Hypothesis proteins travel to the oocyte, we need

Hypothesis
1) To
investigate the Postal (Po) movement from the nurse cells to the
posterior-dorsal (PD) region using microtubules (MTs), as a first step, the expression
of Po in the nurse cells and destination cell (oocyte) should be demonstrated. So,
initially, the three-dimensional
distribution of Po mRNA using a high-resolution fluorescent in situ
hybridization (FISH) in the specimens will be assessed. Several key controls are
required for this experiment. A no-probe sample (probed with hybridization
buffer only) is necessary. In addition, to ensure that the measuring signal is
produced by the probe’s binding specifically
to RNA, the control sample using RNase is required. Besides to determine that
the observed spots are specific to Po and not other RNAs, an ideal negative
control is to test the gene-speci?c probe set in a cell line or tissue. In addition,
positive control probe sets (cataloged
probe) to a gene target that has relatively medium to high abundance, when
performing experiments is required. Finally, to ensure the amount of RNA
present in the samples or degraded a complementary method, such as qPCR can be
run.

Because,
FISH can only detect the steady-state accumulation of Po mRNAs, thus, to
understand how Po proteins travel to the oocyte,
we need to investigate their localization dynamics. To visualize Po protein
transport from nurse cells to their destination, I will use the (live) fluorescent visualization
methods using fluorescently tagged proteins in vivo (fluorophore-labeled molecules i.e. Po specific
antibodies). Besides if we consider the Po mRNA is also co-localized similar to
Po proteins, GFP labeling of tagged
mRNAs, microinjection of fluorescently labeled
mRNAs or molecular beacons can be used to investigate localization. Live-imaging
studies of Po, using injected fluorescent transcripts or GFP-Exu as a
localization factor for Po mRNA can be one of the best methods (Cha et al., 2001; Clark et al., 2007; Mische et al., 2007; Theurkauf and
Hazelrigg, 1998). Using this method, we can localize distributions of Po and
visualize transport of mRNAs in different regions of the oocyte. Therefore, allowing
the dynamic analysis of Po behaviors throughout the process of localization
during oogenesis. Similarly, injection of fluorescently labeled transcripts into
the nurse
cells is a successful strategy for investigating the localization of Po RNAs in
Drosophila oocytes. (Cha et al., 2001; Clark et al.,
2007; Delanoue et al.,
2007; MacDougall et al., 2003; Mische et al., 2007). In this method a control group to check whether the behavior of injected
mRNA and native mRNA is required. Additionally, quantities of added mRNA that might facilitate
detection must be weighed against non-physiological amounts of exogenous RNA.

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In
addition to above-mentioned methods, to demonstrate
the role of MTs in the movement of Po proteins from the nurse cells to the PD
region, I will use the specific mutagenesis approaches (genetically compromised
MT function or structure by targeting tubulin subunits and MT instability) to
disrupt the Po localization without affecting the cytoskeleton. Using these
type of mutants, if there is no proper instability of MT, the localization of
the Po in the PD region of the oocyte will be affected. If this is right and it
seems the localization of Po within the oocyte is MT-dependent, I will also support
this hypothesis using MT depolymerizing drugs (Clark et al. 1994). There are
several tubulin-binding drugs that can alter MT dynamics including the
cancer-fighting taxane class of drugs which block dynamic instability of MT by
stabilizing GDP-bound tubulin in the MT. Also the polymerization inhibitory
drugs such as Colchicine, Vinblastin and Nocodazole can be used (Alberts et al.
4th). However,
the high dosage of these drugs highly affect suppression of MT dynamics, so
optimization of dosage is required. MT staining and or live imaging of MT-associated
proteins can also provide a better support. By co-visualizing MTs labeled with
Tau-GFP together with fluorescent Po proteins, we can demonstrate that Po as
part of the RNP particles travels on
these MT tracks. Because of so many variables affecting the final images, the
proper controls are also essential to be included in these experiments. Negative
controls (no staining with secondary antibody only) and single-fluorophore
controls for each fluorophore, or for combinations of more than 2 fluorophores are required. Additional controls such as the application of antifade reagents to minimize
photobleaching of specimens can be used. A non-reactive dye can be used as a
negative control. Optimal labeling of antibodies for in vivo
applications is required and the available Rapid Antibody Labeling Kits which provide
the rigorous requirements to control the degree of labeling (DOL) will improve
all these findings.

