Patel Prajal H, Barbee Scott A, Blankenship J Todd
Department of Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, Colorado, United States of America.
Molecular and Cellular Biophysics Program, University of Denver, Denver, Colorado, United States of America.
PLoS One. 2016 Mar 1;11(3):e0150291. doi: 10.1371/journal.pone.0150291. eCollection 2016.
Non-translating RNAs that have undergone active translational repression are culled from the cytoplasm into P-bodies for decapping-dependent decay or for sequestration. Organisms that use microRNA-mediated RNA silencing have an additional pathway to remove RNAs from active translation. Consequently, proteins that govern microRNA-mediated silencing, such as GW182/Gw and AGO1, are often associated with the P-bodies of higher eukaryotic organisms. Due to the presence of Gw, these structures have been referred to as GW-bodies. However, several reports have indicated that GW-bodies have different dynamics to P-bodies. Here, we use live imaging to examine GW-body and P-body dynamics in the early Drosophila melanogaster embryo. While P-bodies are present throughout early embryonic development, cytoplasmic GW-bodies only form in significant numbers at the midblastula transition. Unlike P-bodies, which are predominantly cytoplasmic, GW-bodies are present in both nuclei and the cytoplasm. RNA decapping factors such as DCP1, Me31B, and Hpat are not associated with GW-bodies, indicating that P-bodies and GW-bodies are distinct structures. Furthermore, known Gw interactors such as AGO1 and the CCR4-NOT deadenylation complex, which have been shown to be important for Gw function, are also not present in GW-bodies. Use of translational inhibitors puromycin and cycloheximide, which respectively increase or decrease cellular pools of non-translating RNAs, alter GW-body size, underscoring that GW-bodies are composed of non-translating RNAs. Taken together, these data indicate that active translational silencing most likely does not occur in GW-bodies. Instead GW-bodies most likely function as repositories for translationally silenced RNAs. Finally, inhibition of zygotic gene transcription is unable to block the formation of either P-bodies or GW-bodies in the early embryo, suggesting that these structures are composed of maternal RNAs.
经历了主动翻译抑制的非翻译RNA会从细胞质中被挑选出来，进入P小体进行脱帽依赖性降解或隔离。利用微小RNA介导的RNA沉默的生物体还有另一条从活跃翻译中去除RNA的途径。因此，控制微小RNA介导沉默的蛋白质，如GW182/Gw和AGO1，通常与高等真核生物的P小体相关联。由于存在Gw，这些结构被称为GW小体。然而，有几份报告表明GW小体与P小体具有不同的动态变化。在这里，我们使用实时成像来研究早期果蝇胚胎中GW小体和P小体的动态变化。虽然P小体在整个早期胚胎发育过程中都存在，但细胞质GW小体仅在囊胚中期转变时大量形成。与主要位于细胞质中的P小体不同，GW小体存在于细胞核和细胞质中。RNA脱帽因子如DCP1、Me31B和Hpat与GW小体不相关，这表明P小体和GW小体是不同的结构。此外，已知的Gw相互作用蛋白，如AGO1和CCR4-NOT去腺苷酸化复合体，已被证明对Gw功能很重要，它们也不存在于GW小体中。使用翻译抑制剂嘌呤霉素和环己酰亚胺，它们分别增加或减少非翻译RNA的细胞池，会改变GW小体的大小，这突出表明GW小体由非翻译RNA组成。综上所述，这些数据表明活跃的翻译沉默很可能不在GW小体中发生。相反，GW小体很可能作为翻译沉默RNA的储存库发挥作用。最后，抑制合子基因转录无法阻止早期胚胎中P小体或GW小体的形成，这表明这些结构由母体RNA组成。
