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Proteomic analysis of Entamoeba histolytica in vivo
assembled pre-mRNA splicing complexes☆
Jesús Valdésa,⁎, Tomoyoshi Nozakib , Emi Satob , Yoko Chibac , Kumiko Nakada-Tsukuib ,
Nicolás Villegas-Sepúlvedad , Robert Winkler e , Elisa Azuara-Liceaga f ,
María Saraí Mendoza-Figueroaa , Natsuki Watanabe c , Herbert J. Santosb,c,g ,
Yumiko Saito-Nakanob , José Manuel Galindo-Rosalesa
a
Departament of Biochemistry, CINVESTAV, México D.F., Mexico
Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan
c
University of Tsukuba, Graduate School of Life and Environmental Sciences, Tsukuba, Japan
d
Department of Molecular Biomedicine, CINVESTAV, México D.F., Mexico
e
Department of Biotechnology and Biochemistry, CINVESTAV Unidad Irapuato, Irapuato, Guanajuato, Mexico
f
Posgrado en Ciencias Genómicas, UACM, México D.F., Mexico
g
Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines
b
AR TIC LE I N FO
ABS TR ACT
Keywords:
The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000
mRNP
introns and bioinformatic predictions indicate that major and minor spliceosomes occur in
Splicing
Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor
Splicing factors
has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A
Splice sites
and assessed its expression and nuclear localization. Because the snRNP-free U1A form
DExH/A RNA helicases
interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify
Entamoeba histolytica
early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the
precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to
nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected
to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles
(chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with
proteins eluted with MS2–GST–sepharose from nuclear extracts of an MS2 aptamer-tagged
Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins
corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases
required for all stages of splicing, and 12 different splicing-related helicases were identified
also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes
were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some
of these later factors were not previously found in splicing complex analyses.
Biological significance
Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have
not been unraveled, particularly the splicing factors and their activities.
☆ This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013.
⁎ Corresponding author.
E-mail address: [email protected] (J. Valdés).
http://dx.doi.org/10.1016/j.jprot.2014.07.027
1874-3919/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
2
JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X
Considering that many of such introns are located in metabolic genes, the knowledge of the
splicing cues has the potential to be used to attack or control the parasite.
We have found numerous new splicing-related factors which could have therapeutic
benefit. We also detected all the DExH/A RNA helicases involved in splicing and splicing
proofreading control. Still, Entamoeba is very inefficient in splicing fidelity, thus we may
have found a possible model system to study these processes.
This article is part of a Special Issue entitled: Proteomics, mass spectrometry and
peptidomics, Cancun 2013.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
The vast majority of early branching eukaryotes have few or
no introns at all. However, around 3 thousand introns have
been identified within the 9938 reported genes of the protozoa
Entamoeba histolytica [1], and only a few of them have been
validated [2–9].
The introns of Entamoeba possess conserved 5′ (GUUUGU)
and 3′ (UAG) splice sites (ss), respectively, but the branch point
sequences (BS) lack such degree of conservation [8]. These
sites are recognized in a stepwise orderly fashion to assemble
the spliceosome and conform the catalytic site(s) responsible
for the excision of an intron of a pre-mRNA and ligation of its
flanking exons (reviewed in Ref. [10]). First, the U1 small
nuclear ribonucleic particle (snRNP) interacts with the conserved 5′ss of the pre-mRNA whereas both small and large
subunits of the U2 auxiliary factor, U2AF, interact with the 3′ss
forming the spliceosomal E complex. In the next step the U2
snRNP stably interacts with the pre-mRNA's branch site,
leading to the A complex (i.e., prespliceosome). The A complex
then interacts with the preformed U4/U6 · U5 tri-snRNP
particle giving place to the precatalytic B complex, which will
undergo RNP rearrangements, including loss of U1 and U4
snRNPs, thus converted in an activated B* complex which will
carry out the first step of splicing (i.e. a nucleophilic attack of
the BS adenosine at the 5′ss) generating a 3′ exon–lariat intron
intermediary and a cleaved 5′ exon. Finally, the C complex that
catalyzes the second step of splicing, excising the intron and
ligating the 5′ and 3′ exons is formed, thus producing a mature
mRNA in form of a RNP suitable for translation. RNA–RNA
interactions are mainly responsible in spliceosome remodeling and catalytic core conformation [11–13] and numerous
proteins are also required for assembly and splicing catalysis
and splicing fidelity as well [14–16]. Participating factors of
mainly human and yeast have been determined by mass
spectrometry (MS) of in vitro assembled and affinity-purified
spliceosomal complexes of model pre-mRNAs [17–29]. Over
150 proteins have been recruited in such complexes and this
repertoire has been extended when the analysis was carried
out from yeast, human and chicken cellular spliceosomes [30].
