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Published on: June 4, 2019
Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis
Andrew Kenny1, Miles B Morgan1, Sabine Mohr1
1Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.
Abstract:
The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center (MTOC), integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. Here, we use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, we found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the MTOC, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event.
Insights
The oskar mRNA
Area of Science:
- Developmental Biology
- RNA Biology
- Genetics
Background:
- The oskar mRNA plays a crucial role in Drosophila oogenesis, influencing multiple developmental pathways.
- The precise mechanisms by which oskar mRNA executes its diverse functions remain largely uncharacterized.
- Previous attempts to study oskar mRNA function using RNA interference were hampered by ineffective targeting strategies.
Purpose of the Study:
- To investigate the critical temporal requirements of oskar mRNA for specific developmental processes during oogenesis.
- To establish effective RNA interference methods for studying oskar mRNA function in Drosophila.
- To elucidate the noncoding functions of oskar mRNA in karyosome formation, MTOC positioning, and oogenesis progression.
Main Methods:
- Development of novel transgenic short hairpin RNA (shRNA) constructs targeting oskar mRNA.
- Utilized GAL4-UAS system with stage-specific drivers to control oskar mRNA knockdown during different oogenesis phases.
- Phenotypic analysis of oskar mRNA loss-of-function mutants, focusing on karyosome formation, MTOC positioning, and oocyte development.
Main Results:
- Novel shRNAs effectively reduced oskar mRNA levels, recapitulating phenotypes associated with its absence.
- Early oskar mRNA depletion resulted in defects in karyosome formation and microtubule organizing center (MTOC) positioning.
- Later oskar mRNA depletion was essential for preventing premature oogenesis progression, indicating stage-specific requirements.
Conclusions:
- The noncoding function of oskar mRNA is essential for multiple, distinct events throughout Drosophila oogenesis.
- Oskar mRNA's roles are temporally regulated, with different functions being critical at specific developmental stages.
- Effective RNA interference tools are now available for detailed functional analysis of oskar mRNA in Drosophila development.
