The small GTPase Ran is highly conserved in eukaryotic cells
and has roles in the control of nucleocytoplasmic transport
(Melchior et al., 1993; Moore and Blobel, 1993), mitotic
spindle assembly (Carazo-Salas et al., 1999; Kalab et al., 1999;
Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999)
and nuclear envelope assembly (Hetzer et al., 2000; Zhang and
Clarke, 2000). Proper spatial coordination of these processes
relies upon the distribution of GTP- versus GDP-bound forms
of Ran, which is in turn controlled by the localisation of its
regulators (Clarke and Zhang, 2001; Dasso, 2002; Görlich and
Mattaj, 1996; Hetzer et al., 2002). The only known guanine
nucleotide exchange factor (GEF) for Ran, RCC1, is localised
to chromatin throughout the cell division cycle (Bischoff and
Ponstingl, 1991; Moore et al., 2002; Ohtsubo et al., 1989). By
contrast, RanGAP1, the sole GTPase-activating factor that
stimulates GTP hydrolysis by Ran (Bischoff et al., 1994), is
localised to cytoplasm in interphase, in particular the
cytoplasmic face of nuclear pore complexes, and to the mitotic
spindle and kinetochores in mitosis (Joseph et al., 2002;
Mahajan et al., 1997; Matunis et al., 1996). Together with the
concentration of Ran in the nucleus by its active import, the
distinct localisation of its regulators results in a high
concentration of Ran-GTP in the nucleoplasm and a low
concentration of Ran-GDP in the cytoplasm, whereas in
mitosis, Ran-GTP is concentrated around chromosomes
(Caudron et al., 2005; Kalab et al., 2006; Kalab et al., 2002).
Nucleocytoplasmic transport is carried out by the importin
? family of transport proteins, or karyopherins, which carry
cargo across the nuclear envelope via nuclear pores, with
directionality conferred by the high concentration of Ran-GTP
in the nucleoplasm (Görlich and Mattaj, 1996). As import
cargo complexes enter the nucleus through nuclear pore
complexes, Ran-GTP binding to importin ?
disassembly of importin-cargo complexes, releasing cargo into
the nucleoplasm (Stewart, 2007). Conversely, Ran-GTP is an
essential component of export complexes involving the export
receptor, Crm1 (Fornerod et al., 1997). These export
complexes disassemble on entry into the cytoplasm as
RanGAP1 catalyses hydrolysis of GTP by Ran, converting it
into Ran-GDP (Bischoff et al., 1994).
During mitosis, when the nuclear envelope breaks down in
animal cells, similar mechanisms are involved in the
orchestration of spindle assembly by Ran. Ran-GTP generated
around mitotic chromosomes binds to importin ? and causes
the release of spindle assembly factors such as TPX2 from
inhibitory complexes with importins, thereby promoting
microtubule nucleation and stabilisation in the vicinity of
chromosomes (Gruss et al., 2001; Nachury et al., 2001; Wiese
et al., 2001). It has been proposed that a gradient of Ran-GTP
concentration, declining at greater distances from mitotic
chromosomes, provides a spatial signal that is important for
orchestrating activity of different factors required for spindle
assembly (Hetzer et al., 2002).
RCC1 is the guanine nucleotide exchange factor for Ran
GTPase. Generation of Ran-GTP by RCC1 on chromatin
provides a spatial signal that directs nucleocytoplasmic
transport, mitotic spindle assembly and nuclear envelope
formation. We show that RCC1 is expressed in human cells
as at least three isoforms, named RCC1? ?, RCC1? ? and
RCC1? ?, which are expressed at different levels in specific
tissues. The ? ? and ? ? isoforms contain short inserts in their
N-terminal regions (NTRs) that are not present in RCC1? ?.
This region mediates interaction with chromatin, binds
importin ? ?3 and/or importin ? ?, and contains regulatory
phosphorylation sites. RCC1? ? is predominantly localised to
the nucleus and mitotic chromosomes like RCC1? ?.
