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. 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. 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. 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.

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. 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. 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. 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.

In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. Due to the lack of transcription in the fully-grown oocyte, critical oocyte processes including cell cycle progression, chromosome segregation, formation and maintenance of the meiotic metaphase spindles, maternal mRNA recruitment and degradation, fertilization and egg activation are all under post-transcriptional, translational, or post-translational control. Despite advances in understanding mechanisms regulating the translational control of cytoplasmic maternal mRNAs, it is unknown whether localized maternal mRNAs exist in mouse oocytes and what mechanisms are responsible for their control. Maternal mRNAs were isolated from metaphase II (MII) mouse oocytes, microsurgically-removed MII spindle-chromosome complexes, and enucleated MII oocytes and analyzed by cDNA microarray analysis. The analysis identified enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. Maternal mRNAs involved in cellular compartments and processes related to the cytoskeleton, chromatin/nucleus, and cellular signaling were enriched on the MII spindle. Using immunofluorescence and confocal microscopy, MIS18A, a protein encoded by a spindle-localized maternal mRNA, was confirmed to be associated with the MII spindle along with components of the ribosome translational machinery. The key translational regulator, EIF4EBP1, was observed to undergo a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appeared at different positions along the spindle at different stages of meiosis. Overexpression of EIF4EBP1 mutants had a profound effect on the maintenance of MII arrest. Approximately 24% of oocytes expressing a phosphodeficient (Threonine 69 to Alanine) EIF4EBP1 mutant underwent spontaneous activation, suggesting EIF4EBP1 phosphorylation is important for translation of maternal mRNAs and maintenance of MII arrest. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types. / Molecular Biology and Genetics

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Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...