XDazl function in RNA metabolism in Xenopus laevis
von Juliane Pfennig
Datum der mündl. Prüfung:2014-11-26
Betreuer:Prof. Dr. Tomas Pieler
Gutachter:Prof. Dr. Tomas Pieler
Gutachter:Prof. Dr. Michael Kessel
EnglischGerm cell specification in Xenopus laevis depends on the inheritance of maternal determinants from the oocyte in form of mRNAs and proteins. At the onset of zygotic transcription, maternal mRNAs are subjected to miR-mediated decay. Interestingly, germ cell specific mRNAs are excluded from miR-induced degradation. Recent studies have provided insights into how RNA-binding proteins can modulate miR-mediated regulation of RNA stability. In Xenopus embryos for example, ElrB1 and Dead end (XDE) proteins have been demonstrated to protect germ cell specific mRNAs from miR-mediated clearance. Recognition of these mRNAs by miRs relies on target sites within the localization element (LE), a specific region in the 3’ UTR, which is also required for vegetal mRNA transport in the oocyte (Koebernick et al. 2010). In this study, we addressed the role of Deleted in azoospermia-like (XDazl) in the context of germline specific transcript stabilization in Xenopus embryos. The RNA binding protein XDazl is provided maternally and its zygotic transcription starts at stage 17. Ectopic expression of XDazl promotes stabilization of the PGC-specific XDE mRNA as well as that of other germline transcripts. In vitro RNA binding assays indicate direct interaction of XDazl with these transcripts. Moreover, a synergistic effect of XDE and XDazl protein in stabilizing germ cell specific mRNAs was observed. Mapping of the XDazl binding site within the XDE-LE indicated overlapping binding sites with XDE and ElrB1. Degradation of germ cell specific mRNAs is mediated by miRs, since blocking of miR processing leads to enrichment of germline transcripts. XDazl protein binds directly to the LE in its own mRNA, which contains at least three independent XDazl binding sites. The knockdown of XDazl in embryos leads to a reduced number of germ cells at tailbud stage, indicating an important role of XDazl during germ cell formation or maintenance. Immunofluorescence staining of oocytes shows XDazl protein enrichment at the vegetal cortex and in transport particles colocalizing with XDE and XDazl mRNAs, suggesting an early involvement of XDazl in the context of vegetal mRNA transport and/or anchoring.
Keywords: germ cells; Dazl; Xenopus