Die Funktion des Proteins SMG-8 während der Embryonalentwicklung von Caenorhabditis elegans
Evolutionäre Zell- und Entwicklungsbiologie der Tiere
Zusammenfassung der Projektergebnisse
The focus of my research proposal was SMG-8 (Suppressor with Morphological effect on Genitalia), a novel protein that is highly conserved across the animal kingdom but its function has yet to be unraveled. SMG-8 was identified during a genetic screen as a suppressor of FoxA/PHA-4 mutations. FoxA/PHA-4 is the master regulator of foregut development during embryogenesis. However, the underlying mechanism of how SMG-8 modulates FoxA/PHA-4 has yet to be determined. I set out to study how smg-8 and pha-4 interact, and thereby begin to understand the role of smg-8 in development. The first step was to determine the level at which SMG-8 is acting: Does it modify RNA stability, translation, protein stability or degradation? Previous experiments have shown that pha-4;smg-8- mutant embryos have higher levels of PHA-4 protein compared to pha-4 mutants, indicating that smg-8 modulates PHA-4 protein levels, directly or indirectly. I hypothesized that if SMG-8 was affecting PHA-4 protein directly, the amount of pha-4 mRNA in smg-8- mutant and wild-type embryos would be similar. But, if SMG-8 affected pha-4 mRNA, the amount of pha-4 transcript would be higher in smg-8- mutant compared to wild-type embryos. After optimizing the conditions for smFISH (e.g. which stage to use, ensuring reproducibility and testing the effect of reporter genes), I began by showing that the amount of pha-4 mRNA does not differ between smg-8 mutants and wild type embryos, during stages E8 and E16. Previous experiments have shown that the amount of PHA-4 protein is higher in pha-4/smg-8 double mutants compared to pha-4 mutants, suggesting that SMG-8 has a repressive effect on pha- 4. Using a S35-Methionin incorporation assays, I could show that there is no overall increase in protein synthesis in smg-8 mutants compared to wild type animals, suggesting that the differences in the amount of PHA-4 protein in pha-4;smg-8 compared to pha-4 mutants is specific to PHA-4 protein. Furthermore, I compared the amount of pha-4 mRNA in pha-4 mutants vs. pha-4;smg-8 double mutants by smFISH and found that pha-4(q500);smg-8 have significantly more pha-4 transcript than pha-4(q500) embryos, indicating that smg-8 acts on the mRNA level to suppress pha-4. Smg-8 is suggested to be involved in nonsense-mediated decay (NMD), a pathway that leads to the degradation of mRNA molecules with premature stop codons, such as the pha-4(q500) transcript. I compared the amount of pha-4 transcript and protein in pha-4(RNAi) and smg-8;pha- 4(RNAi) embryos to test whether the impairment of the NMD-pathway could be responsible for the decrease in pha-4 mRNA in pha-4(q500) mutants. Surprisingly, while the amount of pha-4 mRNA did not differ, the amount of PHA-4 protein was indeed significantly reduced in pha-4(RNAi) compared to smg-8;pha-4(RNAi) embryos. These results contradict my previous findings and indicate that smg-8 affects PHA-4 protein directly, and does not affect the mRNA, contradicting my previous findings. Finally, I began experimenting with pha-4::gfp reporter constructs to identify the smg-8 sensitive parts of pha-4. I compared pha-4 mRNA levels in pha-4::gfp/pha-4(q500) (created by Hui-Ting Hsu, Mango lab) on control(RNAi) and smg-8(RNAi). Since, the pha-4::gfp transgene rescues the pha-4 phenotype, I did not expect any differences in GFP expression. Unexpectedly, my preliminary analysis showed a significant increase of PHA-4::GFP in smg-8(RNAi) embryos compared to control(RNAi) embryos. This would be the first time, a difference in the amount of PHA-4 protein was observed when comparing smg-8-deficient animals and animals expressing the fulllength pha-4 transcript. Taken together, my data suggest a complex interplay between smg-8 and pha-4. While some results are contradicting, there is mounting evidence that smg-8 is capable to act directly on pha-4 transcripts and thus leading to a reduction of PHA-4 protein. Future experiments will have to determine the molecular mechanism of their interaction and determine the role of smg-8 in embryogenesis.
