Genome wide analysis of maternal RNA decay patterns in Drosopila melanogaster
Zusammenfassung der Projektergebnisse
The first hours of animal development are controlled by the genome of the mother by factors preloaded on the oocyte. Across animal groups this includes a defined number of synchronized cell or nuclear divisions and patterning events; at the molecular level this is reflected by a plethora of posttranscriptional regulatory steps including translation, localization and degradation of selected maternally provided mRNAs. During the maternal to zygotic transition (MZT) control of development is handed over to the awakening zygotic genome. In Drosophila, mRNA turnover during the MZT is accomplished by decay activities encoded by both the maternal and the zygotic genome. The separate contributions of these decay activities to maternal RNA decay, the extent to which they target separate or common mRNA target sets and how decay patterns are integrated with de novo transcription to shape RNA pools during the MZT is widely unclear. Here, we address these questions by performing microarray time courses spanning the MZT in both embryos and unfertilized eggs; RNA level fluctuations in the latter reflect the activity of exclusively maternally controlled processes. For each gene, we analyze individual RNA profiles regarding the separate contributions of (i) maternal provision, (ii) maternal and (iii) zygotic decay activities and (iv) transcription. We find that > 60 % of maternally provided mRNAs are destabilized, 40% of these by a combinatorial action of both maternal and zygotic decay activities. In addition, we also detect wide overlaps of maternal provision and transcription as >20% of all maternally provided mRNAs are topped up by transcription; within this group, mRNAs of circa 400 genes suffer also decay. Hence, maternal and zygotic decay as well as transcription do widely overlap during the MZT in Drosophila. RNA degradation is generally preceded by a lag-phase reflecting the need to activate maternal decay factors preloaded on the egg or to synthesize zygotic decay factors de novo. Maternal decay activities dominate RNA turnover prior to gastrulation as they (i) commence earlier than zygotic activities, (ii) lead to more severe RNA degradation and (iii) as zygotic contributions to combined RNA decay patterns are widely negligible. Analyses of gene function of destabilized RNAs suggests that cell cycle control, translation control and RNA decay mechanisms are regulated at the level of RNA stability and require readjustments at the end of the MZT. Micro-RNA targets are highly enriched in mRNAs suffering both maternal and zygotic decay and we show for targets of the zygotic mir-309 cluster that micro-RNAs widely interact with maternal RNA decay activities. We also reveal interactions of different posttranscriptional regulatory levels: (i) RNA stability and translation control are co-ordinated as stable transcripts are preferentially translated while destabilized transcripts tend to be translationally silent. (ii) linking RNA decay patterns to in situ hybridisation patterns we find striking enrichment of differerent posterior localization patterns for destabilized transcripts. This in line with previous reports describing posterior localization of selected mRNAs via a protection/degradation mechanism. Very recently, we identified signature motifs at the level of RNA secondary structure associated with particular RNA decay patterns; the functional importance of these is currently being investigated. The project was carried out the project essentially as proposed. Capitalizing on new data published a few months after the start of my fellowship, we changed the experimental design to include a time course in embryos. This extended the developmental context of my project and allowed to study both the impact of egg activation and fertilization on RNA stability. To overcome initial challenges with the microarray data analyses of RNA decay data we established a formal co-operation with the group of Dr. Wolfgang Huber at the European Bioinformatics Institute, Hinxton, UK. Ongoing experiments addressing the functionality of recently identified RNA decay signature motifs will be published. Other short term goals following up this study are (i) relating RNA decay patterns to other gene regulatory levels including alternative polyadenylation, (ii) the assessment of validated RNA decay motifs in other developmental contexts including metamorphosis and (iii) addressing whether different decay mechanisms act in a combinatorial or rather redundant manner on maternal transcripts.