Most Entamoeba pre-mRNAs are mono-intronic although
bioinformatic analyses predict that intron retention might be
the main route for alternative splicing events [2,31] in
agreement with the RT-PCR analyses of validated introns
(see above), suggesting that rather than proteome expansion,
splicing might constitute another layer of gene expression
regulation. In spite of this, little is known of the E. histolytica
spliceosomal components, and much less about their functions. Recently, the amoebic U2, U4, U5 and U6 snRNAs have
been identified [2,32]. Furthermore, the Entamoeba major and
minor spliceosomal components [33], as well as DExH/D-box
helicases, some of which are known to be involved in premRNA splicing, and processing [4], have been bioinformatically
deduced. The lack of functional information restricts comparative studies of Entamoeba spliceosome components as much as
our understanding of the lesser conservation of spliceosome
proteins between E. histolytica and humans than between
Entamoeba dispar and humans. Therefore, direct biochemical
approaches are required to characterize the E. histolytica
spliceosomal orthologs that will provide insights into the
pre-mRNA splicing control, and will help to understand
additional gene expression regulation events of E. histolytica
biology.
To search for the E. histolytica RNA-binding early complex
splicing factors in a supra/polyspliceosomal context [34], a
main component of the U1 snRNA, protein U1A was HA
tag-cloned and used in UV cross-linking immunoprecipitation
(CLIP) assays with anti-HA antibodies from nuclear fractions
of HA-U1A and mock amoeba transfectants. The resulting
immunoprecipitates were subjected to MS/MS analysis. This
protein dataset was confirmed and complemented with the
one obtained from immunoprecipitates of amoeba transformed with an aptamer-tagged test intron. Although our
experimental design does not provide stoichiometric data and
may not be sensitive enough to identify low abundance
pre-mRNA-specific factors, it allowed us to probe 36 Entamoeba splicing components (corresponding to 32 cognate splicing
proteins), 12 splicing-related helicases, and 50 nuclear
pre-mRNA-complex components.
2. Materials and methods
2.1. Entamoeba cultures
Trophozoites of E. histolytica strain HM-1:IMSS Cl-6 were
cultured axenically at 35 °C in 13 × 100 mm screw-capped
Pyrex glass tubes or plastic culture flasks in BI-S-33 medium
as previously described [35,36].
2.2. Constructs and amoeba transfectants
The protein coding regions of U1A, RabX13, and RabC1, and the
N-terminus of the human influenza virus NS1 (nNS1) genes
were amplified by PCR from cDNA or genomic DNA (RabX13
clones) using their respective oligonucleotide pairs (5′ → 3′):
U1AF (CACACCCGGGATGGAACAAGAAGAAGATAAAAAAAA)
and U1AR (GTAAC TCGAGTCAATTAGCAAACTGGAGTATTA
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
TTCC); X13WF (TTATCCCGGGAT GGCACAACATGTAAGAA
ATACTAA) and X13WR (AGTTCTCGAGGGATATT AACAACAA
CCAGCTTG); C1F (CACACCCGGGATGGCAGCTAAAGCACACT
ACG) and C1R (GTAACTCGAGTTAGCAGCATCCTTTTCCATTG
TCC); and NS1F (TTATCCCGGGTCCATGCTCATACCCAAGCAG)
and NS1R (GTAACTCGAG TTACCCGGGATAATCTGGAAC). XmaI
and XhoI sites are underlined. For MS2-tagging, a synthetic
gene, flanked by XmaI–XhoI sites and harboring four tandem
repeats of the MS2 aptamer in the middle of the RabX13 intron
was made (GeneScript, USA). The amplified PCR products or
synthetic genes were digested with XmaI and XhoI and ligated
into XmaI/XhoI-digested pEhExHA [37], to produce plasmids
HA-U1A, HA-RabX13, HA-RabC1, HA-nNS1, HA-gRabX13, and
HA-gRabX13-MS2, respectively. The AIG17 construct was described elsewhere [38]. The transformants expressing the above
plasmids were established by liposome-mediated transfection
of the wild-type HM1:IMSS Cl6 strain as previously described
[39].
