Novel functions of Drosophila melanogaster Argonaute2 for miRNA mediated silencing and early embryonic development

Gene silencing mediated by small RNAs is a mechanism of post-transcriptional gene regulation. It is common for many eukaryotic cells. Argonaute proteins are essential components of RNA mediated silencing pathways. Unlike in other model organisms, in Drosophila different Argonaute proteins have been assigned to different RNA silencing pathways. It was shown that Argonaute1 (Ago1) is essential for microRNA (miRNA) mediated silencing while Argonaute2 (Ago2) is required for silencing triggered by small interfering RNAs (siRNAs). This thesis investigated a mutant allele of ago2, the female sterile mutation ago2dop. ago2dop mutations cause developmental defects in cellularisation and microtubule-based transport in early embryos. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. This thesis demonstrates that the GRRs of Ago2 are essential for the normal function of the protein. The ago2dop alleles show an altered number of GRRs. The main question was to find out how Ago2 as the key enzyme of the siRNA mediated silencing pathway is involved in early embryogenesis. This thesis provides evidence that the phenotypes of ago2dop mutant embryos are not a consequence of siRNA mediated silencing. ago2dop mutants are still able to respond to siRNA triggers. Therefore Ago2 must have at least one other important function and might act in another regulatory pathways. This thesis identified a new isoform of Ago2 that lacks the GRRs, which was named Ago2short. The genetics of the ago2dop alleles suggests that mutant Ago2dop protein might negatively interfere with the function of Ago2short. Ago2dop1 also interacts genetically with all other members of the Argonaute protein family. ago2dop1 homozygous embryos that are reduced in ago1, piwi or aubergine (aub) activity show an enhancement of the cellularisation phenotype in early embryos. I propose that the developmental defects are caused by an inability to degrade maternal transcripts at mid-blastula transition (MBT). The MBT is marked by switch from solely maternal to zygotic control of embryogenesis. Downregulation of maternal genes is required at this stage of development. Maternal transcripts might be degraded through a miRNA mediated silencing pathway. In this thesis evidence is presented that miRNA miR-9a might be one possible regulator of maternal transcript degradation. Furthermore genetic analyses demonstrate that Argonaute proteins act in a partial redundant fashion in early development. Ago1 and Ago2 have overlapping functions in the establishment of segment polarity. In protein extracts, Ago1 and Ago2 can be coprecipitated suggesting that they are present in the same protein complex. Genetic analysis revealed that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Taken together, this work demonstrates novel functions for Ago2 and argues strongly against a strict separation of Ago1 and Ago2 functions. Furthermore, the data suggest that different Argonaute proteins might act together to control key steps of embryonic development.