TRANSLATIONAL REGULATION OF SMAUG MRNA

by

Melissa A.Votruba

A thesis submitted in conformity with the requirements

for the degree of Master of Science,

Graduate Department of Molecular Genetics,

University of Toronto

© Copyright by Melissa A. Votruba 2009

TRANSLATIONAL REGULATION OF SMAUG MRNA

Master of Science 2009

Melissa A. Votruba

Graduate Department of Molecular Genetics

University of Toronto

Abstract

In Drosophila, early embryonic development is controlled by maternally loaded RNAs and

proteins. For proper development to occur it is vital these maternal transcripts are post-

transcriptionally regulated. Egg activation triggers many post-transcriptional changes to these

maternal mRNAs, such changes are: translational activation, repression, cytoplasmic

polyadenylation, and mRNA destabilization (Tadros and Lipshitz, 2005). SMAUG, a major post-

transcriptional regulator, has been found to be responsible for the destabilization of two thirds of

the unstable maternal transcripts upon egg activation (Tadros et al., 2007). smg mRNA is

translationally repressed in stage 14 oocytes, but its translation is activated upon egg activation

in a PAN GU kinase dependent manner. smg mRNA is translationally regulated by elements

within the 3’UTR. Here I show that redundant translational repression elements reside in the smg

3’UTR, and PUMILIO mediates repression through one of these elements. I also show that these

elements are sufficient to cause translational repression in stage 14 oocytes. However, other

elements may be required for translational activation in the early embryo. smg mRNA appears to

be regulated post-initiation in stage 14 oocytes in a large repression complex which is similar to

smg mRNA repression in a png mutant.

ii

Acknowledgements

First of all I would like to thank my supervisor, Dr. Howard Lipshitz for all his support,

guidance, and patience. It was a pleasure to work in his lab and have him as a supervisor and role

model. His great sense of humour kept me smiling even during the tough times. Thank you

Howard for the wonderful three year experience! I also would like to thank Wael Tadros for his

tremendous help and leadership. Wael was always ready to help answer any question I had with

patience and expertise. Thanks to Hua Luo for making most of the UGS-deletion constructs and

to Xiao Li who did the computational analysis. Thanks to Heli Veri for her help with the sucrose

gradients. My supervisory committee members, Dr. Craig Smibert and Dr. Anne-Claude Gingras

provided exceptional advice and encouragement. I thank the Smibert lab for providing plasmids,

reagents, and use of laboratory equipment. I am grateful for the time and effort Angelo

Karaiskakis spent ordering supplies and ensuring I had everything I needed to be successful. To

all the members of the Liplab, both past and present, I am greatful for all your kindness, help,

and friendship. It was a pleasure to work with everyone. Most of all I thank my family (Sandy,

Wes, Michael, Sarah, and Jessica) for their constant love, support, and interest in everything I do.

And to my pets (Sneekers, Tasha, Peaches, and Malibu) you make my life so happy just by being

present.

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TABLE OF CONTENTS

Abstract…………………………………………………………………………...………….......ii

Acknowledgements………………………………………………………………………...……iii

Table of Contents……….…………………..…………………………………………………...iv

List of Figures……………………………………………………………………………………vi

CHAPTER 1

INTRODUCION……………………………………………………………………………….…1

1.1 Post-Transcriptional Regulatory Mechanisms…………………………………………......1

1.1a mRNA Stability………………………………………………………………………….........1

1.1b Translational Regulation…………………………………………………………………….4

1.2 Post-Transcriptional Control of Maternal mRNAs in Drosophila…………….…………9

1.3 Translational Repression during Oogenesis and the Early Embryo in Drosophila….....13

1.4 Translational Activation after Egg Activation and during Embryogenesis in

Drosophila……………………………………………………………………………………….14

1.5 Translational Regulation of smg mRNA in Drosophila……………..…………………....16

1.6 Thesis Goals……………………………………...……………………………………….…18

CHAPTER 2

MATERIALS AND METHODS……………………………………………………………..…19

2.1 Fly Strains and Collections……………..………………………………………………….19

2.2 Transgenic Constructs…………………………………………….…………………….….19

2.3 Western Blot Analysis………………………..…………………………………………..…21

2.4 Northern Blot Analysis…………………………………………………..…………………22

iv

2.5 Sucrose Gradients……………………………………………………………………..……22

2.6 Computational Analysis………………………………………………………………....…23

CHAPTER 3

RESULTS……………………………………………………………………………………..…24

3.1 Redundant Translational Repression Cis Elements Reside in the smaug 3’UTR............24

3.2 Computational Analysis Identifies an Evolutionary Conserved PUM-like Binding Site

in the smaug 3’UTR.....................................................................................................................30

3.3 PUMILIO Represses smaug mRNA Translation During Oogenesis Through the 400-785

Region……………………………………………………………………………………………31

3.4 The smaug 3’UTR 400-785 Region is Sufficient to Cause Translational Repression in

Stage 14 Oocytes……..………………………………………………………………………….34

3.5 Smaug mRNA is Repressed Before Translation Initiation ………………………...........37

3.6 In png Mutant Embryo smaug mRNA does not Shift out of the Pellet………...……..…38

CHAPTER 4

DISCUSSION AND FUTURE EXPERIMENTS………….……………………………………41

4.1 Mapping of Redundant Translational Repressive Cis Elements………...………………41

4.2 PUM Represses smg Translation Through the 400-600 Region ……….……………..…42

4.3 The 400-785 Region is Sufficient to Cause Translational Repression in Stage 14

Oocytes………..………………………………………………………………………………....44

4.4 smaug mRNA is Repressed Before Translation Initiation…………………………….....44

4.5 Hypothesized Models of Translational Regulation Mediated by the PAN GU

Kinase……………………………………………………………………………………………45

v

4.6 Finding Direct Targets of the PAN GU Kinase Involved in smg Translation…………..47

4.7 Generalized vs. Specific Translational Repression During Oogenesis……………..……48

4.8 Generalized vs Specific Translational Activation in the Embryo……….……….….…..50

REFERENCES………………………………………………………………………………….51

LIST OF FIGURES

Figure 1-1. Translational regulation of mRNAs in Xenopus…………………………….……6

Figure 1-2. Establishment of the anterior-posterior axis in Drosophila embryos………..…12

Figure 1- 3. The smg 3’UTR regulates smg mRNA translation……………………...……...17

Figure 2-1. Primers used to make UGS deletion constructs…………………………………20

Figure 2-2. Primers used to make smg 3’UTR 400-785 insert…………………………….…21

Figure 3-1. Deletions made in smaug 3’UTR to identify translational regulatory cis

element(s)………………………………………………………………………………………..25

Figure 3-2. Analysis used to determine if deletions remove translational regulatory cis-

element(s)……………………………………..…………………………………………………27

Figure 3-3. Mapping of translational repression elements in the smaug 3’UTR…………...29

Figure 3-4. Redundant translational repressive cis elements in the 400-785 region in smaug

3’UTR………………………………………………………………………………………...….30

Figure 3-5. Computational analysis finds an evolutionary conserved Pumilio-like binding

site in smaug 3’UTR within the 400-785 base pair region………………………….……...…31

vi

Figure 3-6. Pumilio represses smaug mRNA translation in the 400-600 smaug 3’UTR

Region……………………………………………………………………………………………33

Figure 3-7. Model of redundant translational repression on smaug mRNA by PUM and

repressor(s) X…………………………………………………………………………………...34

Figure 3-8. smaug 3’UTR 400-785 region is sufficient to cause translational repression of

Luciferase protein in stage 14 oocytes………………………………………………………....36

Figure 3-9. Sucrose gradients reveal shift of RNA upon egg activation……………..…..…38

Figure 3-10. Sucrose gradients reveal in 0-2 hour png mutant embryo smg mRNA remains

in heavy pellet region and does not shift to polysomes as in wild-type embryos……...…....40

Figure 4-1. The smg 3’UTR and 5’UTR resemble endogenous smg regulation…………....42

Figure 4-2. Models depicting translational regulation of smaug mRNA mediated by the

PAN GU Kinase…………………………………………………………………………………47

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1

CHAPTER 1

INTRODUCTION

In all animals early development is controlled by maternally loaded RNAs and proteins.

For proper early development to occur it is vital that maternal mRNAs are strictly post-

transcriptionally regulated. This regulation can occur in the cytoplasm where mechanisms can

control the localization, translation, and stability of maternal mRNAs (Tadros and Lipshitz,

2005). This thesis will focus on a pathway in Drosophila via which this regulation of maternal

mRNAs occurs.

1.1 Post-Transcriptional Regulatory Mechanisms

Post-transcriptional regulatory mechanisms relevant to this thesis can be divided into two

categories. One is the regulation of mRNA stability and the other is translational regulation.

1.1a mRNA Stability

There are several known mechanisms of eukaryotic mRNA decay (Day and Tuite, 1998).

The major mRNA decay pathway is initiated by shortening of the poly(A) tail. Shortening of the

poly(A) tail is followed by decapping and subsequent 5‟→3‟ exonucleolytic degradation of the

mRNA. An additional pathway involves deadenylation followed by 3‟→5‟ decay that does not

involve decapping. There are three known eukaryotic deadenylases: the CCR4-NOT

deadenylase, the PAN2/PAN3 deadenylase, and the PARN deadenylase (Coller and Parker,

2004). In Drosophila, only the CCR4-NOT deadenylase and the PAN2/PAN3 have been

identified while PARN homologs are not present (Semotok and Lipshitz, 2007; Temme et al.,

2004).

2

After a transcript is deadenylated the next step involved in the degradation pathway can

involve decapping of the transcript (Coller and Parker, 2004; Semotok and Lipshitz, 2007).

Decapping is carried out by two enzymes: DCP2 which acts as the catalytic subunit, and DCP1,

the enhancer enzyme of DCP2. These enzymes function by catalyzing the hydrolysis of the 5‟-

m7GpppN cap. Other enzymes are also involved in this decapping process, and function as

enhancers to the decapping process. After decapping of the transcript, a 5‟-m7GDP and a 5‟-

monophosphate mRNA body are left. The next step in this pathway is the 5‟→3‟

exoribonucleolytic degradation of the transcript, which is catalyzed by a highly conserved

exoribonuclease, XRN1 (Hsu and Stevens, 1993; Muhlrad et al., 1994). XRN1function has not

been established yet in Drosophila, although pacman, the XRN1 Drosophila homolog, can

rescue xrn1Δ in yeast strains, suggesting conservation within this function (Till et al., 1998).

After deadenylation, mRNAs can also be degraded in a 3‟→5‟ direction, which is

catalyzed by the exosome (Anderson and Parker, 1998; Coller and Parker, 2004; Muhlrad et al.,

1995). The exosome is a large complex made of 3‟to 5‟ exonucleases and functions in many

RNA degradation and processing events, in both the nucleus and cytoplasm. In yeast, the 3‟ to 5‟

mRNA degradation pathway is slower then the decapping dependent 5‟ to 3‟ mRNA degradation

pathway (Cao and Parker, 2001). Involved in this pathway are the cytoplasmic exosomes

components, which are the RNase PH-like subunits RRP41/SKI6, RRP42, RRP43, RRP45,

RRP46, and MTR3 (Semotok and Lipshitz, 2007). The exosome also functions with the SKI

complex, which is made of SKI2, SKI3, and SKI8 RNA helicases.

Transcripts can also be degraded in a deadenylation-independent manner through the

action of various endoribonucleases (Semotok and Lipshitz, 2007). Examples are seen in

Xenopus with polysomal RNase 1, which destabilizes albumin and vitellogen liver mRNAs

3

(Chernokalskaya et al., 1998; Cunningham et al., 2000; Semotok and Lipshitz, 2007). Another

example is seen in mammalian erythroid-enriched endoribonuclease, which targets α-globin

mRNA for decay during erythroid differentiation (Rodgers et al., 2002; Semotok and Lipshitz,

2007).

