Báo cáo khoa học: Functional association of human Ki-1⁄57 with pre-mRNA splicing events docx

Functional association of human Ki-1
⁄
57 with pre-mRNA
splicing events
Gustavo C. Bressan
1,2
, Alexandre J. C. Quaresma
1
, Eduardo C. Moraes
1,2
, Adriana O. Manfiolli
3
,
Dario O. Passos
1
, Marcelo D. Gomes
3
and Jo
¨
rg Kobarg
1,2
1 Laborato
´
rio Nacional de Luz Sı
´
ncrotron, Campinas, SP, Brasil
2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil
3 Departamento de Bioquı
´
mica e Imunologia, Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜ o Paulo, Ribeira˜ o Preto,
Brasil

Keywords
Arg methylation; nuclear bodies;
protein–protein interaction; RNA binding;
splicing
Correspondence
J. Kobarg, Laborato
´
rio Nacional de Luz
Sı
´
ncrotron, Centro de Biologia Molecular
Estrutural, Rua Giuseppe Ma
´
ximo Scolfaro
10.000, C.P. 6192, 13084-971 Campinas,
SP, Brasil
Fax: +55 19 3512 1006
Tel: +55 19 3512 1125
E-mail:
(Received 12 April 2009, revised 8 May
2009, accepted 13 May 2009)
doi:10.1111/j.1742-4658.2009.07092.x
The cytoplasmic and nuclear protein Ki-1 ⁄ 57 was first identified in malig-
nant cells from Hodgkin’s lymphoma. Despite studies showing its phos-
phorylation, arginine methylation, and interaction with several regulatory
proteins, the functional role of Ki-1 ⁄ 57 in human cells remains to be
determined. Here, we investigated the relationship of Ki-1 ⁄ 57 with RNA
functions. Through immunoprecipitation assays, we verified the associa-
tion of Ki-1 ⁄ 57 with the endogenous splicing proteins hnRNPQ and
SFRS9 in HeLa cell extracts. We also found that recombinant Ki-1 ⁄ 57

was able to bind to a poly-U RNA probe in electrophoretic mobility shift
assays. In a classic splicing test, we showed that Ki-1 ⁄ 57 can modify the
splicing site selection of the adenoviral E1A minigene in a dose-dependent
manner. Further confocal and fluorescence microscopy analysis revealed
the localization of enhanced green fluorescent protein–Ki-1 ⁄ 57 to nuclear
bodies involved in RNA processing and or small nuclear ribonucleo-
protein assembly, depending on the cellular methylation status and its
N-terminal region. In summary, our findings suggest that Ki-1 ⁄ 57 is
probably involved in cellular events related to RNA functions, such
as pre-mRNA splicing.
Structured digital abstract
l
MINT-7041074: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SF2P32 (uni-
protkb:
Q07021)bytwo hybrid (MI:0018)
l
MINT-7041232: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni-
protkb:
Q13242)bypull down (MI:0096)
l
MINT-7041203: P80-Coilin (uniprotkb:P38432) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041217: SMN (uniprotkb:Q16637) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041189: SC-35 (uniprotkb:Q01130) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(

MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041169: NPM (uniprotkb:P06748) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041249: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni-
protkb:
O60506)bypull down (MI:0096)
Abbreviations
Adox, adenosine-2¢,3¢-dialdehyde; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; GEMS, Gemini of
coiled bodies; GST, glutathione S-transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; SMN, survival of motor neurons; snRNP,
small nuclear ribonucleoprotein; SR protein, Ser ⁄ Arg protein.
3770 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
Introduction
Ki-1 was the first monoclonal antibody used in the
specific detection of the malignant Hodgkin and
Sternberg–Reed cells in Hodgkin lymphoma [1]. It
has been demonstrated that Ki-1 binds to the
120 kDa lymphocyte costimulatory molecule CD30
on the Hodgkin cell’s surface [1,2]. However, it has
been noticed that there is a cross-reaction of Ki-1
with a functionally and structurally uncharacterized
intracellular phosphoprotein antigen of 57 kDa,
termed Ki-1 ⁄ 57 [3]. Although its relationship with
Hodgkin disease has not been confirmed, initial stud-
ies revealed that Ki-1 ⁄ 57 is associated with Ser ⁄ Thr
protein kinase activity when isolated from tumor cells
[4] and localizes to both the cytoplasm and the
nucleus, where it could be found at the nuclear pores

and in several nuclear structures [2]. Ki-1 ⁄ 57 was
found to associate with intracellular hyaluronic acid
and other negatively charged molecules in vitro, and
was therefore also named hyaluronic acid-binding
protein 4 [5].
Another human protein, CGI-55, shares 40.7% iden-
tity and 67.4% similarity with Ki-1 ⁄ 57, suggesting that
they could be paralogs and have similar or redundant
functions in human cells. CGI-55 is also a
nucleus ⁄ cytoplasmic shuttling protein [6] and, because
it was described as a protein able to bind to the
3¢-UTR region of the mRNA encoding the type 1 plas-
minogen activator inhibitor, it was also named plas-
minogen activator inhibitor RNA-binding protein 1
[7]. We have recently found that Ki-1 ⁄ 57 and CGI-55
have overlapping interacting protein partners. Among
them are the chromatin remodeling factor chromo-heli-
case DNA-binding domain protein 3 [8], DAXX, and
Topors [6,9]. This suggests that the nuclear functions
of both proteins may be related to transcriptional
activity. Despite the fact that these proteins share
reasonable sequence similarity, Ki-1 ⁄ 57, but not CGI-
55, interacts with the transcription factor MEF2C [10],
p53 [9], and the signaling ⁄ scaffold receptor of activated
protein kinase C (RACK1) [11,12]. Both Ki-1 ⁄ 57 and
CGI-55 mRNAs show ubiquitous expression in all
human tissues tested, and elevated expression in the
heart, muscle, and liver [8]. Ki-1 ⁄ 57 is also expressed
at higher levels in the brain [8].
Both Ki-1 ⁄ 57 and CGI-55 interact with and are