However, compared to RCC1? ?, RCC1? ? has a greatly
reduced interaction with an importin ? ?3-? ? and a stronger
interaction with chromatin that is mediated by the
extended NTR. RCC1? ? is also the isoform that is most
highly phosphorylated at serine 11 in mitosis. Unlike
RCC1? ?, RCC1? ? supports cell proliferation in tsBN2 cells
more efficiently when serine 11 is mutated to non-
phosphorylatable alanine. Phosphorylation of RCC1? ?
therefore specifically controls its function during mitosis.
These results show that human RCC1 isoforms have
distinct chromatin binding properties, different molecular
interactions, and are
phosphorylation, as determined by their different NTRs.
selectively regulated by
Supplementary material available online at
Key words: RCC1, Ran, Nuclear transport, Mitosis
RCC1 isoforms differ in their affinity for chromatin,
molecular interactions and regulation by
Fiona E. Hood and Paul R. Clarke*
Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK
*Author for correspondence (e-mail: email@example.com)
Accepted 30 July 2007
Journal of Cell Science 120, 3436-3445 Published by The Company of Biologists 2007
Journal of Cell Science
JCS ePress online publication date 11 September 2007
Regulation of RCC1 isoforms
The localisation of RCC1 to chromatin is crucial for the
spatial organisation of Ran-GTP and is therefore important for
the functions of Ran throughout the cell division cycle. The
crystal structure of RCC1 reveals it to form a seven-bladed
propeller, from the seven characteristic RCC1 sequence repeats
(Renault et al., 1998). The association of RCC1 with chromatin
involves interactions between its core region and histones H2A
and H2B (Nemergut et al., 2001). The binding of RCC1 to
chromatin in vitro is promoted by nucleotide-free or GDP-
bound Ran, and inhibited by Ran-GTP (Li et al., 2003; Zhang
et al., 2002). Ran interacts separately with histones H3 and H4
(Bilbao-Cortes et al., 2002) and the affinity of RCC1 with
chromatin is reduced in a D182A mutant (Moore et al., 2002;
Hutchins et al., 2004), which disrupts the interaction with Ran
(Azuma et al., 1999). However, the localisation of RCC1 to
chromatin is critically dependent on a flexible N-terminal
region (NTR) (Moore et al., 2002) which is likely to extend
beyond the core structure (Renault et al., 1998). Deletion of the
NTR disperses RCC1 from mitotic chromosomes and causes a
variety of spindle abnormalities consistent with disruption of
the proper spatial organisation of Ran-GTP (Moore et al.,
2002). The NTR is required for binding of RCC1 directly to
DNA in vitro via residues 21-25 (Seino et al., 1992), although
it is not known if this binding occurs during the interaction with
The NTR of RCC1 also mediates interactions with other
proteins and is the target for post-translational modification.
There are two clusters of basic residues in the NTR that form
a bipartite nuclear localisation signal (NLS) that binds to
importin ? via the adaptor importin ?3 (Kohler et al., 1999;
Nemergut and Macara, 2000; Talcott and Moore, 2000).
Lysines 21 and 22 in the second cluster are essential for
importin ?3 binding whereas the first cluster strengthens the
interaction (Friedrich et al., 2006). The N-terminal methionine
of RCC1 is removed and the ?-amino group of serine 2 is
mono-, di- or tri-methylated, which is required for interaction
with chromatin, but does not appear to be regulated during the
cell cycle (Chen et al., 2007). By contrast, phosphorylation of
serines 2 and 11 by CDK1-cyclin B1 during mitosis disrupts
importin binding and regulates the dynamic interaction with
mitotic chromosomes (Hutchins et al., 2004; Li and Zheng,
Although animal cells contain only one RCC1 gene, human
and hamster cells express RCC1 proteins of different lengths
that are probably generated by alternative mRNA splicing.