Another decay pathway is a specialized pathway responsible for the rapid decay of

aberrant mRNAs called the “mRNA surveillance” pathway (Coller and Parker, 2004; Semotok

and Lipshitz, 2007). This pathway is responsible for degrading mRNAs which contain a pre-

mature translation stop codon (Cao and Parker, 2003). This is a quick functioning pathway, in

which mRNAs are decapped without prior poly(A) tail shortening (Coller and Parker, 2004).

Functioning in this pathway is a complex of proteins that include: UPF1, UPF2, and UPF3

(Semotok and Lipshitz, 2007). This pathway reduces the amount of truncated protein in the cell,

which could have a negative effect on many cellular processes (Cao and Parker, 2003). The

mRNA surveillance pathway also functions by monitoring the absence of a stop codon within an

mRNA. In this situation the mRNA is targeted to the cytoplasmic exosome by an adaptor protein

Ski7p (Frischmeyer et al., 2002; van Hoof et al., 2002).

Recently found is the involvement of microRNAs in post-transcriptional gene regulation.

MicroRNAs are approximately 20 nucleotide long non-coding RNAs that are known to regulate

approximately 30% of all protein coding genes (Filipowicz et al., 2008). MicroRNAs are

involved in both RNA stability and translational regulation by base-pairing with mRNAs. They

function in miRNP complexes which contain Argonaute family proteins. In this section of the

introduction only their role in RNA stability will be discussed. MicroRNA transcript

destabilization is best understood in Drosophila S2 cells. In this pathway the P-body protein

GW182 interacts with miRNP Argonaute1 and binds to the mRNA within the 3‟UTR. This

4

binding marks the mRNA for degradation and recruits the CCR4-NOT deadenylase to the

mRNA. During embryogenesis in zebrafish, the miRNA miR-430 is responsible for the

destabilization of hundreds of maternal mRNAs by promoting their deadenylation and

subsequent decay (Giraldez et al., 2006). Also, the miR-309 cluster in Drosophila has been

found to play a role in the destabilization of a subset of maternal transcripts (Bushati et al.,

2008).

1.1b Translational Regulation

The closed loop model proposes that mRNAs are in circularized structures when they are

translated. The circularized structure is formed by linkage between the 5‟ and 3‟ ends of the

transcript (Johnstone and Lasko, 2001). The closed loop model consists of the eIF4E protein

binding to the 5‟ cap (m7G) of the mRNA. The eIF4E also binds to the eIF4G through a

conserved consensus binding sequence. eIF4G interacts with poly(A) binding protein (PABP),

and PABP binds to long poly (A) tails. This pre-initiation complex then recruits the 40S

ribosomal subunit to start scanning the mRNA and to begin translation. It is not known exactly

why an mRNA is circularized during translation; however, hypothesized benefits are: the

circularized structure could promote re-initiation of ribosomes, this structure could act to protect

the mRNA from destabilization, and lastly the circularized structure could prevent translation of

truncated transcripts.

Translational repression usually occurs at the level of initiation in which trans acting

factors bind to specific elements within the non-coding region of a transcript (Johnstone and

Lasko, 2001). Trans acting factors are most commonly known to bind the 3‟untranslated region

(UTR) of a transcript to function in translational repression, but in some cases they are known to

bind the 5‟UTR. The 3‟UTR binding factors function to inhibit initiation by affecting the

5

interactions between the 5‟cap, eIF4G, eIF4E or interactions between the eIF4G and PABP. A

general model of translational repression at the level of initiation is termed mRNA masking. The

term mRNA masking refers to an mRNA that is concealed in an mRNP particle that prevents the

translation apparatus from accessing the mRNA (Johnstone and Lasko, 2001). Proteins known

to be involved in mRNA masking in Xenopus are the Y-box proteins (Johnstone and Lasko,

2001; Matsumoto and Wolffe, 1998). In Xenopus oocytes, the Y-box protein FRGY2 is highly

abundant and is known to mask mRNAs. DEAD-box helicases are also reported to be involved

in mRNA masking (Minshall et al., 2001; Tadros and Lipshitz, 2005) In Xenopus, the DEAD-

box helicase Xp54 is known to oligomerize on masked mRNAs and represses translation

(Minshall and Standart, 2004; Minshall et al., 2001). Cytoplasmic polyadenylation element

binding protein (CPEB) is known to bind a cis element in the 3‟UTR termed the cytoplasmic

polyadenylation element (CPE). Repression occurs when Maskin binds both CPEB and eIF4E in

a bridge like structure to inhibit the binding of the eIF4E to the eIF4G, and thus translation

initiation is prevented (Figure 1-1). Unmasking occurs upon oocyte maturation when Aurora/Eg2

phosphorylates CPEB. Once phosphorylated, CPEB binds to cytoplasmic polyadenylation

specificity factor (CPSF), which recruits poly(A) polymerase and promotes polyadenylation.

6

Figure 1-1. Translational Regulation of mRNAs in Xenopus.

Top is a masked transcript in an Xenopus oocyte. The CPE has bound CPEB which is in a tight

bridge like structure with Maskin and eIF4E. Bottom translationally active transcript in the

closed loop structure. Aurora/Eg2 phosphorylates CPEB which causes binding of the CPSF. The

CPSF recruits poly(A) polymerase causing polyadenylation. PABP binds the poly(A) tail and

causes the eIF4E to interact with the eIF4G. The 40S ribosomal subunit is recruited (redrawn,

after Tadros and Lipshitz, 2005).

Another form of translational repression is accomplished by deadenylation. In both

vertebrates and invertebrates regulation of poly(A) tail lengths in the cytoplasm is an important

translational regulatory mechanism (Johnstone and Lasko, 2001). Repressed mRNAs are found

7

to have short poly(A) tails, while translationally activated mRNAs have extended poly(A) tails.

In Xenopus oocytes, there are no known cis elements required for deadenylation. It is

hypothesized that in oocytes, when cytoplasmic polyadenylation signals are not present mRNAs

are deadenylated. However, in the Xenopus early embryos cis acting elements required for

deadenylation are present (Richter, 1999). Many of these cis regulatory elements are AU-rich

elements called AREs that contain the sequence AUUA found to mediate deadenylation. For

example, Cdk2 mRNA is polyadenylated at maturation via CPE-directed mechanisms, but is

deadenylated after fertilization. Two sequences found within the 3‟UTR of Cdk2 mRNA are

responsible for deadenylation (Richter, 1999; Stebbins-Boaz and Richter, 1994). Other RNAs

such as Eg2, Eg5 and c-mos found in the embryo contain a different sequence required for

deadenylation. This sequence is a 17 nucleotide sequence called the embryonic deadenylation

element, or EDEN. The EDEN sequence is able to promote deadenylation of a reporter RNA

(Johnstone and Lasko, 2001; Paillard et al., 1998) . The trans factor found to bind the EDEN

sequence is EDEN-BP. The EDEN-BP is able to oligomerize, and inhibition of oligomerization

prevents the binding of EDEN-BP to its target, and therefore deadenylation is inhibited (Cosson

et al., 2006). Another trans acting factor that can promote deadenylation is the poly(A)-specific

RNase (PARN), which is known to function in a CPEB-PARN complex. This complex is

believed to promote deadenylation by either removing adenosines from the poly(A) tail or by

blocking factors known to promote polyadenylation from accessing the 3‟UTR (Copeland and

Wormington, 2001; Radford et al., 2008).

As mentioned above microRNAs also play a major role in translational regulation.

Interestingly, microRNAs have been found to regulate translation at multiple steps (Filipowicz et

al., 2008). MicroRNAs can regulate translation initiation by inhibition of the 5‟ cap, they can

8

regulate translation by preventing 60S subunit joining, and they have been found to also regulate

translation at a post-initiation step. In general, microRNAs function in translational repression

via miRNP complexes. These complexes contain proteins from the Argonaute family as

mentioned above. These miRNP complexes then bind to regions in the 3‟UTR of the target

transcript to promote translational repression. Early studies done in C. elegans showed that the

lin-4 miRNA represses its target mRNA, lin-14, at a step post-initiation (Olsen and Ambros,

1999). In the presence of lin-4 miRNA, lin-14 expression is repressed; however, repressed lin-14

mRNA is found on polysomes. Also, human let-7a miRNA was found to block protein

production on actively translating polysomes (Nottrott et al., 2006). In HeLa cells, a construct

containing the let-7a binding sites had dramatically reduced expression when compared to

expression of a similar construct, but lacking the let-7a binding sites. Sucrose gradients revealed

that both mRNAs, with and without the binding sites, sediment in the polysome region.

Puromycin treatment disrupted polysomes and mRNAs were shifted to lighter fractions in

gradients for both constructs. In contrast, it has been found that some reporter mRNAs which

contain an internal ribosome entry site instead of a regular cap cannot be repressed by miRNAs.

These findings suggest the idea that miRNAs can repress translation either pre or post-initiation

depending on the context (Filipowicz et al., 2008).

In Drosophila, miR2 was shown by Thermann and Hentze (2007), to inhibit translation

at the level of initiation. They showed that miR2 induces the formation of dense heavy miRNPs

referred to as „pseudo-polysomes‟ even when polyribosome formation and 60S ribosomal

subunit joining was blocked. In addition, an mRNA containing an ApppG instead of an mGpppG

cap was able to escape miR2 translational repression, indicating that repression was at the level

of initiation.

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1.2 Post-Transcriptional Control of Maternal mRNAs in Drosophila

In all animals early embryonic development is controlled maternally. During oogenesis

the developing oocyte is loaded with maternal mRNAs and proteins through cytoplasmic bridges

known as ring canals (Semotok and Lipshitz, 2007). During late stage oogenesis and early

embryogenesis transcription does not occur and, therefore, strict posttranscriptional regulation of

these maternal mRNAs is required for proper control of gene expression and development

(reviewed in Tadros and Lipshitz, 2005). In Drosophila, once the developing oocyte reaches

maturity, it leaves the ovary and moves down the oviduct and into the uterus. This triggers egg

activation, which in turn triggers many posttranscriptional events such as cytoplasmic

polyadenylation, translational activation, and mRNA destabilization.

Examples of mRNA destabilization in Drosophila are seen with the maternal transcripts

Hsp83, nanos, and string (Bashirullah et al., 1999). All of these transcripts are highly abundant

after egg activation and eliminated by the midblastula transition (MBT), the stage at which

developmental control is transferred to zygotic factors. One of the hypothesized purposes of

maternal transcription destabilization is to allow transfer to zygotic control of development.

Bashirullah et al. (1999) found that the joint action of two RNA degradation pathways ensures

the degradation of maternal transcripts by the MBT. The first RNA degradation pathway is

controlled by maternal factors and is triggered upon egg activation. The second pathway begins

two hours after fertilization and is controlled by zygotic factors. Recently it has been found that

the zinc finger protein, Zelda, is involved in the activation of the early zygotic genome. Zelda

functions by binding to TAGteam sites within the early transcribed genes to activate

transcription. It is also believed that Zelda may also play a role in maternal transcript

destabilization during the maternal-zygotic transition (Liang et al., 2008)

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SMAUG protein has been found to be a major factor required for maternal mRNA

degradation in Drosophila (Tadros et al., 2007). SMG protein was first identified as a

translational regulator of unlocalized nanos mRNA in the early embryo (Dahanukar et al., 1999;

Smibert et al., 1999). The first transcript found to be destabilized by SMG protein is maternal

Hsp 83 mRNA (Semotok et al., 2005; 2008). In wild type newly laid embryos Hsp 83 is

abundant and distributed throughout the entire embryo, but by 2-3 hours after egg activation it is

eliminated from the bulk cytoplasm. However, in a smg mutant 2-3 hours after egg activation,

Hsp 83 mRNA remains abundant throughout the entire embryo. Semotok et al. (2005) showed

that SMG functions to destabilize Hsp83 mRNA by recruiting the CCR4/POP2/NOT

deadenylase. Taking a more genome wide approach to study transcript destabilization, Tadros et

al. (2007) followed 5097 maternal transcripts from 0-6 hours after egg activation, and found that

1069 maternal transcripts were destabilized during this time period. More importantly, they

found that 712 were SMG dependent for destabilization. In addition, SMG is involved in the

activation of zygotic transcription, likely because it is responsible for destabilizing maternal

transcripts that inhibit zygotic transcription (Benoit et al., 2009). In addition to SMG, miR-309

was found to play a major role in the destabilization of maternal transcripts. In a miR-309

mutant, 410 maternal transcripts are up regulated (Bushati et al., 2008). Interestingly, expression

of miR-309 was found to be SMG dependent (Benoit et al., 2009).

