Jpn. J. Infect. Dis., 61, 95-99, 2008
Original Article
Detection and Localization of Small Nuclear Ribonucleoproteins (snRNPs)
in Trypanosomatids Using Anti-m3G Antibodies
Maira Cegatti Bosetto, Renato Arruda Mortara1, Daniela Luz Ambrósio, Gabriela Duó Passerini,
Marco Túlio Alves da Silva, Erica Sayuri Okuda and Regina Maria Barretto Cicarelli*
Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista,
and 1Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina,
Universidade Federal de São Paulo , São Paulo, Brasil
(Received June 6, 2007. Accepted December 3, 2007)
SUMMARY: This work reports for the first time the identification and immunolocalization, by confocal and
conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following
trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania
major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites,
abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the
snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was
also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.
Although nematodes, trematodes, and euglenoids all carry
out both cis- and trans-splicing (12), the notion has developed over time that trypanosomes lack intervening sequences
and consequently the machinery to carry out cis-splicing
(13). This assumption was based on observations that all
trypanosomatid genes characterized thus far have no introns,
and trypanosomes appear to lack a homologue of the U1
snRNA, which plays a crucial role in the initial recognition
of the 5´ splice site (14).
Recently, intervening sequences were described in the PAP
gene of Trypanosoma brucei and Trypanosoma cruzi, and a
candidate T. brucei U1 small nuclear RNA (snRNA) gene
was characterized by Djikeng et al. (15), demonstrating that
both cis- and trans-splicing can occur in these organisms,
with a prevalence of trans-splicing (13,15,16). Hannon and
co-workers (10) also demonstrated that the U1 snRNP is not
essential for trans-splicing, but only for cis-splicing, as in
nematodes. It has been clearly established that cis-splicing is
also present in trypanosomatid protozoa, albeit at low levels.
In most eukaryotic organisms, all snRNA genes are transcribed by RNA polymerase II to produce primary transcripts
with a 7-methylguanosine (m7G) cap at their 5´ end and short
extensions at the 3´ end. The precursors are exported from
the nucleus to the cytoplasm, where they assemble into a stable
core RNP particle by binding to common proteins, and, as an
essential maturation step, the m7G cap is hypermethylated to
2,2,7-trimethylguanosine (m3G or TMG). This m3G cap and
the common protein ligations compose the signal to nuclear
reimportation of uridine-rich snRNPs (UsnRNPs) (17).
Despite the evolutionary conservation of the secondary
structure, the SL RNA sequence is not conserved among
different organisms capable of processing their mRNAs by
trans-splicing. However, within the trypanosomatids, the exon
sequence shows a considerable degree of conservation at the
primary sequence level. One characteristic feature of the SL
RNA is the presence of a hypermodified cap structure, which
is referred to as cap 4 because the four nucleotides after the
m7G are modified (18,19).
The aim of this study was to identify by conventional and
INTRODUCTION
Among the important factors for eukaryotic cell function,
there are complexes composed of small ribonucleic acids
(RNAs) and proteins. These complexes in turn form small
ribonucleoproteins (RNPs) particles with short RNAs (approximately 300 nucleotides), and tightly complexed proteins
may appear in different sizes and shapes. RNAs are abundant (approximately 107 copies per mammalian cell, similar
to ribosomes) and highly conserved from yeast to humans;
describing their cellular roles has remained an important challenge for molecular biologists. In mammalian cells, small
nuclear ribonucleoproteins (snRNPs) play an essential role
in pre-mRNA processing, primarily in splicing (1-3).
In trypanosomes, Leishmania, and related species, all RNAs
are processed via an unusual mechanism known as transsplicing. Two independent transcript regions, and not a single
pre-mRNA, are joined to consistently form a mature mRNA
with the same initial sequence (approximately 40 nucleotides),
designated as the spliced leader (SL) (4-9).
The trans-splicing reaction in Ascaris lumbricoides has
revealed an important functional base pairing between SL RNA
intron sequences and U6 RNA. This matching occurs at the
Sm binding site region and adjacent nucleotides in SL RNA
and other sequences close to the Sm binding site in U6 RNA
that are complementary in U2 snRNA (10,11); an equivalent
complementarity has not been identified in the cis-splicing
process. The presence of this extensive complementarity most
likely represents a specific trans-splicing modification that
allows exclusion of the U1 and U5 snRNA interaction at the
5´ exon, since the splicing process uses a single leader-intron
RNA sequence for all reactions. In cis-splicing, many 5´ exonintron sequences must be processed by the spliceosome.
