Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes

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.In most eukaryotic cells the centrosome, composed of centrioles and associated pericentriolar material (PCM), acts as a major microtubule organizing center (MTOC) participating in the organization of both the interphase cytoskeleton and the mitotic spindle. In addition, the centriole component of the centrosome can function as a basal body that organizes the formation of a cilium while in many cases the associated PCM continues to operate as a cytoplasmic MTOC. This cilium can be one of two types, a motile cilium with a 9+2 arrangement of microtubules or non-motile (primary) cilia with 9+0 arrangement of microtubules (reviewed in [1]). Most vertebrate cells contain a single non-motile primary cilium that is assembled in