methylated by the protein arginine methyltransferase
PRMT1 [13]. This enzyme is responsible for the meth-
ylation of more than 85% of the cellular protein
substrates [14], and targets the arginines embedded in
typical Arg ⁄ Gly-rich motifs (RG ⁄ RGG ⁄ RXR) [15].
These are conserved motifs in many RNA-binding pro-
teins, and have been reported to mediate RNA binding
[16,17].
In previous yeast two-hybrid screenings, we found
the interaction of Ki-1 ⁄ 57 with several RNA-binding
proteins [9] (unpublished observations). Functionally,
most of these identified RNA-binding proteins are
involved in pre-mRNA splicing regulation, pointing to
a role of Ki-1 ⁄ 57 in pre-mRNA splicing. Here, we
show the first functional signatures for Ki-1⁄ 57 in
human cells, mainly those concerning its possible
involvement in mechanisms of splice regulation.
Results
Protein–protein association analysis
In yeast two-hybrid analyses using Ki-1⁄ 57 and
PRMT1 as baits, we found common RNA-binding
proteins that are functionally associated with each
other in the context of pre-mRNA splicing regulation
(Fig. 1). The splicing proteins SF2p32, YB-1 [9] and
SFRS9 (unpublished observation) were found as posi-
tive prey clones when we used the N-terminus of
Ki-1 ⁄ 57 as bait in our screens (Fig. 1). On the basis of
the functional interconnections between the Ki-1 ⁄ 57-
interacting and PRMT1-interacting proteins (Fig. 1),
we reasoned that heterogeneous nuclear ribonucleo-

protein hnRNPQ could also be functionally related to
Ki-1 ⁄ 57. hnRNPQ has been reported to be associated
with the regulation of pre-mRNA splicing [18], and
has been previously found to be a novel interacting
l
MINT-7041065: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni-
protkb:
Q13242)bytwo hybrid (MI:0018)
l
MINT-7041069: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with YB1 (uni-
protkb:
P67809)bytwo hybrid (MI:0018)
l
MINT-7041079: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with HNRPQ
(uniprotkb:
O60506)bytwo hybrid (MI:0018)
l
MINT-7041087: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0218) with HNRPQ3
(uniprotkb:
O60506-1), HNRPQ2 (uniprotkb:O60506-2) and HNRPQ-1 (uniprotkb:O60506-3)
by anti bait coimmunoprecipitation (
MI:0006)
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3771
partner and target for Arg methylation by PRMT1
[13,19].
Aiming to confirm the endogenous association of
Ki-1 ⁄ 57 with proteins involved in splicing regulation,
we performed immunoprecipitation assays from HeLa
cell extracts. We confirmed such an association

when we immunoprecipitated Ki-1⁄ 57 (and detected
SFRRS1 ⁄ 9 and hnRNPQ isoforms) (Fig. 2A), and also
when we immunoprecipitated SFRS1 ⁄ 9 or hnRNPQ
isoforms (and detected Ki-1 ⁄ 57) (Fig. 2B,C), suggest-
ing that these proteins might form complexes in vivo.
The pan-antibody against hnRNPQ recognizes the
isoforms hnRNPQ1, hnRNPQ2, and hnRNPQ3, and
the antibody against SFRS1 ⁄ 9 recognizes the splicing
factors SFRS9 and SFRS1. The latter is also known as
SF2 ⁄ ASF, and its regulatory subunit, called SF2p32
[20], was also found to be a Ki-1 ⁄ 57-interacting part-
ner (Fig. 1) [9].
Next, we performed pull-down assays with recom-
binant proteins to test the interaction of Ki-1 ⁄ 57 with
SFRS9 and hnRNPQ in vitro. We found that the bacu-
lovirus 6· His–SFRS9 was pulled down by the
bacterial glutathione S-transferase (GST)–Ki-1⁄ 57
(Fig. 2D). Similarly, the bacterial GST–hnRNPQ
(1–443) was also pulled down by the bacterial 6· His–
Ki-1 ⁄ 57 (Fig. 2E). These results suggest that the inter-
action of Ki-1 ⁄ 57 with these splicing proteins occurs
directly and specifically, as no interaction was observed
with GST alone.
Moreover, we also detected the three hnRNPQ iso-
forms when we immunoprecipitated SFRS1 ⁄ 9,
although we did not observe direct in vitro binding
activity between these proteins in our experimental
conditions in pull-down assays (data not shown). This
suggests that these proteins may form part of the same
Ki-1 ⁄ 57-associated complex, although they might not

all interact directly with each other.
Yeast two-hybrid mapping assays
Next, we were interested in knowing the regions of
Ki-1 ⁄ 57 necessary for its interaction with splicing
regulatory proteins. Several N-terminal and C-terminal
Ki-1 ⁄ 57 truncated forms fused to the LexA DNA-
binding domain (Fig. 2F) were cotransformed with
constructs encoding the Ki-1⁄ 57-interacting proteins
fused to a GAL4 activation domain, to test their abil-
ity to interact with each other. Only the full-length
and N-terminal Ki-1 ⁄ 57 constructs were able to inter-
act with the splicing proteins SFRS9, SF2p32, and
YB-1 (Fig. 2G, columns 1–3). This suggests that the
interaction of Ki-1 ⁄ 57 with these molecules may occur
predominantly through its N-terminal region. This
pattern was not verified for hnRNPQ (Fig. 2G, col-
umn 4), as we only observed its interaction with the
full-length Ki-1 ⁄ 57 construct. On the other hand, this
finding may explain why we were not able to identify
hnRNPQ in our yeast two-hybrid screens, where only
the truncated forms of the N-terminus and C-terminus
of Ki-1 ⁄ 57 were used as ‘baits’.
RNA-binding activity of Ki-1
⁄
57 in vitro
Although Ki-1 ⁄ 57 does not have any classic RNA-bind-
ing domains in its amino acid sequence, it has several
Arg ⁄ Gly-rich clusters (RGG-box) (Fig. 3A). The RGG
motif’s importance for the interaction of many
RNA-binding proteins with RNA has already been