*Corresponding author: Mailing address: Departamento de Ciências
Biológicas, Faculdade de Ciências Farmacêuticas, Universidade
Estadual Paulista, Rodovia Araraquara-Jaú, km 01, 14801-902,
Araraquara, São Paulo, Brasil. Tel: +55-16-3301-6946, Fax: +5516-3301-6940, E-mail: [email protected]
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sulfate solution, and the samples were washed two times with
a 50%-saturated solution; the precipitated IgGs were bound
to protein A-Sepharose (Gibco BRL, Rockville, Md., USA)
during overnight incubation at 4°C. After the complex had
been washed, it was incubated with 10 mM m7G diluted in
NET-2 buffer (Tris-HCl, NaCl, Nonidet P40, Sodium Azide)
for 2 h at 4°C to block the IgG binding site. Twenty-five
micrograms of total RNA were added to the reaction and the
samples were incubated again for 2 h at 4°C under constant
rotation; the samples used for the reaction were washed,
treated with proteinase K (20 mg/mL), and 1% SDS for
15 min at 37°C followed by phenol/chloroform extraction.
The reaction samples were precipitated with 100% ethanol
and submitted to electrophoresis in 10% denaturing urea/
polyacrylamide/TBE buffer gel followed by silver staining.
A control reaction was performed without the blocked antibody.
confocal indirect immunofluorescence the m3G epitopes
present in the RNPs of trypanosomatids, which include T.
cruzi epimastigotes of three different strains, Blastocrithidia
ssp., and Leishmania major promastigotes, thus showing for
the first time the immunolocalization of the snRNPs in these
parasites.
MATERIALS AND METHODS
Parasites: T. cruzi epimastigotes forms (Y, NCS, and
Bolivia strains) and Blastocrithidia ssp. promastigote forms
were grown at 28°C (10 days) in LIT medium (20). The L.
major promastigote forms were kindly provided by Dra. Regina
Dórea (Departamento de Ciências Biológicas, Faculdade de
Ciências e Letras, Unesp, Sao Paulo, Brazil).
Indirect immunofluorescence (IIF) reactions: Parasite
cultures were centrifuged for 15 min at 1,000 × g, and the
pellet was washed 3 times with PBS (pH 7.2); after the
last wash, an aliquot was collected for quantitation under a
Neubauer chamber (approximately 106 cells/ml). Cells suspended in PBS (approximately 106 cells/ml) were fixed overnight under agitation with 2% formaldehyde in PBS. Fixed
cells were then washed three times in PBS and 105 cells/ml
suspensions were dropped in predrawn circles on microscopy
slides and left at 37°C to air dry. Immunofluorescence assays
were carried out using rabbit anti-m3G serum (provided by
Dra. Montserrat Bach-Elias, CSIC, Barcelona, Spain) (diluted
1:10), normal human or rabbit serum (diluted 1:80) as a negative control, and chagasic human serum (diluted 1:50) as
a positive control for the parasites on the slides; all sera
were diluted in PBS. T. cruzi-Y strain reactions were also
performed with mouse monoclonal anti-m3G antibodies (ICN
Biomedicals, Costa Mesa, Calif., USA) diluted 1:5, as suggested by the manufacturer. Anti-human IgG (Salck, São
Paulo, Brazil), anti-rabbit IgG (Sigma, St. Louis, Mo., USA),
and anti-mouse IgG (Pierce, Rockford, Ill., USA) FITC conjugates were diluted 1:40, 1:100, and 1:100, respectively, in
0.001% Evans Blue/PBS, as suggested. All incubations with
sera and secondary antibodies were carried out for 30 min at
37°C. After each incubation, the slides were washed in PBS.
All preparations were examined by fluorescence microscopy
(Leitz Dm RXE; Leica, Wetzlar, Germany) using the Leica
DC100 software. The IIF reactions using monoclonal antibody with T. cruzi (Y strain) epimastigotes were also stained
with DAPI (4',6-diamidino-2-phenylindole dihydrochloride;
Molecular Probes, Eugene, Oreg., USA) and imaged by
confocal microscopy using a Zeiss Axiovert 100 microscope
with a 100 × Plan-Apochromatic 1.4 oil immersion objective attached to an MRC-1024UV confocal system (BioRad,
Hercules, Calif., USA). Specificity controls were performed
using competition reactions in which both rabbit and monoclonal antibody anti-m3G were previously and separately
incubated with 7.5 mM 7-methylguanosine (Sigma) in 1M
Tris-HCl pH 7.5 at 4°C for 2 h. The treated antibody solutions were then used as described above. Cross-reactivity
between T. cruzi and insect trypanosomatids has been previously detected in studies of sera from chagasic patients or
from rabbits immunized with different trypanosomatids (21).
