Formation of GW bodies is a consequence of microRNA genesis

Abstract

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.

Figure 1

MicroRNA is present in GW bodies. (A) Transfected let-7 microRNA (miRNA) 3′-labelled with Cy3 was localized to GW bodies (GWBs) in HeLa cells 24 h after transfection. HeLa cells were transiently transfected with Cy3-3′-let-7 after which they were fixed and stained with human anti-GWB serum. Analysis showed that the miRNA (i, arrows) was found within GWBs (ii, arrows). Nuclei were counterstained by 4,6-diamidino-2-phenylindole (DAPI; iii). Note that miRNA-transfected cells often show large cytoplasmic aggregates (i, arrowheads) distinct from GWBs (ii, arrows). The merged images are seen in (iv). Scale bar (i), 10 μm. (B) Biochemical detection of let-7 RNA using an RNase protection assay (upper) and GW182 protein by western blot analysis (lower, lane 2). Lanes 1 and 3 are control immunoprecipitates (IP) with normal human serum (NHS), and total RNA and protein from HeLa cell extracts, respectively.

Figure 2

Loss of GW body staining in Drosha-deficient HeLa cells and transfection of synthetic short interference RNAs rescues GW bodies. HeLa cells transfected with pDrosha-sh plasmid were allowed to grow in the presence of 2 μg/ml puromycin to select for cells that were later transfected with lamin A/C short interference RNA (siRNA). (A) Untreated HeLa cells, (B) Drosha-deficient cells selected for 14–21 days, and (C) these cells 48 h after transfection with siRNA were co-stained with GW182 antibody and Alexa 488-goat anti-human IgG (green, i,iv,vii), and lamin A/C monoclonal antibody and Alexa 350-goat anti-mouse IgG (blue, ii,v,viii). GW bodies (GWBs; arrows) were readily detected in untreated HeLa cells (i). Most of the GWB staining was lost in the Drosha-deficient cells, but dispersed residual GWB staining was still apparent (arrows, iv). Many GWBs were observed in Drosha-deficient cells after transfection with siRNA (arrows, vii). The knockdown of lamin A/C in Drosha-deficient cells transfected with lamin A/C siRNA was effective (viii), with only few cells having detectable lamin A/C (arrowhead). (D) Cy3-labelled siRNA for lamin A/C localized to GWBs in transfected Drosha-deficient cells. Most of the Cy3-siRNA seemed to be aggregated in the cytoplasm but some was detected in GWBs (arrows). Efficient lamin A/C knockdown was observed and was indicated by the absence of lamin A/C staining in the nucleus (dashed circle). Insets are enlarged 1.5-fold and the Cy3 signal is enhanced to show the localization of siRNA to GWBs. Scale bars, 10 μm.

Figure 3

Characterization of Drosha-deficient cells and demonstration of reduced microRNA levels. (A) Reduction of messenger RNA levels of Drosha demonstrated by using reverse transcription–PCR (RT–PCR) to compare untreated HeLa cells and pDrosha-sh-transfected HeLa cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analysis was included for comparison. (B) Reduction of mature microRNA (miRNA) levels demonstrated by using RT–PCR to compare untreated HeLa cells and pDrosha-sh-transfected cells. 5S RNA analysis was included for comparison. (C) Western blot analysis showing a reduction of Drosha protein level in pDrosha-sh-transfected cells (data also shown from duplicate experiment) compared with untreated HeLa cells. GW182, Argonaute 2 (Ago2), Rck/p54 and actin levels are shown for comparison.

Figure 4

Disassembly of GW bodies in HeLa cells resulting from DGCR8 knockdown and reassembly after short interference RNA transfection. A short hairpin RNA plasmid targeting DGCR8 (pDGCR8-sh) was transiently transfected with the green fluorescent protein (GFP) expression plasmid (phrGFP) at a 5:1 molar ratio. After 72 h, transfected cells identified by the coexpression of GFP showed almost complete absence of GW body (GWB) staining, although a few weakly stained GWBs were detectable (arrows, vi). By contrast, GWBs were detected in cells transfected with phrGFP alone (arrows, iii). Transfection of short interference RNA (siRNA) for lamin A/C in DGCR8 knockdown cells resulted in a reappearance of GWBs (arrows, ix) and a reduction in lamin A/C protein in these cells 48 h after siRNA transfection. Scale bar, 10 μm.

Figure 5

GW bodies are foci specific to microRNA processing. In this schematic diagram, primary microRNA (pri-miRNA) nuclear transcripts are cleaved by the Drosha–DGCR8 complex into hairpin pre-miRNA, which is exported to the cytoplasm by exportin 5. In the cytoplasm, Dicer is responsible for cleavage of pre-miRNA, generating the mature miRNA. On correct interaction and partnering of the miRNA to messenger RNA containing the miRNA-targeted sequence element ‘X' in the presence of Ago2/RISC (RNA-induced silencing complex), cytoplasmic foci are formed as a result of association of other proteins (see text). However, in the absence or low levels of miRNA, as a result of Drosha or DGCR8 protein knockdown, these foci do not form. The transfection of short interference RNA (siRNA) as a surrogate for miRNA leads to the apparent reassembly of GW bodies (GWBs). Ago2, Argonaute 2.