Hypothesis1) Toinvestigate the Postal (Po) movement from the nurse cells to theposterior-dorsal (PD) region using microtubules (MTs), as a first step, the expressionof Po in the nurse cells and destination cell (oocyte) should be demonstrated.
So,initially, the three-dimensionaldistribution of Po mRNA using a high-resolution fluorescent in situhybridization (FISH) in the specimens will be assessed. Several key controls arerequired for this experiment. A no-probe sample (probed with hybridizationbuffer only) is necessary.
In addition, to ensure that the measuring signal isproduced by the probe’s binding specificallyto RNA, the control sample using RNase is required. Besides to determine thatthe observed spots are specific to Po and not other RNAs, an ideal negativecontrol is to test the gene-speci?c probe set in a cell line or tissue. In addition,positive control probe sets (catalogedprobe) to a gene target that has relatively medium to high abundance, whenperforming experiments is required. Finally, to ensure the amount of RNApresent in the samples or degraded a complementary method, such as qPCR can berun. Because,FISH can only detect the steady-state accumulation of Po mRNAs, thus, tounderstand how Po proteins travel to the oocyte,we need to investigate their localization dynamics.
To visualize Po proteintransport from nurse cells to their destination, I will use the (live) fluorescent visualizationmethods using fluorescently tagged proteins in vivo (fluorophore-labeled molecules i.e. Po specificantibodies). Besides if we consider the Po mRNA is also co-localized similar toPo proteins, GFP labeling of taggedmRNAs, microinjection of fluorescently labeledmRNAs or molecular beacons can be used to investigate localization. Live-imagingstudies of Po, using injected fluorescent transcripts or GFP-Exu as alocalization factor for Po mRNA can be one of the best methods (Cha et al.
, 2001; Clark et al., 2007; Mische et al., 2007; Theurkauf andHazelrigg, 1998). Using this method, we can localize distributions of Po andvisualize transport of mRNAs in different regions of the oocyte. Therefore, allowingthe dynamic analysis of Po behaviors throughout the process of localizationduring oogenesis. Similarly, injection of fluorescently labeled transcripts intothe nursecells is a successful strategy for investigating the localization of Po RNAs inDrosophila 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 injectedmRNA and native mRNA is required. Additionally, quantities of added mRNA that might facilitatedetection must be weighed against non-physiological amounts of exogenous RNA. Inaddition to above-mentioned methods, to demonstratethe role of MTs in the movement of Po proteins from the nurse cells to the PDregion, I will use the specific mutagenesis approaches (genetically compromisedMT function or structure by targeting tubulin subunits and MT instability) todisrupt the Po localization without affecting the cytoskeleton. Using thesetype of mutants, if there is no proper instability of MT, the localization ofthe Po in the PD region of the oocyte will be affected. If this is right and itseems the localization of Po within the oocyte is MT-dependent, I will also supportthis hypothesis using MT depolymerizing drugs (Clark et al.
1994). There areseveral tubulin-binding drugs that can alter MT dynamics including thecancer-fighting taxane class of drugs which block dynamic instability of MT bystabilizing GDP-bound tubulin in the MT. Also the polymerization inhibitorydrugs 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, sooptimization of dosage is required. MT staining and or live imaging of MT-associatedproteins can also provide a better support. By co-visualizing MTs labeled withTau-GFP together with fluorescent Po proteins, we can demonstrate that Po aspart of the RNP particles travels onthese MT tracks. Because of so many variables affecting the final images, theproper controls are also essential to be included in these experiments.
Negativecontrols (no staining with secondary antibody only) and single-fluorophorecontrols for each fluorophore, or for combinations of more than 2 fluorophores are required. Additional controls such as the application of antifade reagents to minimizephotobleaching of specimens can be used. A non-reactive dye can be used as anegative control.
Optimal labeling of antibodies for in vivoapplications is required and the available Rapid Antibody Labeling Kits which providethe rigorous requirements to control the degree of labeling (DOL) will improveall these findings.Hypothesis2) Asdemonstrated in other studies, 3′ UTRs of the mRNAs (regulatory regions) bindsproteins that regulate functions such as repression of translation 12. Todemonstrate that Po, as an unknown protein binds to only a few mRNAs atdistinct regions within their 3′ UTRs, I will use CLIP (cross-linking andimmunoprecipitation) to identify targets of Poas RNA-binding protein. This method combines UV cross-linking withimmunoprecipitation (IP) to analyzeprotein interactions with RNAs 1234. Using CLIP, RNAbinding protein binding sites on a genome-wide scalecan be found 45. In this approach, the in vivo cross-linking of RNA-protein complexesusing the UV light will be performed.
