Project

Discipline

Microtubule transport; Cbp; RNPs; mRNA localization; Drosophila; BicD, Bic-D

Lead
Der Transport von mRNAs in spezifische zelluläre Kompartimente dient der räumlichen (und zeitlichen) Kontrolle der Aktivität von Genen und ihren Produkten. Wir studieren im Drosophila Modellsystem die Komponenten, welche am Transport und der Translationskontrolle von mRNAs beteiligt sind und wir studieren die mRNAs, die auf diese Weise reguliert werden.
Lay summary
Genetisch Information muss zeitlich und räumlich kontrolliert umgesetzt werden. Wir haben ein zelluläres Transportsystem entdeckt, dass die mRNA, die Information der Gene, an die korrekte Stelle in der Zelle bringt, wo diese zum richtigen Zeitpunkt aktiviert wird und ihre Funktion kontrolliert erfüllen kann. Eine neu entdeckte Komponente von diesem Transportsystem hat auch weitere Funktionen bei der Synthese der mRNA und bei der Verteidigung des Erbgutes gegen parasitierende Elemente. Wir wollen die Funktionen dieses neuen Elementes nun im Detail studieren. Dieses mRNA Transportsystem kommt in unterschiedlicher Zusammensetzung vor und übt je nachdem verschiede Funktionen aus. Wir haben schon einige der transportierten mRNAs identifiziert und wollen nun herausfinden, welche Komplexe mit welchen mRNAs assoziieren und was deren Funktionen sind. In mehreren Fällen konnten wir die Lokalisation schon mit einer lokalen Funktion korrelieren. Es scheint also, als würden die mRNAs wirklich von unserem Transportsystem an den Ort gebracht, wo sie dann zum gegebenen Zeitpunkt aktiv sein müssen. Wir wollen dies nun an speziellen Beispielen noch klar demonstrieren. Es ist technisch sehr schwierig diese Transportvorgänge direkt in der lebenden Zelle zu zeigen. Die meisten Daten beruhen daher auf Experimenten, die interpretiert werden müssen und wir schliessen dann aus den Resultaten indirekt, dass diese Vorgänge so ablaufen. Da die technischen Hilfsmittel immer besser werden, wird es uns vielleicht aber doch bald möglich, diese Vorgänge im lebenden Tier oder Organ sichtbar zu machen, zu filmen und damit direkt zu zeigen. Hierbei handelt es sich um Grundlagenforschung, die auch für die medizinische Forschung relevant ist. Die Arbeiten werden im Drosophila Modellsystem ausgeführt.

Direct link to Lay Summary

Last update: 03.06.2014

Active participation

Self-organised

Number	Title	Start	Funding scheme

A large proportion of the cellular mRNAs exhibit specific distribution patterns that relate to the local function of the proteins they encode. The BicD/ Egl transport machinery localizes many mRNAs to their target site by attaching the cargo to the negative-end directed microtubule motor dynein. Surprisingly, BicD and dynein also transport vesicles and large organelles. We have dissected this transport system using genetic and biochemical tools, allowing us to learn much about composition and dynamics of the different BicD complexes and about the function of some of the BicD-associated proteins. Recently we focused on the role of Cbp80 in oogenesis and found that it acts together with Piwi and P-body components in repressing mRNAs that are being localized. We also identified the transcriptome that associates with BicD/ Egl. During the upcoming granting period we will focus on the analysis of the BicD/ RNA binding protein complexes and on the novel functions of Cbp80.Objective 1: The cytoplasmic functions of Cbp80Cbp80 is known to function in the nucleus, but we uncovered its additional activities in the cytoplasm, where it represses mRNAs that are on the move to their target location. We therefore want to find out to what mRNAs Cbp80 is bound to in the cytoplasm of syncytial Drosophila embryos. For this purpose we will isolate cytoplasmic fractions, purify Cbp80-tag complexes and determine their RNA content with the RNA-IP-Seq (RIPSeq) protocol we established successfully for BicD/ Egl::GFP complexes. We will take advantage of the local Illumina high throughput sequencing facility and the Bernese Bioinformatics group. Using the top ranking mRNAs associated with cytoplasmic Cbp80 we will test whether they are normally translationally repressed, localized, degraded at a specific time point, or whether they display another specific feature. This should reveal the functions of cytoplasmic Cbp80 that we will then test experimentally.Objective 2: Role of Cbp80 in the piRNA pathway We found that Cbp80 interacts with Piwi and it seems to function to localize it into the nucleus, which is essential to start the primary piRNA biogenesis cycle that causes transcriptional silencing of transposable elements (TEs). A role of Cbp80 in the piRNA pathway had so far not been described. To assess transcritptional silencing of TEs we will therefore compare the chromatin structure of the germ line nuclei in the “TE grave yard” regions with and without knock down of Cbp80. Objective 3: Correlating complexes with mRNA targets and RBPs BicD interacts with many different RNA-binding proteins (RBPs) and with some of them in a mutually exclusive manner. We have identified the Egl targets, and will next identify the targets of the other RBPs. We then want to compare the different data sets to learn more about the different complexes. Aside from BicD, Egl and Cbp80 targets, these are mainly FMRP and IMP targets.Objective 4: Role of early apical localization and early embryonic gene expression controlThe Egl targets revealed interesting novel questions. Acf1 (ATP-dependent chromatin assembly factor 1) mRNA localizes to the apical cortex of young embryos. It encodes a nucleosome-remodeling factor involved in chromatin assembly and gene repression. Its protein expression is strictly controlled, localizing to the pericentric heterochromatin. Interestingly, the blastodermal heterochromatin assembles at the apical pole of the nuclei, where Acf1 protein accumulates and adjacent to the cytoplasm where its mRNA is localized! Furthermore, this heterochromatin formation needs Acf1, and FMRP is also involved in this process. We will test whether Acf1 mRNA localization is important for this localized heterochromatin formation.The second gene of interest is sry-a. The protein it encodes is apically localized and essential for cellularization. There are only 4 zygotic genes known to induce cellularization and it seems that their expression is tightly controlled to ensure it does happen only after 13 nuclear division cycles. This led to our hypothesis that we may be able to cause insect embryos to cellularize prematurely if we express these genes prematurely. We will test this hypothesis with simple experiments. Objective 5: live analysis of BicD-dependent transport, cytoplasmic streaming and sortingExperiments addressing the transport dynamics of native mRNAs expressed during oogenesis are technically challenging. We plan two different types of experiments, one to analyze pairwise the distribution of mRNAs that may be co-transported in one complex and one to analyze co-transport of two different protein / organelle cargos that show similar localization patterns.We also identified an earlier fast cytoplasmic streaming event that is involved in establishing the earliest polarities in the female germ line of Drosophila. Because this process seems to be very important for polarity formation we will study it in greater detail.