reported [16,17]. The two major Ki-1 ⁄ 57 RGG-boxes
located at its C-terminal region are highly similar to
those of its putative paralog, CGI-55 (Fig. 3A). The
exact role of CGI-55 in human cells also remains
unknown, but it was found to bind to the 3¢-UTR of
type 1 plasminogen activator inhibitor mRNA [7].
To test whether Ki-1 ⁄ 57 also binds RNA, we per-
formed electrophoretic mobility shift assays (EMSAs).
We found that the recombinant GST–Ki-1 ⁄ 57 bound
Fig. 1. Functional interconnections of Ki-1 ⁄ 57 with splicing regula-
tory proteins through direct physical interactions or participation in
common protein complexes. Black bold lines: experiments
described in this article (see Results for details). Dotted lines: previ-
ously published findings; PRMT1 is found in the same complex
with SF2p32 [48], SFRS9 is associated with SF2p32 [20], and YB-1
and SFRS9 interact with each other [21]. Thin dotted lines: YB-1
and hnRNPQ are functionally related, as both interact with
hnRNPD ⁄ AUF1 [42]; SFRS9 is highly similar in amino acid
sequence to SFRS1 (SF2 ⁄ ASF). Numbers in square brackets indi-
cate the respective references. The database accession numbers
for the proteins shown are (in UniProt code): Ki-1 ⁄ 57, Q5JVS0;
SFRS9, Q13242; hnRNPQ, O60506; YB-1, P67809; SFRS1,
Q07955; SF2P32, Q07021; PRMT-1, Q99873.
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3772 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
to a U-rich RNA probe (Fig. 3B, lanes 3–6). This was
relatively specific, as we also tested other RNA homo-
polymer probes (poly-A, poly-C, and poly-G), and
found no significant binding activity (data not shown).
These observations suggest that the binding of Ki-1 ⁄ 57

to its putative cellular RNA targets may involve
U-rich regions, instead of A-rich regions as reported
for CGI-55 [7].
As we found the N-terminus to be an important
region for the interaction of Ki-1 ⁄ 57 with its protein
A
B
C
E
H G F
D
Fig. 2. Confirmation of the protein–protein interactions among Ki-1 ⁄ 57 and proteins involved in pre-mRNA splicing. (A–C) Immunoprecipita-
tion assays (IP) of endogenous proteins. HeLa cell extracts were immunoprecipitated with G-Sepharose beads and the indicated antibodies.
The obtained protein complexes were analyzed by western blot (WB) as indicated in the figure panels. Arrows indicate the positions of ana-
lyzed proteins. WL, whole cell lysate. Immunoprecipitation with the indicated control antibodies is shown on the right side. (D, E) In vitro
pull-down assays. Recombinant proteins from bacteria [GST, GST–Ki-1 ⁄ 57, GST–hnRNPQ(1–443), 6· His–Ki-1 ⁄ 57] or baculovirus (6·
His–SFRS9) were loaded onto Ni
2+
–nitrilotriacetic acid (6· His-fusion) or glutathione–Sepharose beads (GST-fusion) and incubated with
supernatants of cell lysates as indicated. Arrows indicate the detected proteins. Arrowheads point to the position of the control protein GST.
The additional bands observed correspond to proteolysis degradation products. (F–H) Yeast two-hybrid mapping of Ki-1 ⁄ 57 regions (F) that
interact with the indicated splicing proteins. Black boxes in the diagrams represent the RGG-box motifs present in the sequence of Ki-1 ⁄ 57
(see also Fig. 3A). L40 yeast cells were cotransformed with the plasmids encoding several Ki-1 ⁄ 57 truncated constructs fused to LexA and
the plasmids encoding the prey proteins fused to the GAL4-activating domain (G). Protein–protein interactions were checked through
analysis of reporter gene activation: b-galactosidase activity or capacity to grow on selective minimal medium (in the absence of the amino
acids Trp, Leu, and His) (not shown). (H) Autoactivation control: inability of full-length Ki-1 ⁄ 57 to activate reporter genes in the absence of its
interacting partners.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3773
partners involved in splicing regulation (Fig. 2G), we

further investigated which regions of Ki-1⁄ 57 were
involved in binding to the poly-U RNA. We observed
that the two smaller RGG-box-containing constructs
6· His–Ki-1 ⁄ 57(151–260) and 6· His–Ki-1 ⁄ 57(261–
413) were able to bind, although weakly, to the poly-
U probe; however, only the larger C-terminal
Ki-1 ⁄ 57(122–413) construct could achieve binding as
strong as that of full-length GST–Ki-1 ⁄ 57. Hence, the
C-terminal region Ki-1 ⁄ 57(122–413) seems to be
necessary and sufficient for efficient interaction
with the RNA poly-U (Fig. 3B, lanes 7–10). The
observed ‘supershifted’ bands seemed to be stronger
upon the incubation of poly-U with increasing quan-
tities of Ki-1 ⁄ 57(1–413) and Ki-1 ⁄ 57(122–413)
(Fig. 3B, lanes 4–10). A 25-mer poly-U molecule is
large enough to bind more than one molecule of
Ki-1 ⁄ 57. Owing to its low expression yield and low
stability in solution, an N-terminal construct, 6·
His–Ki-1 ⁄ 57(1–222), could not be tested in these
EMSA experiments.
Influence of Ki-1
⁄
57 on E1A pre-mRNA splicing
in vivo
The association of Ki-1 ⁄ 57 with splicing proteins
pointed to a possible functional role in pre-mRNA
splicing regulation. Therefore, we investigated whether
Ki-1 ⁄ 57 could modulate the splicing site selection of
the adenoviral E1A test minigene, previously explored
for the Ki-1 ⁄ 57-interacting proteins SFRS9 and YB-1