The cross-linked RNA-protein complexes willbe lysed (purified), and PO will be isolated using RNase enzymes via IP. Likewise, I will use proteinases to remove PO fromthe RNA-protein complexes which allow forthe identification of the cross-linked mRNAs 7. cDNA then can be synthesizedvia RT-PCR and the high-throughput sequencing can be used to map interactionsites ofPO by mRNAs.
Individual nucleotide–resolution cross-linking (iCLIP) followingIP, is also a technique similar to the CLIP that allows for the stringentpurification of linked protein-RNA complexes, using IP followed by SDS-PAGE andmembrane transfer. The radiolabelled protein-RNA complexes are then excisedfrom the membrane, and treated with proteinase to release the RNA. This leavesone or two amino acids at the RNA cross-link site. The RNA is then reversetranscribed using barcoded primers.
iCLIP allows RNA-protein interaction sitesto be identified at a higher resolution. The advantage of this approach is that it captures only intimately associated RNAsand proteins, and so is expected to be highly specific.Todemonstrate that Po protein interacts directly with other proteins as part ofthe mRNP complex, we need to isolate and analyze the composition of mRNPtransport granules. So as a first step, I will purify the fractions of RNAgranules and then I will characterize their protein composition and interactionwith Po protein.
I will use the most common method of purification i.e. the sucrosedensity centrifugation, followed by Immunoprecipitation (IP) of components ofthe particle with specific antibodies. The total protein will be loaded on a sucrose density gradient (variabledepending on the sample) and centrifugedat optimized forces. Next, the highmolecular weight complexes together by the RNA will be disrupted by RNase. Subsequently, Iwill run components on SDS-PAGE, stain,excise and identify protein components using mass spectrometry.
Western blotdetection will be used as well to verify the identity of the antigen. Noteworthyis that some non-ideal sedimentations are still possible when using theabove-mentioned method. The first potential issue is the unwanted aggregationof particles, but this can occur in any centrifugation. A series of controlsare necessary including a positive control by loading a lane on the gel with agiven amount of purified recombinant Po protein to be used as a reference. Alsoto check the specificity of antibody only selects the Po, a negative controli.
e. a reaction using lysate from cells where the protein is either knock-downor not expressed is required. To ensure that antibody has efficiently cross-linked to the beads a useful control would be to load a lanewhere the beads have been denatured torelease the antibodies.Hypothesis 3) During localization, the translation of mRNAs isrepressed. Translationrepressors encompass both cis-acting mRNA regulatory sequences and trans-actingprotein factors. The best-characterized repressors act at the level oftranslation initiation fall into four classes including specific andnonspecific mRNA-binding proteins and CAP binding complexes (eIF4E, EIF4G). To demonstrate that Po as apart of the mRNP complex, and a binding protein that binds to 3′ UTRs or the 3?cis-actingregulatory elements involved in translational repression of target mRNAs duringtheir transport to the PD region of the oocyte,the mutagenesis studies can be performed.
This can be achieved by a Po mutationas one of the involved proteins in preventing the assembly of the pre-initiationcomplex. There are different factors that anchor the pre-initiation complex.Among these factors, the repression of the pre-initiation complex byeIF4E-binding proteins is very common. Po might interact to prevent eIF4Gbinding to eIF4E, thereby repressing translation. Therefore, on the one hand, aPo mutation as one of the binding proteins in cis-acting mRNA regulatorysequences can demonstrate whether Po is involved in the translationalrepression. On the other hand P bodies and their components including RNA bindingproteins and a core set of enzymes such as decapping enzymes (Dcp1, Dcp2) notonly play roles in decapping but also play roles in mRNA translationrepression. Therefore the mutation of decapping enzymes as a translationcontrol element that might interact with Po or Cup (a protein to inhibit theinteraction between eIF4E and eIF4G), would be another way to investigate therole of Po in the translation repression.
If one of the components of decappingenzyme is mutated, the Po mRNAs will not localize to the proper region in theoocyte. P bodies can also interact with the components of the RNA silencingpathways such as Argonaute family of proteins (Ago1-4) and RNA bindingproteins. RNAs are also required for P body formation.
Therefore, treatmentwith RNase results in P body disassembly which can be detected by ISH anddifferential centrifugation. Small interfering RNAs (siRNAs) and microRNAs(miRNAs) also silence mRNA expression through RISC (RNA induced silencingcomplex).