Also important to post-transcriptional control of maternal mRNAs in Drosophila is

translational regulation of maternal mRNAs. For rapid correct embryogenesis to occur it is

important that maternal mRNAs which are synthesized and deposited into the developing oocyte,

to be translationally repressed until egg maturation or fertilization occurs, when these maternal

mRNAs are required (as reviewed in Vardy and Orr-Weaver2007b). These maternal mRNAs are

11

generally kept translationally repressed in oocytes and are found to have short poly(A) tails.

Upon oocyte maturation and egg activation these maternal mRNAs are translated and their

poly(A) tails are dramatically increased in length. Examples of transcripts which are

polyadenylated upon egg activation and are translated, are bcd, torso, Toll, hb and smaug

mRNAs (Tadros and Lipshitz, 2005). In Drosophila, no specific cis acting element involved in

polyadenylation has been identified. However, Orb a Drosophila CPEB homolog, which is an

oocyte-specific RNA-binding protein has been shown to be involved in polyadenylation of

certain mRNAs. An orb mutant in Drosophila oocytes results in certain mRNAs inhibited from

being polyadenylated and thus are not translated (Piccioni et al., 2005b).

Translational regulation of maternal mRNAs also ensures that maternal mRNAs that

regulate patterning in the oocyte and early embryo are translated only when properly localized

within the oocyte or embryo. (Tadros and Lipshitz, 2005; Vardy and Orr-Weaver, 2007b).

Specifically, the anterior-posterior embryo coordinates are established by maternal morphogens

which are set up within the early embryo (Figure1-2). For example, repressed bicoid mRNA is

localized to the anterior of the oocyte and embryo. After egg activation bicoid mRNA is

translated in the anterior of the embryo (Johnstone and Lasko, 2001; Salles et al., 1994).

Localized BCD protein in the anterior of the embryo then represses the translation of caudal

mRNA by binding to a cis element in the 3‟ UTR, called the Bcd binding region, thus CAD

protein is excluded from the anterior, and found in a posterior-anterior gradient within the

embryo (Niessing et al., 2002; Tadros and Lipshitz, 2005; Vardy and Orr-Weaver, 2007b). In the

posterior of the embryo NANOS protein is present, and functions to translationally repress

hunchback mRNA. NOS functions to inhibit hb mRNA translation by the joint binding of PUM

and NOS to the Nanos Response Element (NRE) within the 3‟ UTR of hb mRNA. NOS then

12

recruits BRAT which interacts with d4EHP and binds to the 5‟ cap of hb mRNA, thus repressing

translation of hb mRNA in the posterior (Chagnovich and Lehmann, 2001; Cho et al., 2006;

Vardy and Orr-Weaver, 2007b; Wreden et al., 1997). BRAT is an NHL domain protein and is

recruited to the NOS/PUM complex via its NHL domain. The NHL domain is located in the C-

terminus region and contains a β-propeller domain made of six NHL repeats (Arama et al.,

2000). The NHL repeats consist of approximately 44 amino acids which are rich in gylcine and

hydrophobic residues. The C-terminal end contains a cluster of charged residues and the N-

terminal region of each repeat provides sites for protein interactions.

Figure1-2. Establishment of the anterior-posterior axis in Drosophila embryos.

Localization of maternal mRNAs (grey), regulatory proteins (red), and translated proteins

(green) in the early embryo. CUP protein is represented by yellow. (Reprinted from Trends in

Biology, Vol 17, Vardy and Orr-Weaver, Regulating translation of maternal messages: multiple

repression mechanisms, page 548 , © 2007, with permission from Elsevier).

13

1.3 Translational Repression during Oogenesis and the Early Embryo in Drosophila

During oogenesis and early embryo development many maternal mRNAs are

translationally repressed either until they are localized or for temporal control, in which they are

repressed until a specific developmental time period. CUP protein has been found to act during

both oogenesis and embryogenesis as a translational regulator (Piccioni et al., 2005a). CUP

translational regulation in both the ovary and early embryo occurs at the level of initiation. CUP

was found to interact biochemically with the eIF4E, and thus inhibits the binding of the eIF4E to

the eIF4G required for translation initiation. Specifically, CUP is known to translationally

repress oskar mRNA during its localization in the oocyte along with BRUNO (Chekulaeva et al.,

2006). BRUNO binds to oskar mRNA through the BREs found in the 3‟UTR and recruits CUP

protein. In the embryo, CUP is known to repress nanos mRNA translation in the bulk cytoplasm

with the help of SMG protein to ensure that NOS protein is restricted to the posterior (Dahanukar

et al., 1999; Nelson et al., 2004). Translation is repressed by SMG binding to recognition

elements within the 3‟UTR of nanos mRNA and recruiting the 4E-BP CUP. CUP‟s ability to

interact with both SMG and BRUNO suggests that CUP could play a role in regulating many

maternal mRNAs during oogenesis and embryogenesis. Furthering this idea was the recent

finding that CUP also associates with the adaptor protein Miranda and the mRNA carrier Staufen

during oogenesis. Both Miranda and Staufen are involved in mRNA localization in the oocyte

(Piccioni et al., 2009).

The RNA binding protein PUMILIO is also known to be a translational regulator in the

Drosophila ovary and embryo. As mentioned above PUM regulates hb mRNA translation in the

posterior of the embryo along with NOS, BRAT, and d4EHP. In the embryo PUM plays a role in

patterning and pole cell formation (Vardy and Orr-Weaver, 2007a). PUM and NOS

14

translationally repress cyclin B mRNA in the pole cells (Asaoka-Taguchi et al., 1999; Vardy and

Orr-Weaver, 2007b). Vardy and Orr-weaver (2007a), have shown that PUM also plays a role in

cyclin B mRNA translational repression throughout the entire embryo and not just the posterior.

Vardy and Orr-weaver showed that the PAN GU Kinase is required for Cyclin B expression after

the completion of meiosis. In a png mutant the expression of Cyclin B is dramatically reduced.

However, removing PUM in a png mutant embryo caused the png mutant phenotype to be

suppressed and Cyclin B expression restored. Vardy and Orr-Weaver (2007) believe the PNG

kinase restricts the activity of PUM around the syncytial nuclei and that PUM acts with another

partner to repress translation throughout the embryo, as it does with NOS in the posterior. In

addition, Vardy et al. (2009) showed that PUM also plays a role in the repression of cyclin A

mRNA in the late oocyte and early embryo. In both stage 14 oocytes and early embryos in a png

mutant Cyclin A translation is inhibited. If PUM is removed in a png mutant embryo, expression

of Cyclin A is restored. It is unknown if PUM represses cyclin A mRNA in the oocyte. To further

investigate PUM‟s role in translational repression during embryogenesis Gerber et al. (2006)

conducted a genome wide analysis to identify mRNAs which associate with PUMILIO. In the

embryo they found 165 mRNAs that associate with PUM, and which have a common sequence

motif in the 3‟UTR: of UGUA(A/U/C)AUA. This suggests that PUM could post-

transcriptionally regulate many maternal mRNAs.

1.4 Translational Activation after Egg Activation and during Embryogenesis in Drosophila

Once mRNAs are properly localized within the embryo, and are required for temporal

expression, they are translationally activated. A subset of maternal mRNAs has been shown to be

activated upon egg activation including bcd, nanos, Toll, hunchback, caudal, smaug, torso, and

15

string (Tadros and Lipshitz, 2005). Two consistent features which are most commonly involved

in translational activation is the removal of repression and the extension of the poly(A) tail.

During oogenesis, bcd mRNA is stable and translationally repressed. Egg activation

triggers the polyadenylation and translational activation of bcd mRNA. During oogenesis the bcd

poly(A) tail length is approximately 70nt and reaches a 140nt length by 1-1.5 hours after egg

activation (Salles et al., 1994). Salles et al. (1994) show that the bcdE1

mutant which does not

produce functional BCD protein or anterior structures can be rescued by injection of wild-type

bcd transcripts, but not by bcd mRNA missing 537 nucleotides of the 3‟UTR. They next tested

if in vitro addition of the bcd mRNA missing the 537 nucleotides with the addition of 150-200

adenosines could rescue the bcdE1

mutant. They found only a partial rescue of anterior defects,

suggesting that an element required for translation is missing in this transcript. Juge et al. (2002),

overexpressed PAP in the female germline and saw an increase in poly(A) tail length in bcd

mRNA in both the oocyte and the embryo; however, this increase did not induce bcd mRNA

translation in the oocyte. This would suggest that polyadenylation is not sufficient to activate the

translation of bcd mRNA and other elements are involved.

The PAN GU kinase has recently been shown to play a role in mRNA translational

activation upon egg activation. The PNG kinase is responsible for the continual translation of

cyclin B mRNA and cyclin A mRNA upon egg activation. Cyclin B mRNA is translationally

unmasked in stage 14 oocytes, through an ORB dependent mechanism, that does not require

PNG (Vardy and Orr-Weaver, 2007a). After egg activation in a png mutant there is a dramatic

reduction of Cyclin B protein compared to wild-type, confirming that PNG is required after egg

activation for Cyclin B translation. Cyclin B mRNA poly(A) tails are even further

polyadenylated after egg activation. In a png mutant, poly(A) tails lengths do not increase.

16

Overexpression of PAP in a png mutant does not restore Cyclin B protein levels. Removal of

PUM in a png mutant restores Cyclin B protein levels almost to WT levels but poly(A) tail

lengths are not restored. Vardy and O-Weaver (2007) suggest that polyadenylation does augment

translation, but polyadenylation is not essential, and a fully elongated poly(A) tail is not required

for translation after egg activation to proceed. Instead PNG functions through removal of

repression. In the case of Cyclin A, PNG functions to promote Cyclin A expression in stage 14

oocytes and after egg activation (Vardy et al., 2009). PNG also plays a role in polyadenylation,

as the poly(A) tail of cyclin A mRNA is extended after egg activation. In a png mutant cyclin A

mRNA is translationally repressed in both stage 14 oocytes and activated eggs, while removal of

PUM in a png mutant restores Cyclin A expression in the activated egg.

1.5 Translational Regulation of smg mRNA in Drosophila

SMAUG is a major post-transcriptional regulator involved in both translational regulation

and transcript destabilization; however, SMG protein is itself post-transcriptionally regulated.

smg mRNA is present in both the late oocyte and early embryo, but SMG protein is found only

in the early embryo (Dahanukar et al., 1999; Smibert et al., 1999). The PAN GU kinase was

identified in a genetic screen for maternal effect lethal mutants to be essential for maternal

transcript degradation (Tadros et al., 2003). The PNG kinase complex is composed of three

proteins; PNG, GNU, and PLU (Fenger et al., 2000; Lee et al., 2003). All three proteins are

present in both the ovary and the early embryo (Fenger et al., 2000).