[21]. Depending on the 5¢-splice site selection, the E1A
pre-mRNA may generate five isoforms: 13S, 12S, 11S,
10S, and 9S (Fig. 4A) [22]. These isoforms can be
monitored by RT-PCR followed by agarose gel analy-
sis, where the intensity of each band in the gel directly
correlates with the splicing site selection, which, in
turn, reflects the positive or negative influence of regu-
latory proteins [22,23].
We transiently cotransfected the encoding E1A mini-
gene plasmid with increasing amounts of vectors
expressing the recombinant enhanced green fluorescent
protein (EGFP)–Ki-1 ⁄ 57 in COS7 cells. We observed a
significant effect of EGFP–Ki-1 ⁄ 57 in modifying the
pattern of splicing of E1A mRNA in comparison with
empty pEGFP vector (Fig. 4B). Expression of EGFP–
Ki-1 ⁄ 57 leads to formation of the 10S and 9S iso-
forms, concomitantly with a reduction of 13S isoform
formation, in a dose-dependent way (Fig. 4B, lanes
2–4). This finding strongly suggests the functional
involvement of Ki-1 ⁄ 57 in regulatory mechanisms of
pre-mRNA splicing.
Although we also observed a significant modification
of the E1A mRNA splicing pattern by Ki-1 ⁄ 57(1–222)
and Ki-1 ⁄ 57(122–413), respectively, it only occurred at
the highest plasmid concentrations used (Fig. 4C,D).
Moreover, the effects seemed to be isoform specific for
each Ki-1 ⁄ 57 region, as the formation of 10S mRNA
was only increased by the C-terminal region of
Ki-1 ⁄ 57 although with a lower efficiency in compari-
son with the full length protein (Fig. 4C–E). The influ-

ence of the C-terminal construct Ki-1 ⁄ 57(122–413) on
the generation of the 9S mRNA was also more
pronounced (Fig. 4F).
A
B
Fig. 3. GST–Ki-1 ⁄ 57 binds poly-U RNA in vitro. (A) Schematic view
of the RGG-box motif localization in the sequence of Ki-1 ⁄ 57 and
its paralog CGI-55 (database accession number: Q8NC51). (B, C)
EMSA results. (B) Interaction of Ki-1 ⁄ 57 with poly-U RNA. On top
of the panel: the three different truncated versions of recombinant
Ki-1 ⁄ 57 used in the mapping experiments. Increasing concentra-
tions (0.5, 1, 2 and 4 m
M, respectively) of the recombinant protein
GST–Ki-1 ⁄ 57 and the three 6· His-fused truncated versions of
Ki-1 ⁄ 57 (122–413, 151–260, and 261–413) were incubated with the
32
P-labeled poly-U probe (25-mer) and subjected to native polyacryl-
amide gel (10% gel, with a 29:1 acrylamide:bis-acrylamide ratio)
electrophoresis. The arrow indicates shifted bands and the dashed
arrowheads indicate supershifted bands.
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3774 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
A
B
CE
F
D
Fig. 4. Ki-1 ⁄ 57 influences the splicing pattern of the E1A pre-mRNA. (A) Diagram showing the splicing events that generate the 13S, 12S,
10S and 9S mRNAS of the E1A reporter gene [22,23]. (B–D, G) In vivo splicing assays. COS7 cells were transiently cotransfected with an
E1A minigene encoding plasmid, an empty pEGFP vector and increasing amounts (1·,5lg; 2·,10lg; 3·,15lg) of pEGFP-Ki-1 ⁄ 57 (full

length) or pEGFP–Ki-1 ⁄ 57(1–222) and pEGFP–Ki-1 ⁄ 57(122–413) vectors. The empty pEGFP vector was used to keep constant the DNA con-
centration in each transfection. Splicing activity quantization was performed as described in Experimental procedures. The displayed figures
are representative of at least three independent experiments. Vertical bars in the graphs indicate ± standard deviation. Wherever it exists,
the significance of the difference relative to the control (empty pEGFP vector alone; line 1) is indicated by *P < 0.05. (B–D) Influence of the
overexpression of full-length Ki-1 ⁄ 57 (B) and its N-terminal (C) or C-terminal (D) constructs on E1A splice site selection. Essentially the same
results were obtained in HEK293 cells and when we used a flag-tagged construct of Ki-1 ⁄ 57 (data not shown). (E, F) Treatment ⁄ control band
intensity ratios – comparison of the splicing site selection efficiency of Ki-1 ⁄ 57 and its N-terminal or C-terminal truncated forms. The average
of band intensity values obtained for the isoforms 10S (E) or 9S (F) in (B), (C) and (D) in comparison to the average of the intensities in the
control samples were plotted in the graphs, and represent the fold induction of each isoform in relation to the control. We achieved approxi-
mately 60% transfection efficiency in all experiments performed. Open circles, unspliced pre-mRNA; M, marker.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3775
Effect of SFRS9 on E1A pre-mRNA splicing in the
presence of Ki-1
⁄
57
SFRS9 and many other Ser ⁄ Arg proteins (SR proteins)
are well known as regulators of E1A pre-mRNA splic-
ing [21,24]. Seeking for a possible functional influence
of Ki-1 ⁄ 57 on SFRS9 splicing activity, we performed
splicing assays in which both proteins were coex-
pressed in COS-7 cells.
When we cotransfected the construct EGFP–SFRS9
alone with the pMTE1A vector, we observed a strong
inhibitory effect on the formation of the 12S and 10S
mRNAs (Fig. 5, lanes 2 and 3), but, similarly to what
was found for EGFP–Ki-1 ⁄ 57, we also observed stimu-
latory activity in generating the 9S isoform (Fig. 5,
lanes 2 and 3). This finding may suggest that although
both proteins may act together in selecting the most

distal splice site region that generates the 9S isoform,
they can also be involved in different regulatory splic-
ing mechanisms, as EGFP–Ki-1 ⁄ 57 has an opposite
stimulatory activity in generating the 10S isoform in
comparison with SFRS9, which is inhibitory. Interest-
ingly, upon adding increasing amounts of EGFP–
Ki-1 ⁄ 57 we consistently observed that the inhibitory
effect of EGFP–SFRS9 in selecting the 10S isoform
can be partially reversed.
Colocalization analysis of Ki-1
⁄
57 to nucleoli and
splicing speckles
We have previously shown that, upon treatment with
the inhibitor of methylation adenosine-2¢,3¢-dialdehyde
(Adox), the endogenous Ki-1 ⁄ 57, instead of showing a
uniform nuclear ⁄ cytoplasmic distribution, relocalizes
predominantly to the nucleus, where it appears as
nuclear dots [13]. As the results that we present here
pointed to involvement of Ki-1 ⁄ 57 with RNA-binding
proteins related to RNA ⁄ mRNA processing, and as
most of the so far characterized nuclear subdomains
are sites for RNA maturation and processing [25], we
decided to investigate, through confocal microscopy
analysis, the identity of the nuclear substructures
where Ki-1 ⁄ 57 is present in Adox-treated cells.
We tested two lineages of adherent cells, COS7 and
HEK293, and found, in both of them, that upon Adox
treatment the recombinant EGFP–Ki-1⁄ 57 displayed
similar nuclear relocalization, at several dots, as

