title: Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Alberta, Canada.
authors: Joanna J
authors: JJ
authors: Moser
authors: Marvin J
authors: MJ
authors: Fritzler
authors: Jerome B
authors: JB
authors: Rattner
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D001253:Astrocytes / Q000648:ultrastructure; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D054468:Axoneme / Q000235:genetics* / Q000648:ultrastructure; D002478:Cells, Cultured; D002502:Centrioles / Q000378:metabolism / Q000648:ultrastructure*; D002923:Cilia / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008938:Mitosis / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; C474043:DGCR8 protein, human; D011506:Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C465663:TNRC6A protein, human; C517918:DROSHA protein, human; D043244:Ribonuclease III