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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.
iii
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
vii
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.
9
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)
10
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
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