Tadros et al. (2007) showed that all three proteins in the PAN GU kinase complex are

required for SMG protein translation and that the PNG Kinase functions through the smg 3‟UTR.

Using a transgene which contains the GFP ORF under the control of the smg 3‟UTR regulatory

17

elements, Tadros et al. ( 2007) showed that PNG regulates smg mRNA translation via elements

in the 3‟UTR (Figure 1-3).

Figure 1-3. The smg 3’UTR regulates smg mRNA translation.

UGS transgene. UAS-GFP-smg3‟UTR expression driven by Nanos Gal4 VP16 (NGV).

Comparing GFP expression between stage 14 oocytes and 0-3 hour embryos. In WT, GFP

expression is much higher in 0-3 embryos then stage 14 oocytes. In png mutant an increase in

GFP expression is not seen in 0-3 embryos (Reprinted from Developmental Cell, Vol 12, Tadros

et al., 2007, SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and

its translation is activated by the PAN GU kinase, page 148, © 2007, with permission from

Elsevier).

In the oocyte, smg mRNA has short poly(A) tails which are extended by approx 100nt

after egg activation. However, in a png mutant the poly(A) tails are only extended approx 25 nt

after egg activation (Tadros et al., 2007). Over expressing poly(A) polymerase in wild-type early

embryos caused smg poly(A) tails to significantly lengthen and caused a dramatic increase in

SMG protein levels in the early embryo. When adding poly(A) polymerase to png mutant

18

embryos, smg poly(A) tails are increased, but SMG protein is not restored in early embryos.

Therefore, Tadros et al. (2007) concluded that PNG regulates smg mRNA translation via a

mechanism that is independent of its effect on cytoplasmic polyadenylation.

Lastly Tadros et al. (2007) tested 12 known translational repressors for a role in

repression of smg mRNA translation during oogenesis. While no single mutant relieved

repression in stage 14 oocytes, in a pum mutant background increased levels of SMG protein

were found in early embryos. In a png;pum double mutant, however, SMG translation was not

restored. Thus Tadros et al. (2007) postulated that PNG functions to remove redundant

repression by PUM and one or more additional repressors after egg activation to allow smg

mRNA translation.

1.6 Thesis Goals

The goal of my thesis is to understand the mechanism by which smg mRNA is kept

translationally repressed in stage 14 oocytes. Deletions where made in the smg 3‟UTR in the

context of the UAS-GFP-smg 3‟UTR transgene (UGS) to identify regulatory cis elements

involved in translational repression in stage 14 oocytes. Deletion analysis identified redundant

translational repression elements in the 400-785 nt region in the smg 3‟UTR, one element

residing in the 400-600 region and the other residing in the 600-785 region. To identify possible

proteins that bind to identified elements computational analysis was carried out on the 400-785

region. A conserved PUM-like binding site was identified at 466-475 in D. melanogaster 3‟UTR

coordinates. To further investigate PUM translational repression in the 400-600 region I crossed

UGS-600∆785 into a pum mutant background. Western analysis revealed that that all forms of

repression were removed in stage 14 oocytes and PUM does function in this region to mediate

19

repression. In addition, I show that the 400-785 smg 3‟UTR region is sufficient to cause

translational repression in stage 14 oocytes. Interestingly, this region does not allow translation

to occur in the early embryo. To identify at what level of translation these repressors regulate

smg mRNA translation I conducted sucrose gradient analysis. My data suggest that repressed

smg mRNA is regulated pre- initiation in stage 14 oocytes most likely by a heavy repression

complex which contains PUM , and in a png mutant smg mRNA remains repressed in a similar

manner.

CHAPTER 2

MATERIAL AND METHODS

2.1 Fly Strains and Collections

The “wild-type” stock used was w1118

(Tadros et al., 2007). Additional lines were: png50

(Fenger

et al., 2000; Tadros et al., 2007); pum13

and pumMSC

(Barker et al., 1992; Lehmann and Nüsslein-

Volhard, 1987; Wharton et al., 1998); nanos-Gal4-VP16 (NGV), which refers to

P(GAL4::VP16-nos.UTR) (Tadros et al., 2007; Van Doren et al., 1998); maternal tubulin-GAL4

(Martin and St Johnston, 2003; Song et al., 2007). Pum mutants were grown at 25˚C; pum

embryo collections were at 25˚C for 1.5 hours following which embryos were shifted to 18˚C for

30 minutes.

2.2 Transgenic Constructs

UAS-GFP- smg3‟UTR deletion constructs derived from the UAS-GFP-smg3‟UTR (UGS)

construct made by Tadros et al. (2007). The deletion inserts were made by PCR amplifying

regions from the UGS smg 3‟UTR using primers that contained a flanking BsiWI site, or BglII

site, or NheI site (Figure 2-1). To make the 200∆785, 200Δ400, 400Δ600, 600Δ785, and

20

400Δ785 deletion insert, PCR amplified regions were ligated together using BglII. Deletion

inserts were ligated into a cut UGS vector using BsiWI and NheI.

Primer

Position in

smg 3’UTR

Primer Sequence

Flanking

Restriction

Enzyme

785.F 5‟-CGA TCG TAC GGT ATA AAA ACG AAC AAA TG-3‟ BsiWl

401.F 5‟-GGA AGA TCT ACT AAA CTT TAA CAG AAA AGA-3‟ BglII

601.F 5‟-GAA AGA TCT AGC CAA TCA CTC GAT ATG-3‟ BglII

785.F 5‟-GGA AGA TCT GTA TAA AAA CGA ACA AAT G-3‟ BglII

201.F 5‟-CGA TCG TAC GAA TTG AAA AGT GAG AAT TG-3‟ BsiWl

1.F 5‟-CGA TCG TAC GAC CCC AAT CCC AAT CAC AAC ATC-3‟ BsiWl

1266.R 5‟- CTA GCT AGC CTC GGT ATG AAG TTG-3‟ Nhel

400.R 5‟- GGA AGA TCT CTA GTG GTA GTT TCC GCC-3‟ BglII

200.R 5‟-GGA AGA TCT CCC TCT CCA TCC AGC TTT C-3‟ BglII

Figure 2-1. Primers used to make UGS deletion constructs.

F represents forward primer. R represents reverse primer

H114(400-785) construct was made from the H114 construct (contains LUC OFR and α-

tubulin 3‟UTR) in pUASp vector donated from the Smibert lab. The smg 3‟UTR 400-785 region

was PCR amplified from the UGS construct, with primers shown in figure 2-2. The H114

construct was digested with BaMHI and SpeI, and the smg 3‟UTR 400-785 region was inserted

21

into vector beginning of α-tubulin 3‟ UTR (2424nt region). An attB site was inserted at the

BbvCI site at the 5‟ end of the K10 3‟UTR within the vector.

Primer

position in

smg 3’UTR

Primer Sequence

Flanking

Restriction

Enzyme

785 R. 5‟- GGAACTAGTATTTACAATTAGACTACACGTTTTACG-3‟ Spel

401.F 5‟-GGA AGA TCT ACT AAA CTT TAA CAG AAA AGA-3‟ BglII

Figure 2-2. Primers used to make smg 3’UTR 400-785 insert.

R indicates reverse primer. F indicates forward primer.

2.3 Western Blot Analysis

Dechorionated 0-2 hour embryos and stage 14 oocytes were collected and lysed in a RIPA (1%

Triton X,1% deoxycholic acid, 50mM Tris-HCL,150mM NaCl, 5mM EDTA) + protease

inhibitor (Roche Complete Mini) buffer. Extract from approximately seven embryos or seven

stage 14 oocytes per lane was loaded on a polyacrylamide gel (10%). Protein was then

transferred onto a PVDF membrane. Primary antibodies were: Rabbit anti-LUC 1:500 (Cortex

BIOCHEM), guinea pig anti-DDP1 3:20,000 (Nelson et al., 2007; Tadros et al., 2007),

rabbit anti-GFP ab290 1:2,500 (Abcam), and mouse anti-tubulin 1:2000 (Sigma). Secondary

antibodies were: goat anti-rabbit horseradish peroxidase (HRP), goat anti-guinea pig HRP, goat

anti-mouse HRP. All secondary antibodies were used at 1:5,000 (Jackson ImmunoResearch

Laboratories, Inc.). Signals were imaged with FLuorChem using the ECL detection machine.

Software used was Alpha Innotech.

22

2.4 Northern Blot Analysis

RNA was extracted from stage 14 oocytes and 0-2 hour embryos using a modified TRIzol

protocol (Invitrogen) (Tadros et al., 2007). Equal amounts of RNA (50 embryos or oocytes) were

loaded and electrophoresed on a 1% agarose/formaldehyde/MOPs gel and transferred onto a

nylon membrane (Amersham Hybond-N). The blot was pre-hybridized at 65˚C for at least one

hour in a prehyb buffer containing Na Phosphate/SDS/Salmon Sperm DNA/EDTA. After the

prehyb, denatured 32

P-labeled random primed DNA probes were added and allowed to hybridize

overnight at 65˚C. Blots were rinsed three times for 30 minutes each with a high stringency wash

containing 150mM Na Phosphate/0.1% SDS. Blots were exposed to a Molecular Dynamics

phosphor screen and imaged on a Typhoon Phosphoimager. Software used for quantitation was

ImageQuant.

2.5 Sucrose Gradients

0-2 hour embryos were collected, dechorionated, and rinsed with 0.1% Triton in a wash buffer

containing 0.5M NaCl, 25mM MgOAc, 50mM Tris, pH7.5. They were then homogenized on ice

in a lysis buffer containing 0.5M NaCl, 25mM MgAOc, 50mM Tris pH 7.5, 2mg/ml heparin,

0.5mg/ml cycloheximide, 1mM DTT, 50U/ml RNasin, and protease inhibitors. Triton was added

to extract at a 1% concentration. Homogenates were cleared by centrifugation for 10 minutes at

4˚C. 100μl of extract was loaded on top of 5.92ml 15%-45% 5 sucrose gradient in a SW-41

centrifuge tube. The gradient contained 0.5M NaCl, 25mM MgOAc, 50mM Tris pH 7.5. After

centrifugation in a Beckman SW41 rotor for 2.5 hours at 36000 rpm at 4˚C, the gradients were

hand fractionated into 1ml fractions. The pellet was also treated as a fraction and re-suspended in

15% glucose. Stage 14 oocyte gradients were performed in the same way, except that oocytes

were rinsed in 0.1% Triton but not wash buffer.

23

For the EDTA control, wash and lysis buffers contained only 5mM MgOAc. After clearing,

extract was divided into two and 25mM of EDTA was added to one half of the extract and an

equal amount of water was added to the other half.

RNA was extracted from gradient fractions by first adding 20% SDS, 0.5M EDTA, and 20mg/ml

proteinase K followed by a 30 minute digestion took place at room temperature. After an

overnight ethanol/glycogen precipitation, all twelve fractions were subjected to northern blot

analysis.

2.6 Computational Analysis

A text search for all PUM-like sites in the smg 3‟UTR was carried out. The PUM-like sites

searched for were from De Renzis et al. (2007) (UUUUGUU, UUUGUUA, UUUUGUA,

UUUUUGU,UUGUU), Wharton and Struhl (1991) the Nanos Response Element site which

contains Box A (GUUGU) and Box B (AUUGUA) binding sites, and Gerber et al. (2006) PUM

binding sequence motif (UGUAHAUA), which contains the tetranucleotide UGUA found in

mRNAs known to interact with PUF proteins (Gerber et al., 2004). Conservation of identified

PUM-like sites was checked using the UCSC conservation track. This track shows a measure of

evolutionary conservation in twelve Drosophila species, mosquito, honeybee, and red flour

beetle, based on a phylogenetic hidden Markov model (phastCons score). Based on the phastcons

score between the 12 flies, mosquito, honeybee, and beetle, a conserved PUM-like site was

found among 11 fly species.

