A second genetic screen for gurken mRNA mislocalisation reveals novel phenotypes of the piRNA pathway mutants in Drosophila [RNA-seq]
Transposon silencing is essential for germ line development. In Drosophila oogenesis, DNA damage caused by transposon activation affects microtubule-dependent mRNA localisation in the oocyte and impairs embryonic axes formation. Our previous EMS mutagenesis screen for gurken mRNA mislocalisaiton on Chromosome 3L of Drosophila melanogaster identified several piRNA pathway mutants (PMID: 24531791). Here we report the screen for Chromosome 3R. We identified 10 mutation groups disrupting gurken mRNA localisation and other mutations affecting different aspects of oogenesis. We found that mutations in karyopherin-b3 affect localisation and translation of gurken mRNA in a transposon silencing-independent manner. Characterisation of the new mutation in vreteno revealed that the piRNA pathway is essential for the basal stalk development, the process of holding the ovariole and encapsulating the first egg chambers. Females transheterozygous for vreteno and armitage mutations both showed abnormal basal stalks, defective egg chamber formation and loss of germ line cells. We also found that the mutation in the Zinc Finger motif of Spindle-E shows a hypomorphic transposon activation phenotype, consistent with the previous study (PMID: 25239103). Further characterisation showed that the Zinc Finger is required for robust ping-pong piRNA biogenesis and the nuage localisaiton of AGO3, but not of Aubergine, suggesting that it is involved in a specific step of ping-pong biogenesis. Overall design: The study characterises the phenotype of the mutations affecting gurken mRNA localisation in Drosophila oocytes. Of those, we focused on the piRNA pathway mutations and examined their impacts on transposon mRNA expression in the ovaries. We generated poly-A selected RNA sequencing libraries from freshly dissected ovaries and sequenced them by paired-end 150bp mode on Illumina Novaseq.