displayed by the endogenous Ki-1 ⁄ 57 in HeLa cells
[13] (data not shown). We then decided to use the
recombinant EGFP-fused form of Ki-1 ⁄ 57 in our con-
focal analysis, mainly because of the insufficient qual-
ity of the images obtained by labeling the endogenous
Ki-1 ⁄ 57 with monoclonal antibodies. We noticed that
Fig. 5. Effect of Ki-1 ⁄ 57 on SFRS9 activity. COS7 cells were transiently cotransfected with an E1A minigene-encoding plasmid [21,23], an
empty EGFP vector, and increasing amounts of pEGFP vectors encoding full-lengths constructs for SFRS9 or Ki-1 ⁄ 57 (1·,4lg; 2·,8lg; 3·,
12 lg). The empty pEGFP vector was used to keep constant the DNA concentration in each transfection. Splicing activity quantization was
performed as described in Experimental procedures. The displayed figures are representative of at least three independent experiments. Ver-
tical bars in the graphs indicate ± standard deviation. Wherever it exists, the significance of the difference relative to the control (empty
pEGFP vector alone; line 1) is indicated by *P < 0.05. Lanes 2 and 3 display the activity of SFRS9 alone, whereas lines 4–6 (darker gray bars)
show the effect of the increasing amounts of Ki-1 ⁄ 57. The white triangle indicates that the value plotted for the 10S isoform in line 6 is dif-
ferent (P < 0.05) to that in line 4. We achieved approximately 60% transfection efficiency in all experiments performed. Open circles, unsp-
liced pre-mRNA; M, marker. The expression of cotransfected Ki-1 ⁄ 57 and SFRS9 was controlled by RT-PCR and is shown in Fig. S1.
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3776 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
the most evident dot-forming Ki-1 ⁄ 57 in the nuclei of
Adox-treated cells seemed to be related to nucleoli,
mainly because of the well-known large area that this
structure occupies in the cell nucleus.
Although EGFP–Ki-1 ⁄ 57 shows a diffuse distribu-
tion throughout the nucleus, it showed a stronger
signal that colocalizes with the staining of the nucleoli
marker nucleophosmin (Fig. 6Aiii) in Adox-treated
cells. This suggests that the methylation status of
Ki-1 ⁄ 57 is important for its colocalization to this
nuclear subcompartment.
Besides the larger, nucleolar-associated bodies, we
also observed, in the Adox-treated cell nuclei, several

small dot-forming Ki-1 ⁄ 57 domains. Owing to the
interaction of Ki-1 ⁄ 57 with proteins associated with
pre-mRNA splicing, a plausible hypothesis would be
that these regions corresponded to nuclear speckles,
which are nuclear substructures known to be enriched
in small nuclear ribonucleoprotein (snRNP) and many
other transcription-related and pre-mRNA splicing-
related proteins [25,26]. To investigate this possibility,
we studied Ki-1 ⁄ 57’s colocalization with the SR pro-
tein SC-35, a marker protein for splicing speckles [27].
Despite the diffuse distribution of Ki-1 ⁄ 57 in the
nucleus, we saw partial colocalization with the SC-35
dots in nontreated cells (Fig. 6Bjjj). In Adox-treated
cells, we noticed, however, a juxtaposition of the
EGFP–Ki-1 ⁄ 57 and SC-35 nuclear substructures
(Fig. 6Biii). This is an indication that the cellular
methylation status has specific effects on the localiza-
tion of EGFP–Ki-1 ⁄ 57 among different subnuclear
compartments.
Colocalization of EGFP–Ki-1
⁄
57 with Cajal and
Gemini of coiled bodies (GEMS) nuclear bodies
The partial colocalization of EGFP–Ki-1 ⁄ 57 with
splicing speckles in untreated control cells has led us
to test antibodies against molecular marker proteins
for Cajal bodies and GEMS (Gemini of coiled bodies),
both of which are considered to be nuclear compart-
ments involved in snRNP storage and ⁄ or in the assem-
bly of pre-mRNA splicing complexes [25].

Interestingly, we found, through confocal analyses,
that EGFP–Ki-1 ⁄ 57 was again localized in a diffusive
fashion throughout the nucleoplasm, but showed
stronger spotted staining that colocalized with the
Cajal body protein marker p80-coilin in the nucleus of
HEK293 cells treated with Adox (Fig. 7Ai–iii). This
finding may, in addition, strengthen the hypothesis of
the involvement of Ki-1 ⁄ 57 in pre-mRNA processing
events.
The GEMS are regions enriched with the survival of
motor neurons (SMN) protein complexes and are con-
sidered to be Cajal body-like domains [25]. Although
they may be found as distinct structures, they can also
be found colocalized. This suggests that they are
functionally related [28,29]. However, an interesting
particularity of the SMN protein is its demand for the
presence of Arg ⁄ Gly-rich regions in most of its inter-
Fig. 6. Localization of Ki-1 ⁄ 57 to nucleoli
and splicing speckles. HEK293 cells were
transfected with EGFP–Ki-1 ⁄ 57 and treated
or not treated with the methylation inhibitor
Adox. After fixation, the cells were immuno-
stained with the antibodies against the
nuclear proteins nucleophosmin (NPM;
marker protein of nucleoli) or SC-35 (marker
protein of speckles), and analyzed by laser-
scanning confocal microscopy. (A) Partial
colocalization of EGFP–Ki-1 ⁄ 57 to nucleoli
(nucleophosmin) in Adox-treated cells (Aiii),
but not in the control cells (Ajjj). (B) Partial