24

CHAPTER 3

RESULTS

3.1 Redundant Translational Repression Cis Elements Reside in the smaug 3’UTR

To identify the translational regulatory cis element(s) in the smg 3‟UTR a series of non-

overlapping 200bp deletions were produced across the smg 3‟UTR together with two larger

nested deletions, and a deletion that removed all of the 3‟UTR except the polyadenylation signal

(Figure 3-1). The backbone for the deletions was UAS-GFP-smg 3‟UTR from Tadros et al.

(2007) which consists of the full length smg 3‟UTR, and allows PNG dependent GFP translation

after egg activation under the control of cis element(s) found in the smg 3‟UTR. There are three

smg mRNA isoforms that differ in polyadenylation site.. All deletions are present in the smallest

isoform to allow the identification of regulatory element(s) found in all three isoforms.

25

Figure 3-1. Deletions made in smaug 3’UTR to identify translational regulatory cis

element(s).

Diagram depicts deletions made in smaug 3‟UTR. Deletions were made in the UAS-GFP-smg

3’UTR transgene (UGS). The GFP ORF is fused to the smg 3‟UTR with an up stream activating

system. Most deletions are 200 base pairs which expand the smg 3‟UTR region. Two larger

overlapping deletions were also constructed, and one deletion which removed the whole region

was constructed. All deletions were made according to the smallest smaug isoform.

To identify if a deletion has removed an activation or repression element(s) in the smg

3‟UTR , westerns blots were carried out for UAS-GFP -Full length smg 3‟UTR (UGS-FL)

transgene and UAS-GFP-deletion smg 3‟UTR transgenes (UGS-Δ). Expression of transgenes was

driven by the NANOS Gal4-VP16 driver and protein was extracted as described in Methods.

Protein levels were compared between stage 14 oocytes and 0-2 hour embryos for each

transgene. As expected in the control UGS-FL there was a small amount of GFP protein in the

26

stage 14 oocyte, and a large increase in the 0-2 hour embryo (Figure 3-2A) (Tadros et al., 2007).

UGS-600Δ785 represents a deletion in which there is no removal of a repressive cis element,

resembling UGS-FL in the ratio of oocyte to embryo GFP levels (Figure 3-2B). The UGS-

400Δ785 deletion represents a deletion in which a repressive cis element(s) has been removed.

Since GFP protein levels in stage 14 oocytes have increased to the same high level found in 0-2

hour embryos (Figure 3-2C). These results suggest the 400∆785 deletion has removed one or

more elements that repress translation in stage 14 oocytes.

Because transgenes were inserted at random genomic sites by P element transformation,

RNA levels of gfp mRNA were also assessed for each transgene in both stage 14 oocytes and

early embryos to verify that changes in protein level were not due to changes in RNA level

(Figure 3-2A,B,C). For each construct and line, GFP protein level was normalized to RNA level,

and then the oocyte values (protein/RNA) were normalized to embryo values for each deletion.

Quantification using double normalized values (protein/RNA and oocyte/embryo) confirms that

for both UGS-FL and UGS-600Δ785 there was an increase of protein in the embryo, and no

translational regulatory cis element(s) had been removed (Figure 3-2A,B) while, for the UGS-

400Δ785 deletion a repressive regulatory element had been removed, because normalized protein

levels in the stage 14 oocyte had increased to the level in the early embryo (Figure 3-2C).

27

Figure 3-2. Analysis used to determine if deletions remove translational regulatory cis-

element(s). Westerns and Northerns probed for GFP protein or gfp mRNA comparing protein and RNA

levels between stage 14 oocytes and 0-2 hour embryos for UGS- Full Length (FL), UGS-

600Δ785, and UGS-400Δ785 transgenes . Westerns were also probed for DDP1 as a loading

control. Expression of UAS-GFP-smg 3’UTR (with and without deletions) was driven by

NANOS Gal4-VP16 (NGV). (A) In UGS-Full Length (FL) transgene (control) there is an

increase of protein in 0-2 hour embryos when compared to stage 14 oocytes, (B) UGS-600Δ785

is an example of a deletion transgene in which a repression element has not been removed. (C)

UGS-400Δ785 is an example of a deletion transgene in which a repressive regulatory element

has been removed. (A)(B)(C) All transgenes were inserted randomly. To verify changes in

protein was not due to changes in RNA, gfp mRNA was also assessed in both stage 14 oocytes

and 0-2 hour embryos. Northerns were also probed for rpa1 as a loading control. GFP protein

levels were normalized to RNA levels in stage 14 oocytes and 0-2 hour embryos and then oocyte

values were normalized to embryo values among transgenes. Two lines for each transgene were

analyzed. Quantification shows average (protein/RNA and oocyte/embryo) values for the two

lines tested. Error bars were calculated by determining the standard deviation between the

(protein/RNA and oocyte/embryo) values among a transgene.

Such analyses were carried out for each deletion transgene and the results are presented

in figure 8. For the large deletion (1Δ785), which removes almost the entire smg 3‟UTR, as well

28

as the two other large deletions (200Δ785 and 400Δ785), double-normalized GFP expression

levels increased in stage 14 oocytes to levels similar to those in the early embryo (Figure 3-3).

However, for all the smaller deletions (1Δ200, 200Δ400, 400Δ600, 600Δ785), GFP protein

expression in the stage 14 oocyte remained low relative to the early embryo (Figure 3-3).

The fact that 400Δ785 relieved repression while 400Δ600 and 600Δ785 did not suggests

that redundant repression occurs through separate cis elements, one in the 400-600 and the other

in the 600-785 region diagrammed in figure 3-4. However, it remains possible that there is only

one cis regulatory element within the 400-785 region, which spans the 600 nt site such that, each

smaller deletion did not remove the full cis element, which retained repression ability. Data to be

described below are consistent with the former (i.e. redundant element) hypothesis.

29

Figure 3-3. Mapping of translational repression elements in the smaug 3’UTR.

Summary of mapping translational repressive cis element(s) in smg 3‟UTR. Left diagrams

deletions made in smg 3‟UTR in UGS transgene. o=stage 14 oocytes and e= 0-2 hour embryos.

Westerns indicate changes in GFP protein levels between stage 14 oocytes and 0-2 hour embryos

among UGS deletion transgenes. DDP1 used as a loading control. Also shown is a summary of

quantification of oocyte values (protein/RNA) normalized to embryo values (protein/RNA),

where embryo is set to one. Standard deviation was calculated for double normalized value.

Larger deletions indicate repression element(s) has been removed. Smaller deletions indicate no

repression element(s) has been removed. For each transgene two separate lines were tested

except for transgene indicated by * in which only one line was tested.

30

Figure 3-4. Redundant translational repressive cis elements in the 400-785 region in smaug

3’UTR.

Diagram modeling redundant translational repressive cis elements in smg 3‟UTR. UGS-400Δ600

deletion showed no removal of translational repression in stage 14 oocytes. UGS-600Δ785

deletion also showed no removal of translational repression in stage 14 oocytes. However,

removing both elements with the UGS-400Δ785 deletion translational repression was removed in

stage 14 oocytes.

3.2 Computational Analysis Identifies an Evolutionary Conserved PUM-like Binding Site

in the smaug 3’UTR

As described in the introduction, data from Tadros et al. (2007) suggested that the

sequence specific RNA binding protein PUM might be involved in repressing smg translation

during oogenesis. Therefore, computational analysis was carried out on the smg 3‟UTR,

specifically searching nucleotides 400-785 for PUM-like binding sites (see Materials and

Methods Chapter 2). Within the 400-785 base pair region a single evolutionarily conserved

31

PUM-like binding site was found (466-475 in D. melanogaster 3‟UTR coordinates) (Figure 3-5).

The presence of a PUM-like site is consistent with the hypothesis that PUM could repress smg

translation.

Figure 3-5. Computational analysis finds an evolutionary conserved Pumilio-like binding

site in smaug 3’UTR within the 400-785 base pair region. A single conserved PUM-like binding site containing the core tetranucleotide UGUA found

within Gerber‟s et al. (2006) 8nt PUM binding motif , the Box B sequence from the Nanos

Response Element (AUUGUA), and the UGUANWUW PUM- like site found in 12 Drosophila

species (Stark et al., 2007) (Red box). This site is conserved among 11 Drosophila species.

Black represents conserved regions and blue represents non-conserved regions.

3.3 PUMILIO Represses smaug mRNA Translation During Oogenesis Through the 400-600

Region

To further investigate the possibility that PUM translationally represses smg through the

400-600 region we decided to test the UGS-600Δ785 deletion in a pum homozygous mutant. As

controls, the UGS-FL and UGS-400Δ785 transgenes were also tested in a pum homozygous

mutant. We reasoned that if PUM acts through the 400-600 smg 3‟UTR region redundantly with

32

another repressor or repressors in the 600-785 region, then in a pum mutant the UGS-600Δ785

transgene should have all forms of repression removed, and GFP protein expression should

increase in late oocytes to that found in the early embryo. With both the UGS-FL and UGS-

400Δ785 transgenes, GFP expression in pum mutant late oocytes and early embryos should be

similar to levels found in wild-type background. Consistent with this hypothesis, in a pum mutant

the 600Δ785 deletion resulted in high GFP protein levels in late oocytes (Figure 3-6). Thus,

PUM acts through the 400-600 smg 3‟UTR region to inhibit translation redundantly with one or

more repressors that act through the 600-785 region (Figure 3-7).

33

Figure 3-6. Pumilio represses smaug mRNA translation in the 400-600 smaug 3’UTR

region.

To verify Pumilio translational repression in the 400-600 smg 3‟UTR region UGS-FL, UGS-

600Δ785, and UGS-400Δ785 transgenes were tested in a pum homozygous mutant background,

and levels of GFP protein between stage 14 oocytes and 0-2 hour embryos were compared. As a

wild-type control the same transgenes were also tested in a pum heterozygous mutant

background. α-Tubulin was used as a loading control. Expression was driven by the maternal

tubulin-GAL 4 driver. (A) UGS-FL shows expected GFP protein levels between stage 14 oocytes

and 0-2 hour embryos in both wild-type control and pum mutant background. (B) UGS-600Δ785

shows expected GFP protein levels between stage 14 oocytes and 0-2 hour embryos in a wild-

type background. In a pum background however, stage 14 oocyte GFP protein levels have

increased to the high levels of GFP protein found in 0-2 hour embryos. (C) UGS-400Δ785

transgene shows expected GFP protein levels in stage 14 oocytes and 0-2 hour embryos, in both

wild-type control and pum mutant. Protein levels in stage 14 oocytes and 0-2 hour embryos are

similar, because deletion removes redundant repression elements. Westerns have only been

completed once and need to be repeated in order to quantify them.

34

Figure 3-7. Model of redundant translational repression on smaug mRNA by PUM and

repressor(s) X.

PUM binds at the 466-475 region in the smg 3‟UTR to redundantly repress smg mRNA

translation with repressor(s) X in the 600-785 region during oogenesis. Repressors are removed

upon egg activation to allow smg mRNA translation.

3.4 The smaug 3’UTR 400-785 Region is Sufficient to Cause Translational Repression in

Stage 14 Oocytes

To determine if the smg 3‟UTR 400-785 region is sufficient to cause translational

repression in stage 14 oocytes the region was inserted into a transgene containing the Luciferase

ORF and α-Tubulin 3‟UTR ( H114(400-785)). The control transgene lacked the smg 3‟UTR 400-

785 region (H114). In the case of H114, high LUC levels were found in both oocytes and

embryos (Figure 3-8 ). However, for H114(400-785), LUC levels were low in both stage 14

oocytes and early embryos (Figure 3-8). To verify that difference in protein levels was not due to

a difference in RNA stability, northern blot analysis was conducted; double-normalization

showed that the H114(400-785) mRNA was translationally repressed approximately 15 fold in

35

oocytes and 7 fold in embryos relative to H114 alone (Figure 3-8). I conclude that the smg

3‟UTR 400-785 region is sufficient to cause translational repression in the oocyte; however,

additional regions in the smg 3‟UTR appear to be required for high level translation in the

embryo.

