of averages derived from triplicate readings of each reaction.

228 cDNA to a TnT reaction with and without addition of amino acids and tRNAs and measured translation as rluc signal. There was a radical reduction in translation in dialyzed lysates that was rescued by the addition of amino acids (Figure A-13). In lysates also depleted of tRNAs, the reduction in readthrough was even greater but the recovery was also reduced. This may have been an artifact of multiple freeze-thawing of the lysate in between steps that could be optimized.

Concurrent to the dialysis experiments we attempted to visualize translation of the revised PK containing mRNA. We optimized our gel and transfer protocols to visualize small peptides, but we were unable to detect expression of either termination or readthrough products, with either Fluorotech tRNA or radioactive methionine incorporation. We added a flag-tag to the N terminus of the CEMPK construct, but we could still not detect the product, even after Flag-IP of IVT lysates. It is possible that the pseudoknot interferes with translation initiation of CEMPK or there is another aspect of the experimental construct that prevents translation in vivo. We plan to continue work on an experimental construct for which we can verify translation of termination and readthrough products. Our attempts at customizing the IVT lysate will also provide further opportunities for studying recoding.

229