Immunoprecipitation reactions with anti-m3G antibody
blocked by m7G: To further characterize the specificity of
the antibody, immunoprecipitation assays were performed
with the total RNA of T. cruzi (epimastigote form of the Y
strain) and rabbit anti-m3G serum. Briefly, the serum was precipitated with an equal volume of 100%-saturated ammonium
RESULTS
IIF reactions: Figures 1, 2, and 3 present the results of
confocal and conventional IIF reactions with different species of trypanosomatids: T. cruzi (Y, NCS, and Bolivia
strains), Blastocrithidia ssp. (insect parasite), and L. major
promastigote forms, respectively. Positive controls for the
parasites on the microscopy slides were performed with
serum from a chagasic patient (Figs. 2 and 3, panel 1), since
they have cross-reactivity with chagasic sera. Parasites incubated with normal human serum or normal rabbit serum, as
negative controls, showed no detectable fluorescence under
the experimental conditions used in this study (data not
shown). L. major and Blastocrithidia ssp. promastigotes
also reacted with chagasic serum, which is similar to findings previously described for other trypanosomatids (22).
Figure 1 shows the TMG epitopes detected in the T. cruzi
UsnRNPs analyzed by confocal microscopy. In the competition reaction, abrogation of the perinuclear fluorescence
was observed, and only nuclei and kinetoplasts (labeled with
DAPI) were detected (Fig. 1B, in red). Panels C and D present
phase-contrast pictures of the reactions without and with
competition, respectively, to observe the limits of the cellular membrane of the parasites outlined in panels A and B,
respectively. Parasites incubated with anti-m3G antibody
showed perinuclear fluorescence, indicating that the TMG
cap epitopes of the snRNPs were localized predominantly in
discrete regions close to the nuclei (arrows in Fig. 1A). A
similar fluorescence pattern was observed for the three
different parasites after reaction with anti-m3G antibody (Figs.
2 and 3, panel 2). The identity of these epitopes, and hence
the specificity of the rabbit anti-m3G antibody, was ascertained by competition reactions with m7G that block the Ig
binding-sites, thus abolishing fluorescence in all of the parasites tested (Fig. 1B, Figs. 2 and 3, panel 3).
Immunoprecipitation reactions: This reaction revealed
a pattern of bands that represented the immunoprecipitated
snRNAs (Fig. 4B). The TMG antibody reaction was then
blocked with m7G and no bands were detected (Fig. 4A).
These reactions confirmed the specificity of the anti-m3G
antibody used in this study, since the TMG antibodies in which
binding sites were blocked by m7G did not react with the
m3G cap localized in the 5´ ends of the snRNAs. The immunoprecipitated bands shown in Fig. 4B reflect not only antibody
specificity, but also refer to the snRNAs of the trypanosomatid
snRNPs. These results, taken together, provide support for
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the specificity of the TMG antibody and epitope localization.
DISCUSSION
These data confirm the presence of TMG cap epitopes
of UsnRNPs, which were immunolocalized in different
trypanosomatids, independent of the reactivity with chagasic
serum or pathogenicity. This region is a relevant target in
these parasites in particular, and in eukaryotes in general, since
they are markers of 5´ end snRNAs. All mRNA precursors
transcribed by RNA polymerase II have a 5´ cap added by
polymerase-associated enzymes. The 5´ cap protects mRNAs
from exoribonucleases and also plays an important role in
mRNA translation and processing, and therefore also in the
splicing process (23).
In trypanosomatids, the UsnRNAs are transcribed by RNA
polymerase III are also capped at the 5´ end (24). The premRNAs receive this structure during trans-splicing when a
capped SL sequence of 39 nucleotides is transferred, forming SL RNA at the 5´ end of each mRNA. SL RNA is also
transcribed by RNA polymerase II (25).
Reuter et al. (26) studied the localization and distribution
of UsnRNAs during mitosis in mammalian cells by immunofluorescence microscopy with snRNA cap-specific anti-m3G
antibody. Whereas the snRNAs are strictly nuclear at late
prophase, they are distributed throughout the cell plasma at
metaphase and anaphase; snRNAs then reenter the newly
formed nuclei of the two daughter cells at early telophase,
producing speckled nuclear fluorescent patterns typical of
interphase cells. Independent immunofluorescence studies
Fig. 1. Confocal fluorescence microscopy of Trypanosoma cruzi
epimastigotes forms (Y strain). snRNPs were localized by labeling
with monoclonal anti-m3G antibodies. (A) Arrows indicate snRNPs
fluorescence (green) and nuclei and kinetoplasts labeled with DAPI
(red). (B) Same as A, but reaction of anti-m3G serum was abolished
by the competition reaction. (C, D) Corresponding differential interference contrast of the same parasites in A and B. Magnification bar
= 10 μm.
Fig. 2. Subcellular localization of snRNPs epitopes in Trypanosoma cruzi Y (A), NCS (B) and Bolivia (C) strains epimastigotes
forms by conventional immunofluorescence microscopy. 1) chagasic human serum; 2) rabbit anti-m3G serum diluted 1:10,
and 3) rabbit anti-m3G treated with 7-methylguanosine. Arrows indicate reaction with epitopes localized with anti-m3G
serum. Magnification ×1,000.