36

Figure 3-8. smaug 3’UTR 400-785 region is sufficient to cause translational repression of

Luciferase protein in stage 14 oocytes. Western Blot probed for LUC protein, comparing LUC expression between stage 14 oocytes and

0-2 hour embryos in the H114 construct and the H114(400-785) construct. Both constructs

contain the LUC ORF and α-Tubulin 3‟UTR. The H114(400-785) construct also contains the

smg 3‟UTR 400-785. Constructs are driven by NGV. Western also probed for DDP1 as a loading

control. Northern blot probed for luc mRNA and rpa1 mRNA as a loading control. Protein levels

were normalized to RNA levels. Quantification of normalized values reveals a dramatic decrease

in LUC expression in late oocytes and early embryos carrying the H114(400-785) transgene. For

the H114 construct only one line was tested for Western and Northern analysis and error could

not be calculated. For the H114(400-785) construct three lines were tested for Western and

Northern analysis and quantification shows average value (RNA/protein) of the three lines

tested. Error bars show standard deviation among the three lines tested. The H114 stage 14

oocyte luc mRNA lane is under loaded as shown by the level of rpa1 mRNA.

37

3.5 Smaug mRNA is Repressed Before Translation Initiation

To determine whether translational repression of smg mRNA occurs before or after

translation initiation smg mRNA was analyzed using sucrose gradients and northern analysis on

wild-type stage 14 oocyte extract, wild-type 0-2 hour embryo extract, and 0-2 hour png mutant

embryo extract.

In wild-type stage 14 oocytes rRNA is abundant in lighter fractions within gradient, but

shifts to heavier fractions (ie. the polysome region) upon egg activation (Figure 3-9). This

suggests that little or no translation occurs in the stage 14 oocyte and high level translation is

activated upon fertilization. In the stage 14 oocyte almost 50% of the smg mRNA pelleted at the

bottom of the tube, but shifted out of the pellet into the polysome fractions upon fertilization

(Figure 3-9). Addition of EDTA to embryo extract shifted rRNA and smg mRNA into the lighter

fractions, consistent with both the pellet material and the polysomes being Mg++ dependent.

Addition of EDTA also functions as a control to determine which fractions in the gradient

contain polysomes and other heavy complexes. Unlike EDTA, puromycin disrupts only

polysomes and not other large complexes. If puromycin-release experiments show that the

pelleted complexes in stage 14 oocytes are resistant, while the polysome-region complexes in

embryos are sensitive to puromycin, it will be possible to conclude that smg mRNA is repressed

prior to translation initiation and is in a heavy repression complex rather then in a heavy

polysome complex in the pellet (see Discussion). One possible protein contained in this heavy

repression complex is PUM. Future experiments to conclusively demonstrate how smg mRNA is

repressed in stage 14 oocytes are presented in the Discussion. Stage 14 oocyte gradient has been

repeated once with similar results. Wild-type embryo gradient and EDTA gradients have only

been completed once.

38

Figure 3-9. Sucrose gradients reveal shift of RNA upon egg activation.

Wild-type stage 14 oocyte extract and wild-type 0-2 hour embryo extract were prepared and

loaded on top of sucrose gradients and spun for 2.5 hours. o= stage 14 oocytes and e= 0-2 hr

embryos. Gradients were hand fractionated in 1ml fractions and Northern analysis was carried

out. Ribosomal RNA stained by ethidium bromide (A) rRNA in wild-type stage 14 oocytes is

abundant in lighter fractions of gradient and is shifted to heavier fractions upon egg activation.

(B) smg mRNA in wild-type stage 14 oocytes is abundant in pellet fraction of gradient. In 0-2

hour embryos smg mRNA fractionates with polysomes. (C) EDTA was added to embryo extract.

EDTA caused rRNA and smg mRNA to shift from the polysome region to lighter fraction

regions in early embryos.

3.6 In png Mutant Embryo smaug mRNA does not Shift out of the Pellet

Sucrose gradient analyses were also carried out with 0-2 hour png mutant embryo extract.

smg mRNA in png mutant 0-2 hour embryos is mostly in the pellet region (Figure 3-10A). The

profiles of the smg mRNA in the gradients from 0-2 hour png mutant and wild-type oocytes, are

39

similar in that a substantial fraction of the smg mRNA was in the pellet (26% in png vs. 44% in

wild-type stage 14 oocytes; see figure 3-10B. If the pellet is, in both cases, resistant to

puromycin disruption (puromycin causes premature chain release and disrupts only polysomes),

it will be possible to conclude that, in png mutants, the translation repression complex is

maintained post-egg activation. It should be noted that there is more smg mRNA in the lighter

fractions of the gradient in png mutant embryos than in wild-type stage 14 oocytes. This could

mean that in a png mutant repression could also occur via a lighter repression complex which is

not seen in the stage 14 oocyte.

40

Figure 3-10. Sucrose gradients reveal that in 0-2 hour png mutant embryos, smg mRNA

remains in heavy pellet region and does not shift to polysomes as in wild-type embryos.

(A)0-2 hour png mutant embryo extract smg mRNA is abundant in pellet region similar to smg

mRNA expression in stage 14 oocytes. Fraction 11 was accidently not loaded. o=stage 14

oocyte, e= 0-2 hour png mutant embryo. (B) For stage 14 oocytes, wild-type 0-2 hour embryos,

and 0-2 hour png mutant embryos percent of smg mRNA was calculated within each fraction of

gradient. For stage 14 oocytes and png mutant embryos profiles are similar.

41

CHAPTER 4

DISCUSSION AND FUTURE DIRECTIONS

4.1 Mapping of Redundant Translational Repressive Cis Elements

I have mapped redundant translational repressive cis elements in the smg 3‟UTR, one of

which mediates PUM- dependent repression (400-600) and the other of which mediates

repression by one or more additional repressors. These elements function by inhibiting

translation during late oogenesis, and upon egg activation repression mediated through these

elements is removed. Higher resolution mapping of these elements will require the use of smaller

(i.e. 100 base pair) deletions within the 400-785 region. Once smaller elements are mapped, the

next step would be to identify trans factors which bind directly or indirectly to these elements.

To identify factors which bind directly to cis elements a UV cross linking assay could be

performed as described in Nelson et al. (2007) and Smibert et al. (1996), in which a radioactive

labelled RNA probe ( prepared by in vitro transcription in the presence of 32

P-labeled UTP)

corresponding to identified cis elements is incubated in embryo or oocyte extract and exposed

to UV cross linking to identify binding proteins. TRAP-tagging as described in Nelson et al.

(2007) could be performed to identify factors which bind directly or indirectly to cis elements.

TRAP tagging allows the in vitro transcription of RNA (containing sequence of identified cis

elements) with two RNA affinity tags. The first tag is an S1 aptamer which can bind streptavidin

resin and the second tag consist of two MS2 coat protein binding sites.

To further verify the deletion mapping results it would also be beneficial to insert the

deletions into the SGS construct which contains the smg5‟UTR-GFP OFR-smg3‟UTR which

produces no PNG-independent GFP in stage 14 oocytes (Tadros et al., 2007) , thus more closely

resembling endogenous smg regulation (Figure 4-1)

42

Figure 4-1. The smg 3’UTR and 5’UTR regulate smg mRNA translation

SGS transgene containing smg5‟UTR-GFP ORF-smg3‟UTR. Comparing GFP expression

between stage 14 oocytes and 0-3 hour embryos. In WT, No GFP expression in stage 14 oocytes,

but high GFP expression in 0-3 embryos. In png mutant there is no GFP expression in both stage

14 oocytes and 0-3 hour embryos. (Reprinted from Developmental Cell, Vol 12, Tadros et al.,

2007, SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its

translation is activated by the PAN GU kinase, page 148, © 2007, with permission from

Elsevier).

4.2 PUM Represses smg Translation Through the 400-600 Region

Computational analysis of the 400-785 region within the smg 3‟UTR identified a single

evolutionary conserved PUM-like binding site at 466-475 nt. This site contains the UGUA

tetranucleotide required for PUF-family protein binding (Gerber et al., 2004; 2006) as well as

Box B (AUUGUA) which is part of the NRE (Wharton and Struhl, 1991) and the UGUANWUW

PUM- like site found in 12 Drosophila species (Stark et al., 2007) (Red box). Removal of either

PUM or the 400-600 nt region elements in the context of a deletion that removed the 600-785,

derepressed smg mRNA translational repression in the late oocyte. The pum13

allele which was

used for these experiments is a weak temperature sensitive allele that expresses normal levels of

full length protein and allows normal abdominal segmentation at 29˚C but not at 17˚C (Lehmann

43

and Nüsslein-Volhard, 1987; Wharton et al., 1998). Because of the temperature sensitivity

embryos for the pum experiment were laid at 25˚C for 1.5 hours and shifted to 18˚C for 30

minutes. The pum13

mutant contains a single amino acid substitution of Asp for Gly at residue

1330 which is a non-conserved position within repeat 7 of the RNA-binding domain (Wharton et

al., 1998). PUM protein produced from this mutant is able to bind RNA normally and recruit

NOS, but is defective in regulating hb mRNA in embryos (Sonoda and Wharton, 1999; Wharton

et al., 1998). Sonoda and Wharton (2001), showed in a yeast four-hybrid that bait consisting of a

ternary complex containing the RNA-binding domain of PUM, full- length NOS, and NRE

bearing RNA could recruit BRAT, however individual factors from this complex could not.

Thus, BRAT, an NHL domain protein, functions in a complex with PUM and NOS to

translationally repress hb mRNA in the posterior. In a yeast four-hybrid, the pum13

RNA binding

domain could recruit NOS into a ternary complex, but could not recruit BRAT into a quaternary

complex. Thus I tentatively conclude that PUM represses smg mRNA in stage 14 oocytes in a

BRAT-dependent manner. To check for BRAT‟s involvement in translational repression of smg

3‟UTR, the deletions could be tested in a brat mutant background. If the 600Δ785 deletion in a

brat background causes an increase of GFP expression in stage 14 oocytes similar to that seen in

the pum background, I would conclude that BRAT functions in a complex with PUM to repress

smg mRNA in the late oocyte. To verify that PUM binding to the 466-475 nt site has functional

implications, it will be essential to mutate this putative PUM binding site in the context of

600Δ785 to determine if repression of smg mRNA translation fails in the late oocyte.

44

4.3 The 400-785 Region is Sufficient to Cause Translational Repression in Stage 14 Oocytes

I have also shown that the smg 3‟UTR 400-785 region is sufficient to cause translational

repression in stage 14 oocytes of a reporter mRNA containing a LUC ORF and α-Tubulin

3‟UTR. Interestingly, repression largely persisted in the early embryo. These results suggest that

the PNG kinase functions not only via the 400-785 region to relieve repression but also through

another region(s) of the smg 3‟UTR to allow translation upon egg activation. These additional

region(s) most likely function as activation element(s) that recruit factors to smg mRNA which

override translational repression. The smg 3‟UTR could contain multiple such activation

elements, since the deletion analysis did not show any removal of activation elements. To

determine which additional region(s) PNG functions through to allow translational activation

upon egg activation, additional regions from the smg 3‟UTR could be added into the H114(400-

785) construct. It is also possible that luc mRNA is translated in the oocyte in the H114

construct, but in the embryo translation of luc mRNA is inhibited, possibly by elements within

the LUC ORF. LUC protein detected in the 0-2 hour embryo in the H114 construct could

actually be protein carried over from the oocyte. It will therefore also be important to insert the

“FL” (1-785) smg 3‟UTR into the construct to make sure translational activation does occur. It is

also possible that inserting the 400-785 region into the α-Tubulin 3‟UTR disrupted an important

element required for translational activation of this construct.

