Localization of endogenous and exogenous SHQ1. (A,B) Indirect double immunofluorescence of endogenous SHQ1 (panel 1) and nucleolin and coilin (A,B, respectively, panel 2), and corresponding phase contrast and differential interference contrast images (panel 3). Due to the small size of the coilin labeled Cajal bodies, the images in B were acquired in 22 consecutive optical planes of 0.2 μm step size and deconvolved using a Huygens algorithm. Panels 1 and 2 show maximum projections highlighting each Cajal body (squares), which are depicted enlarged in their different individual 0.2 μm planes below, demonstrating that SHQ1 is not enriched (II) and excluded from Cajal bodies (I and III). (C) Overexpressed GFP tagged SHQ1 accumulates in the cytoplasm. (D) Partial shuttling of GFP-SHQ1. Cells cotransfected with GFP-SHQ1 and RFP-NAF1 were treated for 3 h with the CRM1-dependent protein export inhibitor Leptomycin B (LMB), which caused all of RFP-NAF1 and about half of GFP-SHQ1 to accumulate in nuclei, indicating that both proteins shuttle SHQ1, at least to some extent, between nucleus and cytoplasm. Note that even when concentrated in the nucleoplasm both proteins are still excluded from nucleoli, the sites of mature H/ACA RNPs. (E) GFP-SHQ1 is not recruited to the transcription site of an H/ACA RNA. GFP-SHQ1 (panel 2) transfected into our cell line that harbors an array of an H/ACA RNA whose induction is detected by in situ hybridization using a fluorescent probe complementary to an intron of the pre-mRNA containing the H/ACA RNA (panel 1) (Darzacq et al. 2006) is not recruited to the site of transcription (arrow). Bars, 10 μm. Width of enlarged panels, 3.2 μm (B) and 2.6 μm (E).