However, no apparent difference in activity or cellular function
of these novel isoforms compared to the shorter, more abundant
isoform was found (Miyabashira et al., 1994). No further
consideration of the possible importance of different RCC1
isoforms has been made.
Here we show that human cells express at least three
isoforms of RCC1, which we name RCC1?, RCC1? and
RCC1?. Normal human tissues show different levels of
expression of these isoforms. We show that RCC1? has
increased affinity for chromatin in cells, reduced binding to
importins, and is the isoform that is most highly
phosphorylated at serine 11 during mitosis. In a cell
proliferation assay, RCC1? promotes cell proliferation more
efficiently when phosphorylation at serine 11 is abolished by
mutation. This work demonstrates that isoforms of RCC1 in
mammalian cells have different molecular interactions and
divergent mechanisms of regulation, suggesting that they have
distinct functions during the cell division cycle and in
Multiple isoforms of RCC1 are expressed in mammalian
Alignment of the amino acid sequences of mammalian RCC1
molecules derived from cDNA sequences (Fig. 1A) indicates
that three isoforms are expressed in human cells. These
correspond to the originally identified short isoform, a second
isoform with a 31 amino acid insert previously identified by
Miyabashira et al. [(Miyabashira et al., 1994) RCC1-I], and a
third isoform with a 17 amino acid insert that we also identified
by RT-PCR amplification of RCC1 cDNA from HeLa cells. We
named these three isoforms RCC1?, RCC1? and RCC1?,
respectively. The insert in the novel human isoform, RCC1?,
consists of the first 17 residues of the insert in RCC1?. RCC1?
is closely related to the hamster and mouse RCC1-I proteins
previously identified (Miyabashira et al., 1994), and is
predicted in rhesus monkey, whereas RCC1? is predicted in
chimpanzee. This suggests that the expression of all three
isoforms is conserved in primates.
Examination of the genomic sequence of human RCC1
indicates that the latter part of exon 6? that is present in RCC1?
but not RCC1? contains a GT-AG splice donor and acceptor
sites, and also a potential branch site, suggesting that the
differences between all three RCC1 transcripts could be due to
alternative splicing (Fig. 1B). Comparison of the genomic
sequence of mouse with human shows the sequence
conservation of the 6? exon of RCC1 (supplementary material
Fig. S1), including the alternative splice sites identified in
human, indicating that homologues of all three isoforms may
be expressed in non-primate mammals as well as in primates.
An isoform containing a four amino acid insert is found in
Xenopus laevis, indicating that multiple isoforms are also
present in other vertebrates.
To examine expression of RCC1 proteins in human cells,
two antibodies were generated (Fig. 1C). The first antibody,
anti-R-INS, was raised to a peptide corresponding to the insert
region of RCC1?, and so should detect both RCC1? and
RCC1?. The second antibody, anti-R-DIS, was raised to a
peptide corresponding to the region either side of the insert site.
This epitope was predicted to be disrupted when the insert is
present, and therefore should only detect RCC1?. A similar
strategy was used successfully by Miyabashira et al.
(Miyabashira et al., 1994). These antibodies were tested by
immunoblotting of proteins retrieved from HeLa cell extracts
using glutathione S-transferase (GST) expressed as a fusion
with RanT24N, a mutant that forms a stable complex with
RCC1 (Fig. 1D). Anti-R-DIS detected a single band
precipitated specifically by GST-RanT24N that corresponded
to the major polypeptide detected by an antibody raised against
the invariant C terminus of RCC1, consistent with it being
(45 kDa). Anti-R-INS detected two bands
corresponding to the two higher molecular mass bands detected
with the total RCC1 antibody. The migration of these larger
polypeptides is consistent with the predicted sizes of RCC1?
and RCC1? of 46.7 and 48.2 kDa, respectively, indicating that
both are expressed in HeLa cells, with RCC1? being expressed
at a higher level than RCC1?. In addition, a very minor band
Journal of Cell Science
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