4.4 smaug mRNA is Repressed Before Translation Initiation

My sucrose gradient experiments show that smg mRNA resides in the pellet in stage 14

oocytes and then in the polysome region upon egg activation. This is almost certainly a shift onto

polysomes since smg mRNA is translated in early embryos (Benoit et al., 2009; Tadros et al.,

45

2007). Consistent with the pellet representing a translational repression complex, in png mutant

smg mRNA remains in the pellet. To verify this hypothesis, a puromycin control needs to be

completed for each time point and genotype. Puromycin disrupts polysomes but not other heavy

mRNP complexes. Puromycin structure is similar to the aminoacyl-tRNA and therefore,

competes for entry into the A site and can become part of the growing peptide chain, this leads to

premature release of incomplete polypeptide chains (Azzam and Algranati, 1973; Blobel and

Sabatini, 1971). It would also be beneficial once trans factors from cis element mapping are

identified ( mentioned above) to determine if they are also present in the wild-type stage 14

oocyte pellet and png mutant pellet.

A further test would use wild-type stage 14 oocyte extract and png mutant embryo extract

from transgenics for UGS-400Δ785. A prediction is that the UGS-400∆785 mRNA would be

absent from the pellet in wild-type stage 14 oocytes and 0-2 hour png mutant embryos. It would

also be interesting to test the UGS-600Δ785 construct in the pum mutant background as well as

H115(400-785); the prediction is that UGS-600∆785 will be absent from the pellet in pum

mutants while H115(400-785) will be present in the pellet in wild-type oocytes.

4.5 Hypothesized Models of Translational Regulation Mediated by the PAN GU Kinase

Based on my results two possible models are hypothesized. In one, PAN GU Kinase

functions after egg activation to inhibit all repressors that bind the 400-785 region, thus allowing

smg mRNA translation in the embryo (Figure 4-2A). One of these repressors is PUM, which

functions through the 400-600 region. An alternate hypothesis is the PAN GU Kinase inhibits

repressor(s) that act through the 600-785 region, while a different protein functions to relieve

repression by PUM in the 400-600 region. Function of both PAN GU and the other protein

46

would be required after egg activation to allow smg translation (Figure 4-2B). Both models

provide an explanation for why Tadros et al. (2007) were unable to identify a single repressor

mutation of which suppressed png defects in smg mRNA translation in embryos or that, singly

mutated resulted in smg translation in stage 14 oocytes.

To test which model is correct, the UGS-600Δ785 and the UGS-400Δ600 deletions will

need to be tested in a png mutant background. If PNG functions to remove repression through

both the 400-600 and 600-785 regions, then when UGS-600Δ785 is tested in a png mutant, GFP

levels should remain low in both late oocytes and early embryos. This is because the repressor in

the 400-600 region (PUM/BRAT) is not removed in a png background. If PNG functions to

remove repression only through the 600-785 region, then when UGS-600Δ785 is tested in a png

mutant translational repression should still occur in the late oocyte, but translation should

proceed in the embryo. A third possible model: that PNG functions to remove repression via the

400-600 region (PUM) while a different protein relieves repression via the 600-785 region, is

unlikely because Tadros et al. (2007) showed that removing PUM in a png mutant does not

restore smg mRNA translation in early embryos.

47

Figure 4-2. Models depicting translational regulation of smaug mRNA mediated by the

PAN GU Kinase.

Two hypothesized models in which the PAN GU Kinase regulates smg mRNA translation

through removal of repression. (A) PNG mediates smg mRNA translation by inhibiting both

redundant repressors, PUM and repressor(s) X after egg activation. (B) PNG regulates smg

mRNA translation by inhibiting repression from repressor(s) X while ? inhibits repression by

PUM after egg activation.

4.6 Finding Direct Targets of the PAN GU Kinase Involved in smg Translation

To identify direct targets of the PNG kinase it would be beneficial to determine the

phosphorylation status of the identified trans factors mentioned above and determine if the PNG

kinase is directly responsible for their phosphorylation. In a genome-scale screen for PNG kinase

substrates Lee et al. (2005) did not identify PUM in the screen. To determine if the PNG kinase

is responsible for the phosphorylation of identified trans factors changes in phosphorylation

48

status in wild type and png mutant embryos and oocytes need to be observed. In addition, the in

vitro kinase assay (Lee et al., 2003) can be used to determine if the PNG kinase can directly

phosphorylate any of the identified trans factors. Factors which are phosphorylated by the PNG

kinase are likely to be direct targets. The next step would be to determine if phosphorylation of

these targets by PNG plays a role in smg translation ie. mutate the site of phosphorylation and

determine if smg mRNA is repressed in embryos.

4.7 Generalized vs. Specific Translational Repression During Oogenesis

PUM is known to function as a post-transcriptional regulator during Drosophila

oogenesis and embryogenesis. There are several known examples of PUM mediated

translational repression during early embryogenesis (reviewed in Vardy and Orr-Weaver 2007).

PUM regulates translation of hb mRNA in the posterior of the embryo along with NANOS,

BRAT and d4EHP. In addition, cyclin B mRNA is translationally repressed in the pole cells by

PUM and NANOS, via recruitment of the CCR4/POP2/NOT deadenylase complex. In the ovary

PUM is required for the maintenance of germline stem cell self renewal and to inhibit cytoblast

differentiation (Forbes and Lehmann, 1998; Szakmary et al., 2005). It has been hypothesized by

Szakmary et al. (2005) that in the ovary PUM and NOS function together to translationally

repress mRNAs that are involved in differentiation. It has also been hypothesized that PUM and

NOS function with a miRNA complex to inhibit translation (Shen and Xie, 2008). My data has

identified an additional function of PUM as a translational repressor of smg mRNA during late

oogenesis, likely in collaboration with BRAT. This is similar to PUM function in the embryo, in

which it functions with NOS and BRAT to inhibit translation (Sonoda and Wharton, 2001). It

will be interesting to investigate if PUM functions to inhibit translation of multiple mRNAs in

49

the late oocyte and not just smg mRNA. Another recent example of possible PUM-mediated

translational repression in the late oocyte is that of Cyclin A (Vardy et al., 2009). In this case the

PNG GU Kinase antagonizes the translational repression by PUM during early embryo

development to allow Cyclin A expression. In stage 14 oocytes PNG is also required for Cyclin

A expression. It has, however, not yet been tested whether in a png mutant, removing PUM

expression restores Cyclin A expression in stage 14 oocytes.

My preliminary sucrose gradient analysis suggests that there are relatively few polysomes

in stage 14 oocytes, suggesting that there is general repression of translation. It will be

interesting to determine whether PUM functions to inhibit translation of multiple mRNAs during

late oogenesis or it is more specific in its effects.

My sucrose gradient data also suggest that smg maybe in a large mRNP that may

represent a “repression complex”. It recently has been found that repressed oskar mRNA in the

oocyte is contained in a heavy RNP particle (Besse et al., 2009) containing the nucleo-

cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP which is

needed both for oskar mRNA transport in the oocyte and for translational silencing of

unlocalized oskar mRNA. PTB binds to the oskar 3‟UTR and is required for oligomerization of

oskar mRNA. The oskar RNP complex most likely functions by blocking the initiation

machinery from accessing the mRNA until it is localized to the posterior pole. I propose that smg

mRNA in stage 14 oocytes is also in a repression complex and PUM and BRAT are two possible

proteins found within this complex. I also propose that this repression complex blocks the

translation initiation machinery from accessing the mRNA until egg activation.

50

4.8 Generalized vs Specific Translational Activation in the Embryo

Many mRNAs are cytoplasmically polyadenylated in early Drosophila embryos (Salles et

al., 1994). smg mRNA polyadenylation is seen upon egg activation (Tadros et al., 2007) as is

polyadenylation of cyclin A and cyclin B mRNAs (Vardy and Orr-Weaver, 2007a; Vardy et al.,

2009). This is consistent with a general mechanism of translational activation in the early

embryo in which mRNAs are polyadenylated to promote translation. In png mutants,

polyadenylation of smg, cyc A, and cyc B is compromised and they are not translated (Tadros et

al., 2007; Vardy and Orr-Weaver, 2007a; Vardy et al., 2009). Over expression of poly(A)

polymerase (PAP) in png mutants caused smg and cyclin poly(A) tails to lengthen, but SMG

translation was not restored. These results suggest that PNG function is required for

polyadenylation of its target transcripts, but that PNG promotes translation independent of

polyadenylation, possibly by antagonizing the function of repressors such as PUM. These

findings argue that activation of translation requires more than just extended poly(A) tails. PUM

regulates hb mRNA in the posterior of the embryo in both a poly(A) dependent and independent

manner. (Chagnovich and Lehmann, 2001; Wreden et al., 1997). In the case of smg mRNA it

will be interesting to assess the relationship between relief of repression and polyadenylation. It

is possible that there are multiple mechanisms to ensure proper translational activation in the

embryo, some poly(A) dependent and the others poly(A) independent.

51

References

Anderson, J.S., and Parker, R.P. (1998). The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex. EMBO J 17, 1497-1506.

Arama, E., Dickman, D., Kimchie, Z., Shearn, A., and Lev, Z. (2000). Mutations in the beta-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain. Oncogene 19, 3706-3716.

Asaoka-Taguchi, M., Yamada, M., Nakamura, A., Hanyu, K., and Kobayashi, S. (1999). Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos. Nature Cell Biol 1, 431-437.

Azzam, M.E., and Algranati, I.D. (1973). Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes. Proc Natl Acad Sci U S A 70, 3866-3869.

Barker, D.D., Wang, C., Moore, J., Dickinson, L.K., and Lehmann, R. (1992). Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos. Genes Dev 6, 2312-2326.

Bashirullah, A., Halsell, S.R., Cooperstock, R.L., Kloc, M., Karaiskakis, A., Fisher, W.W., Fu, W., Hamilton, J.K., Etkin, L.D., and Lipshitz, H.D. (1999). Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster. Embo J 18, 2610-2620.

Benoit, B., He, C.H., Zhang, F., Votruba, S.M., Tadros, W., Westwood, J.T., Smibert, C.A., Lipshitz, H.D., and Theurkauf, W.E. (2009). An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition. Development (Cambridge, England) 136, 923-932.

Besse, F., Lopez de Quinto, S., Marchand, V., Trucco, A., and Ephrussi, A. (2009). Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation. Genes Dev 23, 195-207.

Blobel, G., and Sabatini, D. (1971). Dissociation of mammalian polyribosomes into subunits by puromycin. Proc Natl Acad Sci U S A 68, 390-394.

Bushati, N., Stark, A., Brennecke, J., and Cohen, S.M. (2008). Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr Biol 18, 501-506.

Cao, D., and Parker, R. (2001). Computational modeling of eukaryotic mRNA turnover. RNA 7, 1192-1212.

Cao, D., and Parker, R. (2003). Computational modeling and experimental analysis of nonsense-mediated decay in yeast. Cell 113, 533-545.

Chagnovich, D., and Lehmann, R. (2001). Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo. Proc Natl Acad Sci U S A 98, 11359-11364.

52

Chekulaeva, M., Hentze, M.W., and Ephrussi, A. (2006). Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124, 521-533.

Chernokalskaya, E., Dubell, A.N., Cunningham, K.S., Hanson, M.N., Dompenciel, R.E., and Schoenberg, D.R. (1998). A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family. RNA 4, 1537-1548.

