Moser Joanna J, Fritzler Marvin J, Rattner Jerome B
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Alberta, Canada.
BMC Cell Biol. 2011 Aug 31;12:37. doi: 10.1186/1471-2121-12-37.
In most cells, the centriolar component of the centrosome can function as a basal body supporting the formation of a primary cilium, a non-motile sensory organelle that monitors information from the extracellular matrix and relays stimuli into the cell via associated signaling pathways. Defects in the formation and function of primary cilia underlie multiple human diseases and are hallmarks of malignancy. The RNA silencing pathway is involved in the post-transcriptional silencing of > 50% of mRNA that occurs within GW/P bodies. GW/P bodies are found throughout the cytoplasm and previously published live cell imaging data suggested that in a malignant cell type (U2OS), two GW/P bodies reside at the centrosome during interphase. This led us to investigate if a similar relationship exists in primary cells and if the inhibition of the miRNA pathway impairs primary cilium formation.
Two GW/P bodies as marked by GW182 and hAgo2 colocalized to the basal body of primary human astrocytes as well as human synoviocytes during interphase and specifically with the distal end of the basal body in the pericentriolar region. Since it is technically challenging to examine the two centrosomal GW/P bodies in isolation, we investigated the potential relationship between the global population of GW/P bodies and primary ciliogenesis. Astrocytes were transfected with siRNA directed to GW182 and hAgo2 and unlike control astrocytes, a primary cilium was no longer associated with the centrosome as detected in indirect immunofluorescence assays. Ultrastructural analysis of siRNA transfected astrocytes revealed that knock down of GW182, hAgo2, Drosha and DGCR8 mRNA did not affect the appearance of the earliest stage of ciliogenesis but did prevent the formation and elongation of the ciliary axoneme.
This study confirms and extends a previously published report that GW/P bodies reside at the centrosome in U2OS cells and documents that GW/P bodies are resident at the centrosome in diverse non-malignant cells. Further, our study demonstrates that repression of key effector proteins in the post-transcriptional miRNA pathway impairs primary cilium formation.
在大多数细胞中，中心体的中心粒成分可作为基体，支持初级纤毛的形成。初级纤毛是一种非运动性的感觉细胞器，可监测来自细胞外基质的信息，并通过相关信号通路将刺激传递到细胞内。初级纤毛形成和功能的缺陷是多种人类疾病的基础，也是恶性肿瘤的标志。RNA沉默途径参与了GW/P小体中超过50%的mRNA的转录后沉默。GW/P小体遍布细胞质，先前发表的活细胞成像数据表明，在一种恶性细胞类型（U2OS）中，两个GW/P小体在间期位于中心体。这促使我们研究在原代细胞中是否存在类似的关系，以及miRNA途径的抑制是否会损害初级纤毛的形成。
以GW182和hAgo2标记的两个GW/P小体在间期与原代人星形胶质细胞以及人滑膜细胞的基体共定位，并且特别与中心粒周围区域基体的远端共定位。由于单独检查两个中心体GW/P小体在技术上具有挑战性，我们研究了GW/P小体的整体群体与初级纤毛发生之间的潜在关系。用针对GW182和hAgo2的siRNA转染星形胶质细胞，与对照星形胶质细胞不同，在间接免疫荧光试验中检测到初级纤毛不再与中心体相关。对siRNA转染的星形胶质细胞的超微结构分析表明，敲低GW182、hAgo2、Drosha和DGCR8 mRNA不影响纤毛发生最早阶段的外观，但确实阻止了纤毛轴丝 的形成和延长。
本研究证实并扩展了先前发表的一份报告，即GW/P小体存在于U2OS细胞的中心体中，并记录了GW/P小体存在于多种非恶性细胞的中心体中。此外，我们的研究表明，转录后miRNA途径中关键效应蛋白的抑制会损害初级纤毛的形成。
