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First published online 1 September 2005
doi: 10.1242/jcs.02545

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RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

¹ Istituto di Biologia e Patologia Molecolari CNR, Universita' di Roma `La Sapienza', P. le A. Moro, 5, 00185 Roma, Italy
<sup>2</sup> Laboratory `B', Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy
³ Department of Experimental Medicine, Via Vetoio, Coppito 2, University of L'Aquila, 67100 L'Aquila, Italy
⁴ Department of Biotechnology and Biosciences, University of Milano Bicocca, Piazza della Scienza 2, 20146 Milano, Italy
⁵ AIRC-Roman Oncogenomic Center-(ROC), Via delle Messi d'Oro 156, 00158 Rome, Italy

View larger version (47K): [in a new window]   Fig. 1. RPB3 subcellular localization. (A) Endogenous RPB3 in cycling (GM) and differentiated (DM) myogenic C2C7 cells. (B) Endogenous RPB3 in HeLa cells. (C) Transfected Myc-RPB3 in cycling (GM) and differentiated (DM) myogenic C2C7 cells. (D) Endogenous RPB11 in cycling (GM) and differentiated (DM) myogenic C2C7 cells. Indirect immunofluorescence (A-D) was performed with the indicated antibodies. Hoechst-stained nuclei relative to the same fields are shown. (E) Western-blot analysis of subcellular cytoplasmic and nuclear fractions derived from GM and DM myogenic C2C7 cells. The blot was incubated with anti-RPB3 antibody to determine nuclear/cytoplasmic localization. To verify fraction quality, the same extracts were blotted and incubated with anti-Sp1 antibody.

View larger version (27K): [in a new window]   Fig. 2. RPB3 interacts with HCR. (A) AH109 yeast cells were co-transformed with the indicated constructs and plated onto SD medium lacking leucine and tryptophan (-LW) to verify the expression of both bait (W+) and prey (L+) plasmids, or onto medium lacking leucine, tryptophan, histidine and adenine (-LWHA) to examine the interaction between bait and prey proteins. (B) Y187 yeast cells were co-transformed with the indicated constructs and assayed for ß-galactosidase activity. (C) In-vitro-translated and S35-labelled HCR was subjected to GST pull-down analysis using GST or GST-RPB3 beads. (D) Whole-cell extracts of NIH 3T3 cells transfected with both Flag-HCR and Myc-RPB3, or with Flag-HCR and empty Myc-tag vectors (Myc-Vector) were immunoprecipitated with anti-Flag monoclonal antibody and analysed by western blot using an anti-Myc monoclonal antibody.

View larger version (55K): [in a new window]   Fig. 3. HCR expression and localization in C2C7 cells. (A) Total lysates from C2C7 myogenic cells incubated in differentiation medium (DM) for the indicated times or maintained in growth medium (GM) were analysed by western blot using the indicated antibodies. -Tubulin was used to normalize the amount of protein extracts loaded in each lane. (B, top) Indirect immunofluorescence of HCR performed using purified anti-HCR rabbit polyclonal antibody in cycling (GM) and differentiated (DM) myogenic C2C7 cells. (B, bottom) Hoechst-stained nuclei of the same fields as in B, top. (C) Colocalization of overexpressed EGFP-RPB3 and Flag-HCR proteins. (left) Fluorescence of EGFP-RPB3 fusion protein; (middle) indirect immunofluorescence using anti-Flag monoclonal antibody; (right) merge of EGFP-RPB3 and Flag-HCR fluorescence signals. (D) Colocalization of endogenous RPB3 and HCR proteins. (Green) Indirect immunofluorescence using rabbit polyclonal antibody against RPB3; (red) indirect immunofluorescence using mouse polyclonal antibody against HCR; (yellow) merge of RPB3 and HCR indirect immunofluorescence signals; (blue) Hoechst-stained nuclei relative to the same field.

View larger version (39K): [in a new window]   Fig. 4. The -like-1 RPB3 domain is involved in both cytoplasmic localization and HCR contact. (A) EGFP-RPB3 full-length and relative nested deletion constructs. (B) Subcellular localization of EGFP-RPB3 and indicated deletion construct, overexpressed in cycling (GM) myogenic C2C7 cells. (C) Whole-cell extracts of NIH 3T3 mouse fibroblasts transfected with both Flag-HCR and EGFP-RPB3 deletion constructs were immunoprecipitated with anti-Flag monoclonal antibody and analysed by western blot using anti-EGFP monoclonal antibody. The asterisks indicate a heavy-chain Ig band (top) and a non-specific band (bottom); both partially cover the signal corresponding to the RPB3-a deletion mutant (lane 3).

View larger version (33K): [in a new window]   Fig. 5. HCR RNAi affects EGFP-RPB3 subcellular localization. (A) Western blot analysis of cycling (GM) myogenic C2C7 cells transfected with pSuper RNAi empty vector and with increasing DNA amounts of pSuper-HCR interference vector. (B) Fluorescence of EGFP-RPB3 fusion protein co-expressed in cycling (GM) myogenic C2C7 cells, either with empty pSuper vector (top) or with pSuper-HCR interference vector (bottom). All nuclei are Hoechst stained.