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.

Figure 1

RNA silencing proteins, GW182 and hAgo2, localize to the centrosome/basal body in human astrocytes throughout interphase. (A) Astrocytes double labeled with mouse anti-acetylated tubulin (red) and human GW182 antiserum 18033 (green) and counterstained with DAPI (blue). Left panel illustrates two daughter cells. Cells were seeded at a low density to allow the identification of daughter cells following the completion of mitosis (early G1). Each cell contains a centrosome with associated GW/P bodies (arrows). Centre panel illustrates a cell in G1/S phase (the period of primary cilium expression). One of the two GW/P bodies is demonstrated in this focal plane at the basal body (arrow). Right panel illustrates a late S/G2 cell with prominent chromatin condensation (DAPI) and a centrosome with two GW/P bodies (arrow). (B) GW182 and (C) hAgo2 localization to the centrosome and the basal body of primary cilia was examined by IIF using 18033 and mouse monoclonal 4F9 to recombinant hAgo2 [29] (green). Nuclei were marked by DAPI. The inset box in (B) shows the location of GW182 marked by mouse monoclonal 4B6 antibody and (C) hAgo2 mouse monoclonal antibody relative to the primary cilia. All IIF scale bars = 15 μm. (D) GW182 localizes to the pericentriolar region in immunoelectron micrographs. Arrows indicate the immunogold stained GW/P body foci. EM scale bars = 100 nm.

Figure 2

SiRNA knockdown of RNAi components, GW182, hAgo2, Drosha and DGCR8, inhibits ciliogenesis in human astrocytes. (A) The primary cilium was examined by IIF using antibodies to glu tubulin (white) in astrocytes transfected with no siRNA/Lipofectamine, scrambled siRNA and si-GW182 and double stained for GW bodies using mouse monoclonal 4B6 antibody to GW182 (green). The primary cilium as marked by glu-tubulin (white) was also examined in astrocytes transfected with si-hAgo2 and double stained with mouse monoclonal 4F9 antibody to hAgo2 (green). Nuclei were stained by DAPI. The merge + DAPI images were enlarged to focus on the cilium and centrioles relative to GW182 and hAgo2 staining. Scale bar = 15 μm. (B) The percentage of astrocytes containing primary cilia were quantitated from 5 independent IIF experiments containing > 250 cells described in (A). Bars represent the standard deviation of the mean. (C) Ultrastructural analysis of control astrocytes, scale bar = 400 nm, si-GW182, si-hAgo2, si-Drosha and si-DGCR8 treated astrocytes scale bars = 500 nm. (D) Human astrocytes were transfected for 30 hours with 100 nM siRNA and Lipofectamine 2000 (Invitrogen), lysed and processed for western blot analysis. The cells in the no siRNA/Lipofectamine lane were treated with Lipofectamine 2000 only (no siRNA) and the control lane represented untreated cells. (E) Cell cycle analysis showing the percentage of human astrocyte cells in G1, S and G2 phase during normal growth conditions (control) and under 100 nM siRNA transfected conditions.