Hypothesis
2) As
demonstrated in other studies, 3′ UTRs of the mRNAs (regulatory regions) binds
proteins that regulate functions such as repression of translation 12. To
demonstrate that Po, as an unknown protein binds to only a few mRNAs at
distinct regions within their 3′ UTRs, I will use CLIP (cross-linking and
immunoprecipitation) to identify targets of Po
as RNA-binding protein. This method combines UV cross-linking with
immunoprecipitation (IP) to analyze
protein interactions with RNAs 1234. Using CLIP, RNA
binding protein binding sites on a genome-wide scale
can be found 45. In this approach, the in vivo cross-linking of RNA-protein complexes
using the UV light will be performed. The cross-linked RNA-protein complexes will
be lysed (purified), and PO will be isolated using RNase enzymes via IP. Likewise, I will use proteinases to remove PO from
the RNA-protein complexes which allow for
the identification of the cross-linked mRNAs 7. cDNA then can be synthesized
via RT-PCR and the high-throughput sequencing can be used to map interaction
sites of
PO by mRNAs. Individual nucleotide–resolution cross-linking (iCLIP) following
IP, is also a technique similar to the CLIP that allows for the stringent
purification of linked protein-RNA complexes, using IP followed by SDS-PAGE and
membrane transfer. The radiolabelled protein-RNA complexes are then excised
from the membrane, and treated with proteinase to release the RNA. This leaves
one or two amino acids at the RNA cross-link site. The RNA is then reverse
transcribed using barcoded primers. iCLIP allows RNA-protein interaction sites
to be identified at a higher resolution. The advantage of this approach is that it captures only intimately associated RNAs
and proteins, and so is expected to be highly specific.

To
demonstrate that Po protein interacts directly with other proteins as part of
the mRNP complex, we need to isolate and analyze the composition of mRNP
transport granules. So as a first step, I will purify the fractions of RNA
granules and then I will characterize their protein composition and interaction
with Po protein. I will use the most common method of purification i.e. the sucrose
density centrifugation, followed by Immunoprecipitation (IP) of components of
the particle with specific antibodies. The total protein will be loaded on a sucrose density gradient (variable
depending on the sample) and centrifuged
at optimized forces. Next, the high
molecular weight complexes together by the RNA will be disrupted by RNase. Subsequently, I
will run components on SDS-PAGE, stain,
excise and identify protein components using mass spectrometry. Western blot
detection will be used as well to verify the identity of the antigen. Noteworthy
is that some non-ideal sedimentations are still possible when using the
above-mentioned method. The first potential issue is the unwanted aggregation
of particles, but this can occur in any centrifugation. A series of controls
are necessary including a positive control by loading a lane on the gel with a
given amount of purified recombinant Po protein to be used as a reference. Also
to check the specificity of antibody only selects the Po, a negative control
i.e. a reaction using lysate from cells where the protein is either knock-down
or not expressed is required. To ensure that antibody has efficiently cross-linked to the beads a useful control would be to load a lane
where the beads have been denatured to
release the antibodies.

Hypothesis 3) During localization, the translation of mRNAs is
repressed. Translation
repressors encompass both cis-acting mRNA regulatory sequences and trans-acting
protein factors. The best-characterized repressors act at the level of
translation initiation fall into four classes including specific and
nonspecific mRNA-binding proteins and CAP binding complexes (eIF4E, EIF4G). To demonstrate that Po as a
part of the mRNP complex, and a binding protein that binds to 3′ UTRs or the 3?cis-acting
regulatory elements involved in translational repression of target mRNAs during
their transport to the PD region of the oocyte,
the mutagenesis studies can be performed. This can be achieved by a Po mutation
as one of the involved proteins in preventing the assembly of the pre-initiation
complex. There are different factors that anchor the pre-initiation complex.
Among these factors, the repression of the pre-initiation complex by
eIF4E-binding proteins is very common. Po might interact to prevent eIF4G
binding to eIF4E, thereby repressing translation. Therefore, on the one hand, a
Po mutation as one of the binding proteins in cis-acting mRNA regulatory
sequences can demonstrate whether Po is involved in the translational
repression. On the other hand P bodies and their components including RNA binding
proteins and a core set of enzymes such as decapping enzymes (Dcp1, Dcp2) not
only play roles in decapping but also play roles in mRNA translation
repression. Therefore the mutation of decapping enzymes as a translation
control element that might interact with Po or Cup (a protein to inhibit the
interaction between eIF4E and eIF4G), would be another way to investigate the
role of Po in the translation repression. If one of the components of decapping
enzyme is mutated, the Po mRNAs will not localize to the proper region in the
oocyte. P bodies can also interact with the components of the RNA silencing
pathways such as Argonaute family of proteins (Ago1-4) and RNA binding
proteins. RNAs are also required for P body formation. Therefore, treatment
with RNase results in P body disassembly which can be detected by ISH and
differential centrifugation. Small interfering RNAs (siRNAs) and microRNAs
(miRNAs) also silence mRNA expression through RISC (RNA induced silencing
complex).