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First published online 12 September 2007
doi: 10.1242/jcs.009092

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RCC1 isoforms differ in their affinity for chromatin, molecular interactions and regulation by phosphorylation

Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK

View larger version (47K): [in this window] [in a new window]   Fig. 1. Three RCC1 transcript variants are expressed in humans. (A) Linear representation of RCC1 protein domains (not to scale), showing sequence alignment of the NTR (residues 1-27) of human RCC1 protein with insert-containing RCC1 isoforms from: human (hs); chimpanzee (pt); rhesus monkey (macm); golden hamster (ma); mouse (mm); and African clawed frog (xl). RCC1 homologues in species other than human are not shown. Insert sequences are shown in bold. Phosphorylation sites (serines 2 and 11) are indicated by circles labelled P. (B) Schematic showing how alternative mRNA splicing around and within exon 6' could generate the known RCC1 transcript variants. (C) Isoform-specific antibodies were generated to peptides corresponding to RCC1 sequences. Anti-R-INS should detect insert-containing isoforms, whereas the anti-R-DIS epitope should be disrupted by them. (D) Precipitation of RCC1 proteins from asynchronous HeLa cell extracts by GST-RanT24N or GST as a control, with analysis by immunoblotting with RCC1 isoform-specific antibodies. (E) Immunoblotting of human cultured cell lysates. (F) Immunoblotting of normal human tissue lysates on an Instablot (IMGENEX) membrane (Cambridge Bioscience), which is pre-loaded with SDS-PAGE-separated normal tissue lysates (20 µg protein/lane). The blot was probed using anti-R-INS (upper panel), then re-probed using anti-RCC1 (lower panel). RCC1 isoforms are indicated, with the question mark signifying a possible RCC1 band of unknown identity. Molecular mass (in kDa) is indicated on the left of the immunoblots.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Localisation of GFP-RCC1 and GFP-RCC1 in U2OS cells. (A) Representative deconvolved images of different cell cycle stages of live cells transiently co-transfected with GFP-RCC1 or GFP-RCC1 and RFP-histone H2B. Bars, 12 µm. (B) Proportion of live cells transiently transfected with each GFP-RCC1 isoform with each pattern of localisation (left), shown as mean percentages from three experiments ± s.d., with over 1200 cells for each parameter. Examples are shown (right). Bar, 20 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 3. RCC1 has a more stable interaction with chromatin in U2OS cells than RCC1. (A) FRAP of live interphase cells expressing GFP-RCC1 isoforms. Example images from one photobleach are shown, with the bleach spot indicated by an arrow. Mean half-times of recovery for each isoform in seconds are shown, ± s.d., calculated from 90 cells for GFP-RCC1 and 63 cells for GFP-RCC1 over three separate experiments. (B) Subcellular fractionation. Soluble nuclear markers are Ran, Crm1 and importin ; cytoplasmic marker is GAPDH; and insoluble nuclear marker is lamin B. (C) Localisation of GFP-GST-NTR (GG-NTR), GFP-GST-NTR (GG-NTR) and GFP-GST constructs in fixed cells. Values shown are from >22 mitotic cells for each construct counted over four experiments. Bar, 16.3 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 4. RCC1 interacts less well with importins than RCC1. (A) Endogenous importin precipitated from asynchronous HeLa extracts using beads coupled to GST-RCC1, GST-RCC1 or GST, with or without His6-importin 3 (Imp 3). (B) Endogenous RCC1 precipitated from asynchronous HeLa extracts using beads coupled to GST-importin (Imp ) or GST, with or without His6-importin 3. (C) Binding of untagged RCC1, RCC1 or GST to the chromatin pellet from subcellular fractionation of HeLa cells, with His6-importin 3 and GST-importin , or with GST as a control. (D) Endogenous RCC1 isoforms from asynchronous HeLa extracts bound by DNA-cellulose, with or without His6-importin 3 and GST-importin . Molecular mass (in kDa) is indicated on the left.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Phosphorylation of RCC1 isoforms. (A) Subcellular fractionation of mitotic (M) and asynchronous (A) HeLa cells. Soluble nuclear markers are Ran, Crm1 and importin ; cytoplasmic marker is GAPDH; insoluble nuclear marker is lamin B; and mitotic chromatin marker is phospho-S10 histone H3. (B) Alignment of the total RCC1 and pS11 RCC1 from SDS-PAGE and immunoblotting of fractionated chromatin pellet run in triplicate. (C) Phosphorylation of untagged RCC1 and RCC1 by p13Suc1 precipitates of CDKs from mitotic HeLa extract, or GS4B precipitates as a control. Molecular mass (in kDa) is indicated on the left.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Proliferation of tsBN2 cells expressing RCC1 isoforms. GFP-RCC1 and GFP-RCC1, and their respective S11A non-phosphorylatable mutants, were transiently expressed in tsBN2 cells. Following transfection, cells were split equally between dishes and kept at the permissive temperature (32°C) for 24 hours (day 0), then shifted to the restrictive temperature (39.7°C) for the time periods indicated. The number of GFP-positive cells (A) and DAPI-positive cells (B) is expressed as a percentage of the number of each present at day 0. Mean of four values for each data point ± s.d. is shown. About 60 GFP-positive cells and more than 150 DAPI-positive cells were counted for each data point at day 0. (C) Number of DAPI-positive cells expressed as a multiple of those initially transfected at day 0. In order to account for the few cells that survive at the restrictive temperature in the absence of transfected RCC1, the mean percentage of surviving DAPI-positive cells from an untransfected control were first subtracted from the mean percentage of total DAPI-positive cells in each transfected sample. The resultant value was then divided by the mean percentage of transfected cells determined at day 0.