colocalization of EGFP–Ki-1 ⁄ 57 to speckles
(SC-35) only in the control cells (Bjjj). The
Adox treatment seems to cause only a
close juxtaposition between the speckles
and the Ki-1 ⁄ 57-associated substructure
(Biii, inset). Bars: 5 lm. Figure insets
emphasize colocalizations or close juxtaposi-
tion of structures.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3777
acting partners [30], suggesting the possible requirement
of Arg methylation for these protein–protein associa-
tions. Through confocal analyses, we observed the
partial colocalization of Ki-1⁄ 57 with SMN ⁄ GEMS
in Adox-treated cells (Fig. 7Biii). Despite the diffuse
nuclear distribution of EGFP–Ki-1 ⁄ 57, the majority
of the red GEMS spots coincide with spots of
brighter EGFP–Ki-1 ⁄ 57 staining (Fig. 7Bi–ii). This
finding unveils a novel nuclear body with which
Ki-1 ⁄ 57 is associated in HEK293 cells treated with
Adox. We, like others before us, observed also that
Cajal bodies colocalize with nucleoli in some cells [31]
(not shown).
Subcellular localization of Ki-1
⁄
57 truncated
forms
In order to obtain further clues on the functions of
different regions of the Ki-1 ⁄ 57 amino acid sequence
in human cells, we fused to EGFP the various trun-

cated forms used in the mapping studies described
before (Figs 2G, 3B and 4C–D). Through fluorescence
microscopy analysis of transfected HEK293 cells, we
observed that all tested C-terminal constructs showed
similar nuclear and cytoplasmic localization as
observed for full-length Ki-1 ⁄ 57 (Fig. 8D–F). In turn,
the N-terminal construct displayed an exclusively
nuclear localization that, after careful analysis, could
be found in a few regions consisting of nuclear bodies
(Fig. 8C). This may suggest that the targeting of
Ki-1 ⁄ 57 to nuclear subdomains requires its N-terminal
region. On the other hand, when we treated the
HEK293 cells with Adox, we observed a small but sig-
nificant change in the localization of the C-terminal
construct. In the majority of analyzed cells Ki-1 ⁄
57(122–413) was seen more predominantly in the
nuclear compartment, in contrast to the diffusely
nuclear ⁄ cytoplasmic distribution observed in control
cells (compare panels I and D in Fig. 8). It is interest-
ing to observe that, upon Adox treatment, the N-ter-
minal construct showed pronounced relocalization
from the nucleoplasm to several well-defined nuclear
bodies (Fig. 8H), as observed for the full-length
Ki-1 ⁄ 57 construct (Fig. 8G). More than 90% and 98%
of the cells transfected with full-length EGFP–Ki-1 ⁄ 57
(Figs 6 and 7) and with EGFP–Ki-1 ⁄ 57(1–222) (not
shown), respectively, were found in the nucleus at
nuclear substructures upon Adox treatment (Fig. 8L).
This suggests that the C-terminus of Ki-1 ⁄ 57 is not a
required region for its association with these nuclear

subdomains, and therefore suggests that some signal
for localization control may exist at the N-terminus.
This localization may occur via protein–protein inter-
actions, as suggested by our mapping results with the
Ki-1 ⁄ 57-interacting proteins involved in pre-mRNA
splicing (Fig. 2G).
Discussion
We here describe Ki-1 ⁄ 57 as a novel human protein
that is functionally related to regulatory events of pre-
mRNA processing. From a wider point of view, the
Fig. 7. EGFP–Ki-1 ⁄ 57 is found in Cajal
bodies and GEMS upon Adox treatment.
HEK293 cells transfected with EGFP–
Ki-1 ⁄ 57 were treated or not treated with
the inhibitor of methylation Adox. The fixed
cells were stained with antibodies against
p80-coilin, which label Cajal bodies (A), or
against SMN, a marker for GEMS (B),
and analyzed by laser-scanning confocal
microscopy. Bars: 5 lm. Figure insets
focus on colocalizations or point out high-
magnification images of the selected
structures.
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3778 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
involvement of Ki-1⁄ 57 in modulating the splicing site
selection of the E1A pre-mRNA reported here may
unify its roles in the two main functional ‘worlds’ of
the nuclear context: RNA metabolism and trans-
criptional regulation. It is well known that in eukary-

otes, transcription and pre-mRNA maturation events
(5¢-capping, splicing, 3¢-end processing and polyadeny-
lation) occur cotranscriptionally and that the machin-
eries responsible for these activities are functionally
and physically associated [31,32]. Furthermore, it could
be speculated that not only the processing ⁄ maturation,
but also the expression, of some subsets of mRNA
may be regulated by Ki-1 ⁄ 57 in a defined cellular
context. Several of the identified Ki-1 ⁄ 57-interacting
proteins are involved in transcriptional control, such
as CHD3, RACK1, p53 and others p53-associated
proteins [8,9,11], thereby reinforcing a putative tran-
scriptional regulation role for Ki-1 ⁄ 57.
Here, as well as in a previous study, Ki-1 ⁄ 57 has
been observed as dot-like structures in the cell nucleus
[2,13]. Growing evidence points to important roles of
these nuclear subdomains, not only as storage spaces
but also as dynamic structures involved in RNA tran-
scription, processing, and maturation [25,27,33,34]. We
further showed the importance of methylation for its
localization at distinct nuclear ‘spots’, and showed that
in control cells, but not in Adox-treated cells, EGFP–
Ki-1 ⁄ 57 partially localizes to nuclear speckles, whereas
in Adox-treated cells it partially localizes to GEMS,
Cajal bodies, and nucleoli. The nuclear speckles are
known to be storage places for pre-mRNA splicing
complexes and, in turn, Cajal bodies and GEMS are
regions involved in snRNA modification, snRNP bio-
genesis, and trafficking of small nucleolar RNPs (small
nucleolar RNPs) ⁄ snRNPs to nucleoli or speckles,

respectively [25]. Therefore, the methylation process
may be an important step in the migration of Ki-1 ⁄ 57
in ‘assembled’ RNP complexes at Cajal ⁄ GEMS bodies
to nuclear speckles, from where they could be recruited
to transcriptionally active sites, which are regions that
are likely to involve synchronous splicing activity.
Interestingly, the methylation effects on snRNP
assembly in both cytoplasmic and nuclear phases have
been demonstrated in other studies. Gonsalvez et al.
[35] have shown that in cells treated with the methyla-
tion inhibitor 5¢-deoxy-5¢-(methylthio)adenosine, the
process of snRNP assembly in the cytoplasmic compart-
ment is disrupted. Similarly, the Arg methylation of the
100
50
0
Nuclear - with dots
Number of adox treated
cells (%)
Adox + Control
Nuclear/Cytopl. -
without dots
n = 3
Ki-1/57 Ki-1/57(1–222)
Ki-1/57(1–413)
B
A
L
CDEF
GH I JK