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Previous studies already have used specific antibodies for
m3G for immunofluorescence microscopy detection of the
reactivity of m3G-containing cap structures of the snRNAs
U1 to U5 in situ and thereby for detection of the nuclear
matrix localization of the snRNPs in interphase cells (27).
Immunofluorescence microscopy was also used by Bridge
et al. (28) to investigate the effects of adenovirus infection
on the nuclear organization of splicing snRNPs in HeLa cells;
the results showed that splicing snRNPs were localized
at the center of viral DNA synthesis, as determined using
specific antibodies against common snRNP antigens and the
snRNA-specific m3G 5´ cap structure. Also using immunofluorescence, Potashkin et al. (29) employed specific antibodies for the m3G-cap structure of snRNAs to examine the
distribution and localization of snRNPs particles within the
yeast cell nucleus of Saccharomyces cerevisiae. It was found
that most of the abundant snRNAs were localized to portions
of the nucleus previously referred to as the “nucleolus”. These
findings were in contrast to the results obtained with mammalian cells, in which most of the abundant snRNAs were
present in a non-nucleolar network within the non-chromatincontaining regions of the nucleoplasm (29).
In the present study, we confirmed the presence of TMG
epitopes in different trypanosomatids; specific localization
of these epitopes close to the nuclei was confirmed after competition reactions with m7G were carried out using either
polyclonal or monoclonal anti-m3G antibodies. To date, several studies have highlighted the importance of the TMG
cap structure in UsnRNAs, particularly with respect to the
distribution of these structures inside cells.
Using specific polyclonal antibodies, Palfi and Bindereif
(30) described the localization of and characterizied several
common snRNPs in T. brucei. Based on immunofluorescence
microscopy, snRNPs proteins in these trypanosomes appear
to be predominantly localized in the nucleus, with a fraction
of proteins residing in the cytoplasm. This differential distribution might reflect an ordered assembly pathway for snRNPs
that involves both cytoplasmic and nuclear stages, similar to
the cytoplasmic-nuclear assembly pathways proposed for the
mammalian U2snRNP.
The present results clearly demonstrated for the first time
direct evidence of the presence of UsnRNAs in the cytoplasmic region near the nucleus, which most likely suggests
the nuclear reimporting of UsnRNPs in these parasites. The
predominance of fluorescence in the cytoplasm was shown
to be due to the parasite treatment before IIF reaction (e.g., in
the absence of Triton X-100 pretreatment). The specificity
of the antibody was demonstrated by immunoprecipitation
assay, as shown in Fig. 4, since antibody competition with
m7G confirmed that the snRNP bands disappeared.
The results demonstrated that the accurate modification of
the entire cap structure requires that SL RNA is in the SL
RNP complex, and SL RNA capping takes place in the
nucleus. Recently, it was suggested that SL RNA might also
be found in the cytoplasm. Treatment with the karyopherinspecific inhibitor leptomycin B eliminated the cytoplasmic
SL RNA and exerted an influence on modification at the +4
position and 3´ end formation, suggesting that the cytoplasmic
stage is necessary for SL RNA biogenesis (18). Our studies
are now focused on the characterization of all UsnRNPs in
various strains of T. cruzi in order to evaluate the role in transsplicing reactions (manuscript submitted).
Fig. 3. Subcellular localization of snRNPs epitopes in Blastocrithidia
ssp. (A) and Leishmania major (B) promastigote forms. 1) chagasic
human serum; 2) rabbit anti-m3G serum diluted 1:10, and 3) rabbit
anti-m3G treated with 7-methylguanosine. Arrows indicate reaction
with epitopes localized with anti-m3G serum. Magnification ×1,000.
Fig. 4. Immunoprecipitation reaction using Trypanosoma cruzi (Y
strain) total RNA and anti-m3G antibody with (A) and without (B) 7methylguanosine electrophoreses in 10% urea/polyacrylamide gel/
TBE [1X]. The asteriscs indicate UsnRNAs and SL precipitated by
trimethylguanosine antibody. MW, molecular weight in base pairs.
with anti-(U1)RNP auto antibodies, which react specifically
with proteins unique to the U1 snRNP species, showed
the same distribution of snRNP antigens during mitosis, as
was observed with the snRNA-specific anti-m3G antibody.
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ACKNOWLEDGMENTS
To Dra. Montserrat Bach-Elias (CSIC, Barcelona, Spain) for donating
the anti-m3G polyclonal antibody and Dra. Regina C. C. Dórea for providing
Leishmania major cultures.
This work was supported by FAPESP (proc. 99/11393-4) and PADC/
FCF-UNESP (proc. 2002/12-I) and also supported by CAPES.
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