Cho, P.F., Gamberi, C., Cho-Park, Y.A., Cho-Park, I.B., Lasko, P., and Sonenberg, N. (2006). Cap-dependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos. Curr Biol 16, 2035-2041.

Coller, J., and Parker, R. (2004). Eukaryotic mRNA decapping. Annu Rev Biochem 73, 861-890.

Copeland, P.R., and Wormington, M. (2001). The mechanism and regulation of deadenylation: identification and characterization of Xenopus PARN. RNA 7, 875-886.

Cosson, B., Gautier-Courteille, C., Maniey, D., Ait-Ahmed, O., Lesimple, M., Osborne, H.B., and Paillard, L. (2006). Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding. Biol Cell 98, 653-665.

Cunningham, K.S., Dodson, R.E., Nagel, M.A., Shapiro, D.J., and Schoenberg, D.R. (2000). Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3'-untranslated region by the mRNA endonuclease polysomal ribonuclease 1. Proc Natl Acad Sci U S A 97, 12498-12502.

Dahanukar, A., Walker, J.A., and Wharton, R.P. (1999). Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol Cell 4, 209-218.

Day, D.A., and Tuite, M.F. (1998). Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview. J Endocrinol 157, 361-371.

De Renzis, S., Elemento, O., Tavazoie, S., and Wieschaus, E.F. (2007). Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo. PLoS Biol 5, e117.

Fenger, D.D., Carminati, J.L., Burney-Sigman, D.L., Kashevsky, H., Dines, J.L., Elfring, L.K., and Orr-Weaver, T.L. (2000). PAN GU: a protein kinase that inhibits S phase and promotes mitosis in early Drosophila development. Development (Cambridge, England) 127, 4763-4774.

Filipowicz, W., Bhattacharyya, S.N., and Sonenberg, N. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9, 102-114.

Forbes, A., and Lehmann, R. (1998). Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development (Cambridge, England) 125, 679-690.

Frischmeyer, P.A., van Hoof, A., O'Donnell, K., Guerrerio, A.L., Parker, R., and Dietz, H.C. (2002). An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295, 2258-2261.

Gerber, A.P., Herschlag, D., and Brown, P.O. (2004). Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol 2, E79.

53

Gerber, A.P., Luschnig, S., Krasnow, M.A., Brown, P.O., and Herschlag, D. (2006). Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci U S A 103, 4487-4492.

Giraldez, A.J., Mishima, Y., Rihel, J., Grocock, R.J., Van Dongen, S., Inoue, K., Enright, A.J., and Schier, A.F. (2006). Zebrafish MiR-430 Promotes Deadenylation and Clearance of Maternal mRNAs. Science.

Hsu, C.L., and Stevens, A. (1993). Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure. Mol Cell Biol 13, 4826-4835.

Johnstone, O., and Lasko, P. (2001). Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu Rev Genet 35, 365-406.

Juge, F., Zaessinger, S., Temme, C., Wahle, E., and Simonelig, M. (2002). Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila. EMBO J 21, 6603-6613.

Lee, L.A., Lee, E., Anderson, M.A., Vardy, L., Tahinci, E., Ali, S.M., Kashevsky, H., Benasutti, M., Kirschner, M.W., and Orr-Weaver, T.L. (2005). Drosophila genome-scale screen for PAN GU kinase substrates identifies Mat89Bb as a cell cycle regulator. Dev Cell 8, 435-442.

Lee, L.A., Van Hoewyk, D., and Orr-Weaver, T.L. (2003). The Drosophila cell cycle kinase PAN GU forms an active complex with PLUTONIUM and GNU to regulate embryonic divisions. Genes Dev 17, 2979-2991.

Lehmann, R., and Nüsslein-Volhard, C. (1987). Involvement of the pumilio gene in the transport of an

abdominal signal in the Drosophila embryo. Nature 329, 167-170.

Liang, H.L., Nien, C.Y., Liu, H.Y., Metzstein, M.M., Kirov, N., and Rushlow, C. (2008). The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila. Nature 456, 400-403.

Martin, S.G., and St Johnston, D. (2003). A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity. Nature 421, 379-384.

Matsumoto, K., and Wolffe, A.P. (1998). Gene regulation by Y-box proteins: coupling control of transcription and translation. Trends Cell Biol 8, 318-323.

Minshall, N., and Standart, N. (2004). The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer. Nucleic Acids Res 32, 1325-1334.

Minshall, N., Thom, G., and Standart, N. (2001). A conserved role of a DEAD box helicase in mRNA masking. RNA 7, 1728-1742.

Muhlrad, D., Decker, C.J., and Parker, R. (1994). Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes Dev 8, 855-866.

Muhlrad, D., Decker, C.J., and Parker, R. (1995). Turnover mechanisms of the stable yeast PGK1 mRNA. Mol Cell Biol 15, 2145-2156.

54

Nelson, M.R., Leidal, A.M., and Smibert, C.A. (2004). Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression. Embo J 23, 150-159.

Nelson, M.R., Luo, H., Vari, H.K., Cox, B.J., Simmonds, A.J., Krause, H.M., Lipshitz, H.D., and Smibert, C.A. (2007). A multiprotein complex that mediates translational enhancement in Drosophila. J Biol Chem 282, 34031-34038.

Niessing, D., Blanke, S., and Jackle, H. (2002). Bicoid associates with the 5'-cap-bound complex of caudal mRNA and represses translation. Genes Dev 16, 2576-2582.

Nottrott, S., Simard, M.J., and Richter, J.D. (2006). Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nature structural & molecular biology 13, 1108-1114.

Olsen, P.H., and Ambros, V. (1999). The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 216, 671-680.

Paillard, L., Omilli, F., Legagneux, V., Bassez, T., Maniey, D., and Osborne, H.B. (1998). EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos. EMBO J 17, 278-287.

Piccioni, F., Ottone, C., Brescia, P., Pisa, V., Siciliano, G., Galasso, A., Gigliotti, S., Graziani, F., and Verrotti, A.C. (2009). The translational repressor Cup associates with the adaptor protein Miranda and the mRNA carrier Staufen at multiple time-points during Drosophila oogenesis. Gene 428, 47-52.

Piccioni, F., Zappavigna, V., and Verrotti, A.C. (2005a). A cup full of functions. RNA Biol 2, 125-128.

Piccioni, F., Zappavigna, V., and Verrotti, A.C. (2005b). Translational regulation during oogenesis and early development: the cap-poly(A) tail relationship. C R Biol 328, 863-881.

Radford, H.E., Meijer, H.A., and de Moor, C.H. (2008). Translational control by cytoplasmic polyadenylation in Xenopus oocytes. Biochim Biophys Acta 1779, 217-229.

Richter, J.D. (1999). Cytoplasmic polyadenylation in development and beyond. Microbiol Mol Biol Rev 63, 446-456.

Rodgers, N.D., Wang, Z., and Kiledjian, M. (2002). Characterization and purification of a mammalian endoribonuclease specific for the alpha -globin mRNA. J Biol Chem 277, 2597-2604.

Salles, F.J., Lieberfarb, M.E., Wreden, C., Gergen, J.P., and Strickland, S. (1994). Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266, 1996-1999.

Semotok, J.L., Cooperstock, R.L., Pinder, B.D., Vari, H.K., Lipshitz, H.D., and Smibert, C.A. (2005). Smaug recruits the CCR4/POP2/NOT deadenylase complex to trigger maternal transcript localization in the early Drosophila embryo. Curr Biol 15, 284-294.

Semotok, J.L., and Lipshitz, H.D. (2007). Regulation and function of maternal mRNA destabilization during early Drosophila development. Differentiation 75, 482-506.

55

Semotok, J.L., Luo, H., Cooperstock, R.L., Karaiskakis, A., Vari, H.K., Smibert, C.A., and Lipshitz, H.D. (2008). Drosophila maternal Hsp83 mRNA destabilization is directed by multiple SMAUG recognition elements in the open reading frame. Mol Cell Biol 28, 6757-6772.

Shen, R., and Xie, T. (2008). Stem cell self-renewal versus differentiation: tumor suppressor Mei-P26 and miRNAs control the balance. Cell Res 18, 713-715.

Smibert, C.A., Lie, Y.S., Shillinglaw, W., Henzel, W.J., and Macdonald, P.M. (1999). Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. Rna 5, 1535-1547.

Smibert, C.A., Wilson, J.E., Kerr, K., and Macdonald, P.M. (1996). Smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo. Genes and Development 10, 2600-2609.

Song, Y., Fee, L., Lee, T.H., and Wharton, R.P. (2007). The molecular chaperone Hsp90 is required for mRNA localization in Drosophila melanogaster embryos. Genetics 176, 2213-2222.

Sonoda, J., and Wharton, R.P. (1999). Recruitment of Nanos to hunchback mRNA by Pumilio. Genes Dev 13, 2704-2712.

Sonoda, J., and Wharton, R.P. (2001). Drosophila Brain Tumor is a translational repressor. Genes Dev 15, 762-773.

Stark, A., Lin, M.F., Kheradpour, P., Pedersen, J.S., Parts, L., Carlson, J.W., Crosby, M.A., Rasmussen, M.D., Roy, S., Deoras, A.N., et al. (2007). Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. Nature 450, 219-232.

Stebbins-Boaz, B., and Richter, J.D. (1994). Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development. Mol Cell Biol 14, 5870-5880.

Szakmary, A., Cox, D.N., Wang, Z., and Lin, H. (2005). Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation. Curr Biol 15, 171-178.

Tadros, W., Goldman, A.L., Babak, T., Menzies, F., Vardy, L., Orr-Weaver, T., Hughes, T.R., Westwood, J.T., Smibert, C.A., and Lipshitz, H.D. (2007). SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its translation is activated by the PAN GU kinase. Dev Cell 12, 143-155.

Tadros, W., Houston, S.A., Bashirullah, A., Cooperstock, R.L., Semotok, J.L., Reed, B.H., and Lipshitz, H.D. (2003). Regulation of maternal transcript destabilization during egg activation in Drosophila. Genetics 164, 989-1001.

Tadros, W., and Lipshitz, H.D. (2005). Setting the stage for development: mRNA translation and stability during oocyte maturation and egg activation in Drosophila. Dev Dyn 232, 593-608.

Temme, C., Zaessinger, S., Meyer, S., Simonelig, M., and Wahle, E. (2004). A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J 23, 2862-2871.

Thermann, R., and Hentze, M.W. (2007). Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875-878.

56

Till, D.D., Linz, B., Seago, J.E., Elgar, S.J., Marujo, P.E., Elias, M.L., Arraiano, C.M., McClellan, J.A., McCarthy, J.E., and Newbury, S.F. (1998). Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster. Mech Dev 79, 51-55.

Van Doren, M., Williamson, A.L., and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr Biol 8, 243-246.

van Hoof, A., Frischmeyer, P.A., Dietz, H.C., and Parker, R. (2002). Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science 295, 2262-2264.

Vardy, L., and Orr-Weaver, T.L. (2007a). The Drosophila PNG kinase complex regulates the translation of cyclin B. Dev Cell 12, 157-166.

Vardy, L., and Orr-Weaver, T.L. (2007b). Regulating translation of maternal messages: multiple repression mechanisms. Trends Cell Biol 17, 547-554.

Vardy, L., Pesin, J.A., and Orr-Weaver, T.L. (2009). Regulation of Cyclin A protein in meiosis and early embryogenesis. Proc Natl Acad Sci U S A.

Wharton, R.P., Sonoda, J., Lee, T., Patterson, M., and Murata, Y. (1998). The Pumilio RNA-binding domain is also a translational regulator. Mol Cell 1, 863-872.

Wharton, R.P., and Struhl, G. (1991). RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos. Cell 67, 955-967.

Wreden, C., Verrotti, A.C., Schisa, J.A., Lieberfarb, M.E., and Strickland, S. (1997). Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development (Cambridge, England) 124, 3015-3023.