Ki-1/57(1–222) Ki-1/57(122–413) Ki-1/57(151–263) Ki-1/57(264–413)
Fig. 8. The localization of Ki-1 ⁄ 57 to nuclear
bodies depends on its N-terminal region. (A)
Schematic representations of the truncated
Ki-1 ⁄ 57 constructs fused to EGFP, used for
the localization assays in untreated or Adox-
treated HEK293 cells. (B–F) Untreated cells.
The full-length EGFP–Ki-1 ⁄ 57 and its C-ter-
minal constructs (122–413), (151–263) and
(264–413) show a diffuse nuclear and cyto-
plasmic localization (B, D–F), whereas the
N-terminal construct (1–222) shows an
exclusively nuclear localization (C), at dis-
crete nuclear dots (white arrowheads). (G–L)
Adox-treated cells. The full-length EGFP–Ki-
1 ⁄ 57 and its N-terminal construct (1–222)
predominantly show nuclear localization (G,
H), at several nuclear bodies (white arrow-
heads). A small but significant amount of
nuclear relocalization can be observed for
the C-terminal construct (122–413) (I). No
changes were observed for the smaller C-
terminal constructs (151–263) and (264–413)
(J, K). (L) Proportion of Adox-treated cells
containing nuclear bodies. More than 90%
and 98% of the cells transfected with full-
length EGFP–Ki-1 ⁄ 57 and EGFP–Ki-1 ⁄ 57
(1–222), respectively, were found in the
nucleus at several dots. Approximately 100
cells were analyzed in each of three inde-

pendent experiments.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3779
Sm proteins in the nucleus seemed to be important for
subnuclear targeting of snRNPs or for the regulation of
pre-mRNA splicing [36]. Moreover, the methylation has
been shown to be important for the subnuclear relocal-
ization of other proteins not related to snRNPs, includ-
ing MRE11, which is prevented from migrating from
promyelocytic leukemia protein bodies to DNA damage
sites upon inhibition of methylation by Adox or
5¢-deoxy-5¢-(methylthio)adenosine [37].
Endogenous Ki-1 ⁄ 57 can also be found in nucleoli
in HeLa and Hodgkin disease-derived cells [2]. In
eukaryotic cells, the primary function of the nucleoli is
the biogenesis of ribosome subunits through complex
machinery [38]. However, several other RNA modifica-
tions can occur in this nuclear compartment, not only
in the context of rRNA, but also in events related to
snRNA maturation [38]. Similarly to rRNA, several
small nucleolar RNA (small nucleolar RNA)-guided
modifications, such as 2¢-O-ribose methylation and
pseudouridination, are found in snRNAs [39,40].
Consistent with this, it has been reported that several
snRNAs pass through the nucleolus before they reach
their nucleoplasmic destination, where the splicing
itself occurs [40]. Ki-1 ⁄ 57 may be related to maturation
and assembly steps of snRNPs that take place in
nucleoli, Cajal bodies and GEMS before they reach
the nuclear speckles and become available to perform

their splicing activities.
Apart from the presence of the conserved Arg ⁄
Gly-rich clusters in the sequence of Ki-1 ⁄ 57, no other
amino acid sequence signatures are found through the
common computational predictors available on the
internet (data not shown). We did not find any nuclear
localisation signal or nuclear export signal with signifi-
cant scores on these programs, or any known domains
or motifs. Nonetheless, our deletion studies revealed
an interesting pattern of possible functional regions in
Ki-1 ⁄ 57 (Figs 2G and 3B). The Ki-1⁄ 57 C-terminal
region containing the two major RGG-box clusters
seems to be involved in poly-U RNA binding, whereas
the N-terminal region is mainly related to the interac-
tion with the associated pre-mRNA splicing proteins
SFRS9, SFp32, and YB-1. Analyzing the subcellular
localizations of these truncated Ki-1 ⁄ 57 forms in
HEK293 cells treated with the inhibitor of methylation
Adox, we noticed that only the N-terminal construct
was able to localize to nuclear bodies, similarly to
what was observed with the full-length construct.
Therefore, the localization of the N-terminus to
nuclear bodies in Adox-treated cells may be mediated
via protein–protein interactions.
The fact that we found that the N-terminus of
Ki-1 ⁄ 57 seems to functionally mediate protein–protein
interactions with splicing proteins and that the
RG-box-containing C-terminus seems to mediate inter-
actions with RNA is also very interesting; especially in
light of our results obtained with the protein deletions

in the splice assay. The protein with the N-terminal
deletion and that with the C-terminal deletion are less
functional than the full-length protein, suggesting that
both the N-terminal protein-binding domain and the
C-terminal RNA-binding domain are required for
efficient splice regulation (Fig. 4C,D).
In summary, our findings show that Ki-1 ⁄ 57 is prob-
ably a novel human protein involved in mechanisms
related to RNA metabolism, such as pre-mRNA splic-
ing. Further studies are necessary to dissect the molec-
ular mechanisms underlying the regulatory effect of
Ki-1 ⁄ 57 in pre-mRNA splicing, as well as to unveil its
putative endogenous pre-mRNA targets.
Experimental procedures
Plasmids and yeast two-hybrid interaction
analysis
Cloning of the complete cDNA encoding Ki-1 ⁄ 57 or its
truncated constructs into pBTM116 vector has been
described previously [11]. The full-length cDNA for SFRS9
was amplified by PCR from a fetal brain cDNA library
(Clontech, Mountain View, CA, USA) and subcloned in
the vectors pGEM-T easy (Promega, Madison, WI, USA),
pGAD424 (expression in yeast), pEGFPC (expression in
mammals) or pFastBac (baculovirus transfer vector). Clon-
ing of the cDNA encoding hnRNPQ(1–443) into the
pGEX4T1 vector has been described previously [41]. The
pGAD424–hnRNPQ(1–443) construct was obtained by
direct subcloning from the pGEX4T1 vector. The EGFP
fusion constructs were generated by direct subcloning of
the cDNAs coding for Ki-1 ⁄ 57 or its truncated forms

