Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.
G3 (Bethesda). 2021 Dec 8;11(12). doi: 10.1093/g3journal/jkab340.
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.
Oskar 转录本作为一种非编码 RNA，有助于果蝇卵巢中的一系列途径，包括核仁体形成、微管组织中心 (MTOC) 的定位、某些核糖核蛋白颗粒的完整性、滋养细胞分裂的控制、几种蛋白质限制在生殖系中，以及通过卵母细胞发生进展。 Oskar mRNA 如何发挥作用以执行这些功能尚不清楚。在这里，我们使用敲低方法来确定 Oskar 在这三个功能中所需的关键阶段。现有的用于去除生殖系中 Oskar mRNA 的转基因 shRNA 靶向与 Bruno1 蛋白结合的调节位点重叠的序列，以赋予翻译抑制，并在卵母细胞发生过程中无效。针对其他位点的新型转基因 shRNA 能够有效降低 Oskar mRNA 水平，并重现与 mRNA 缺失相关的表型。使用在卵母细胞发生的不同发育阶段具有活性的 GAL4 驱动子，我们发现早期缺失 Oskar mRNA 会导致核仁体形成和 MTOC 定位缺陷，但不会导致卵母细胞发生阻滞。在后期缺失 Oskar mRNA 是防止卵母细胞发生进展所必需的。因此， Oskar mRNA 的非编码功能需要不止一个事件。
