Fig 6. Cyto-CLIP for HNRNPU identifies a direct interaction between HNRNPU and the 3'-UTR of IL-6 mRNA.

(A) The subcellular location of HNRNPU in cells treated with P/I or DMSO for 4 hours was determined by immunofluorescence using the anti-HNRNPU antibody. The nucleus and cytoplasm were visualized with Hoechst and anti-GAPDH staining, respectively. (B) Western blotting of whole lysate (whole) and the cytoplasmic fraction (cyto) with Lamin B1 as a nuclear marker and Actin as a cytoplasmic marker. (C–D) Autoradiogram of the nitrocellulose membrane of labeled ³²P-labeled RNA crosslinked to immunoprecipitation purified HNRNPU from cyto or whole crosslinked lysate treated with low (1:100000) or high (1:100) RNase A using an anti-HNRNPU-specific antibody (C) and detection of isolated HNRNPU by Western blotting on same membrane (D). (E) HNRNPU binds to the 3’UTR of IL-6 mRNA in cytoplasmic fraction. The distribution of HNRNPU whole-CLIP and cyto-CLIP unique tags at the IL-6 locus was visualized with the UCSC Genome Browser. CLIP-tag peaks are shown for whole-CLIP, cyto-CLIP replicate #1, and cyto-CLIP replicate #2. Magnified view of IL-6 3’UTR locus, highlighted in yellow (Top panel), including the HNRNPU binding and constitutive decay element (CDE) underscored in green (Middle panel). 3’-untranslated region (3’-UTR) sequences of human IL-6 mRNA are shown with marks for HNRNPU cyto-CLIP, AU-rich element (ARE) and CDE. (Bottom panel) (F–G) The genomic distribution of HNRNPU whole-CLIP (F) and cyto-CLIP (G) unique tag cluster peaks are shown.