Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA.
Department of Genetics, Davidson Life Sciences Complex, University of Georgia, Athens, GA, USA.
RNA Biol. 2021 Dec;18(12):2376-2389. doi: 10.1080/15476286.2021.1914422. Epub 2021 Apr 27.
The Dynein motor is responsible for the localization of numerous mRNAs within oocytes and embryos. The RNA binding protein, Egalitarian (Egl), is thought to link these various RNA cargoes with Dynein. Although numerous studies have shown that Egl is able to specifically associate with these RNAs, the nature of these interactions has remained elusive. Egl contains a central RNA binding domain that shares limited homology with an exonuclease, yet Egl binds to RNA without degrading it. Mutations have been identified within Egl that disrupt its association with its protein interaction partners, BicaudalD (BicD) and Dynein light chain (Dlc), but no mutants have been described that are specifically defective for RNA binding. In this report, we identified a series of positively charged residues within Egl that are required for RNA binding. Using corresponding RNA binding mutants, we demonstrate that specific RNA cargoes are more reliant on maximal Egl RNA biding activity for their correct localization in comparison to others. We also demonstrate that specification and maintenance of oocyte fate requires maximal Egl RNA binding activity. Even a subtle reduction in Egl's RNA binding activity completely disrupts this process. Our results show that efficient RNA localization at the earliest stages of oogenesis is required for specification of the oocyte and restriction of meiosis to a single cell.
动力蛋白负责将许多 mRNA 定位在卵母细胞和胚胎中。RNA 结合蛋白平等主义者(Egl)被认为将这些不同的 RNA 货物与动力蛋白联系起来。尽管许多研究表明 Egl 能够特异性地与这些 RNA 结合,但这些相互作用的性质仍然难以捉摸。Egl 包含一个中央 RNA 结合结构域,与外切酶具有有限的同源性,但 Egl 结合 RNA 而不降解它。已经在 Egl 中鉴定出突变,这些突变破坏了它与其蛋白相互作用伙伴 BicaudalD (BicD) 和动力蛋白轻链 (Dlc) 的结合,但尚未描述专门针对 RNA 结合缺陷的突变体。在本报告中,我们确定了 Egl 中一系列需要 RNA 结合的正电荷残基。使用相应的 RNA 结合突变体,我们证明了特定的 RNA 货物更依赖于 Egl RNA 结合活性的最大程度来正确定位,而其他 RNA 货物则不然。我们还证明了卵母细胞命运的指定和维持需要 Egl RNA 结合活性的最大化。即使 Egl 的 RNA 结合活性稍有降低,也会完全破坏这个过程。我们的结果表明,卵母细胞指定和减数分裂限制为单个细胞所需的卵子发生早期阶段的有效 RNA 定位。