(obtained from the pBTM vector) into the pEGFPC vector
(Life Technologies Corporation, Carlsbad, CA, USA). The
pACT2 constructs containing the partial cDNAs for YB-1
and SF2p32 correspond to the ‘bait’ plasmid DNAs iso-
lated from the yeast two-hybrid screening previously
reported [9]. The partial or the total cDNA encoding for
Ki-1 ⁄ 57-interacting proteins were fused to GAL4 (pACT or
pGAD vectors) and applied to mapping assays using sev-
eral truncated forms of Ki-1 ⁄ 57 fused to LexA (pBTM116
vector), as described previously [11,12,42].
Cell culture and treatments, total cell lysates,
immunoprecipitations, and preparation of
cytoplasmic and nuclear fractions
Human L540 and HEK293 cells and monkey COS-7 cells
were cultivated under standard conditions as described
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3780 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
previously [11,41]. Transfection was performed by using the
calcium phosphate method. Treatment with 100 lm Adox
was performed as previously described by De Leeuw et al.
[43]. Total lysates were obtained and immunoprecipitated as
described previously [11], and subcellular fractionation of the
L540 cells was performed as previously described [13].
Pull-down assays, western blots, and antibodies
The in vitro pull-down assays and western blots were
performed as previously described [13,41]. The primary anti-
bodies were: anti-green fluorescent protein (rabbit poly-
clonal; Abcam Inc., Cambridge, MA, USA), anti-hnRNPQ
(mAB; Abcam), anti-c-tubulin (mouse mAB; Invitrogen,
Carlsbad, CA, USA), anti-FEZ1 (rabbit polyclonal) [44],

anti-Ki-1 ⁄ 57 (A26) (mouse mAB) [45], or anti-Ki-1 ⁄ 57 (Ki-1)
(mouse mAB) [1]. Additional primary antibodies, purchased
from Sigma-Aldrich (St Louis, MO, USA), were: anti-
SFRS1 ⁄ 9 (rabbit polyclonal), anti-SC-35 (mAb), anti-B23-
nucleophosmin (mAb), anti-p80-coilin (mAb), anti-SMN
(mAb), and anti-glyceraldehyde-3-phosphate dehydrogenase
(mAb). Secondary antibodies conjugated with Alexa-
Fluor488 and AlexaFluor594 were obtained from Invitrogen.
Recombinant protein expression and purification
and EMSAs
GST, GST–hnRNPQ(1–443), GST–Ki-1 ⁄ 57 and the trun-
cated constructs Ki-1 ⁄ 57(122–413), Ki-1 ⁄ 57(151–260) and
Ki-1 ⁄ 57(261–413) fused to 6· His were expressed in
Escherichia coli BL21-CodonPlus-RIL (Stratagene, La Jolla,
CA, USA) and purified in a similar way to that previously
described [42]. Baculovirus production of 6· His–SFRS9 [8]
and the gel shift assay [42,46] were performed as previously
described. The RNA–protein complexes were run out on
nondenaturing 10% polyacrylamide gels in 0.5· TBE at
4 °C. The radioactive bands in the gel were visualized on a
Phosphor imager system (Fuji, Shinjuku-ku, Japan).
In vivo splicing assay
For the in vivo splicing analysis, we transiently transfected
COS-7 cells with the minigene E1A encoding plasmid
pMTE1A [47], in combination with crescent amounts of
Ki-1 ⁄ 57 or SFRS9 constructs. The DNA concentration in
each transfection was kept constant by using the empty vec-
tor pEGFP (Life Technologies Corporation). After 48 h of
transfection, the cells were resuspended in 1 mL of TRizol
reagent (Life Technologies Corporation) for total RNA

extraction according to the manufacturer’s protocol. cDNA
synthesis was performed using oligodT primer (GE Health-
care, Waukesha, WI, USA) and the Moloney murine leuke-
mia virus reverse transcriptase (Life Technologies
Corporation). The PCRs were performed with the prim-
ers 5¢-ATTATCTGCCACGGAAGGTGT-3¢ (sense) and
5¢-GGATAGCAGGCGCCATTTTA-3¢ (antisense), as pre-
viously described [21]. After separation of the amplification
products on 3% agarose gels containing ethidium bromide,
the band intensities were calculated using the software
image j ( National
Institute of Mental Health, Bethesda, MD, USA). The
intensities of all isoforms were summed, set as 100%, and
used to normalize the intensity of each band.
Microscopy analyses
For the subcellular localization assays, HEK293 cells were
grown on glass coverslips with the required culture med-
ium. The cells were fixed with NaCl ⁄ P
i
containing 2%
paraformaldehyde, permeabilized with 0.3% Triton X-100,
and blocked with NaCl ⁄ P
i
⁄ 2% BSA. The primary antibod-
ies were incubated at room temperature in NaCl ⁄ P
i
⁄ 2%
BSA, and then with the Alexa594-coupled secondary anti-
body (Life Technologies Corporation). Coverslips were
mounted with Prolong gold antifade medium containing

4¢,6-diamidino-2-phenylindole (Life Technologies Corpora-
tion). Routinely, cells were examined with a Nikon micro-
scope. For the colocalization assays, samples were analyzed
on a Leica TCS SP5 laser scanning confocal microscope
(Leica Microsystems, Wetzlar, Germany). For quantitative
analysis, three independent slides were examined by fluores-
cence microscopy for the presence of nuclear dots upon
treatment with the inhibitor of methylation Adox. One
hundred cells were counted on each microscope slide across
randomly chosen fields.
Acknowledgements
This work was financially supported by the Fundac¸ a
˜
o
de Amparo a
`
Pesquisa do Estado Sa
˜
o Paulo (FAPESP),
the Conselho Nacional de Pesquisa e Desenvolvimento
(CNPq), and the LNLS. Confocal microscopy was per-
formed at Laborato
´
rio de Microscopia Confocal da
Faculdade de Medicina de Ribeira
˜
o Preto – USP. We
thank M. E. R. Camargo and Z. D. Correa for technical
assistance. We further would like to thank Dr Adrian
Krainer for providing the pMTE1A plasmid.

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Supporting information
The following supplementary material is available:
Fig. S1. RT-PCR control.
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online version of this article.
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G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3783