2.3. Antibodies, western blotting, immunofluorescence, and
recombinant GST–MS2 purification
Protein lysates, protein analysis and immunolocalizations by
confocal microscopy were carried out essentially as described
[40]. For nuclear staining, 4′,6-diamino-2-phenylindole (DAPI)
was included in the mounting medium. The plasmid coding
for the recombinant GST–MS2 protein was a kind gift from Rei
Yoshimoto and Mutsuhito Ohno. Purification of the GST–MS2
protein was carried out essentially as described [41].
2.4. CLIP assays
Amoeba transformants were exposed to UV light in a
Stratalinker® UV Crosslinker 2400 for 30 min. Then immunoprecipitations with anti-HA agarose or MS2–GST–sepharose
were carried out essentially as described [40] with modifications. For nuclear extracts, amoebas were lysed with 2% NP-40
in HEPES+ buffer (10 mM HEPES, 0.15 mM MgCl2, 10 mM KCl,
proteinase inhibitors E64 7 nM and Complete Mini 1 pill,
added with 30 U/mL of RNase inhibitor). Nuclei were pelleted
by centrifugation at 12,000 rcf for 10 min, at 4 °C, and washed
two times in HEPES+ buffer. Nuclear pellet was suspended in
Splicing-PEG Buffer (35 mM KCl, 4 mM MgCl2, 2 mM ATP,
20 mM creatine phosphate, 1.5 mM DTT, and 50 μg/mL
creatine kinase) and lysed by 5 freeze and thaw cycles, and
lysates were clarified by centrifugation at 20,000 rcf for
20 min, at 4 °C. For unspecific protein bead-binding, extracts
were precleared with 50 μL of protein G-sepharose, and
anti-HA agarose or MS2–GST–sepharose was blocked with
0.5 mg of yeast tRNA. Anti-HA agarose or MS2–GST–sepharose
immunoprecipitates were eluted with 20 mg/mL of HA
peptide or elution buffer (20 mM HEPES–KOH pH 7.9, 100 mM
KCl, 0.6% Sarkosyl, 10% Glycerol, 0.1% NP-40, 0.1 mM EDTA,
and 1 mM DTT). Eluates were split into two. The first set of
eluates were incubated with 1 U of RQ1 RNase-free DNase
(Promega) in the appropriate buffer at 37 °C for 30 min, then
deproteinized with 100 μg Proteinase K/0.1% SDS at 50 °C for
1 h. RNA was extracted using TRIzol reagent (Invitrogen)
according to the manufacturer's instructions and samples
were analyzed by RT-PCR. The second set of eluates were
3
treated with 5 μg RNase A and RQ1 RNase-free DNase as
mentioned above. Enriched proteins were concentrated with
10K Microcon® centrifuge filters and analyzed by SDS-PAGE
and MS/MS.
2.5. RT-PCR
The synthesis of cDNA was performed using the SuperScript
III First Strand Synthesis System (Invitrogen) according to the
manufacturer's instructions. U2 snRNA, U6 snRNA, Cdc2 and
actin mRNA molecules were detected with their respective
primer sets: U6f (GGATCCACTTCGGTGGAAAT) and U6r (CTT
CTCGTATGAGCGTGTCATC); U2f (TAACAGATCTATCACCTTC
TCGGCCTTTATG) and U2r (TAACAGATCTTGTTTCCATGCA
CATCCTCG); Cdc2f (GCTGTATTACTTGAACTGAAACATCCT)
and Cdc2r (TCTTCATCACA AAATTCAAATACTAAA); and Actf
(GGGAGACGAAGAAGTTCAAGC) and Actr (TG GATGGGAATA
CAGCTCTTG).
2.6. Protein and MS/MS analyses
Eluted proteins from three independent experiments were
resolved by 4–20% SDS-PAGE. Gels were stained with the
Silver Stain MS kit (Wacko). Duplicate lanes of each protein
sample were concentrated by allowing the samples to run on
the gel for up to approximately 1 cm by electrophoresis. Gels
were stained, lanes were excised and peptides were analyzed
at the W.M. Keck Biomedical Mass Spectrometry Laboratory at
the University of Virginia. Briefly, the solution samples were
transferred to a siliconized tube and washed in 200 μL 50%
methanol. The gel pieces were dehydrated in acetonitrile,
rehydrated in 30 μL of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h.
The DTT solution was removed and the sample alkylated in
30 μL 50 mM iodoacetamide in 0.1 M ammonium bicarbonate
at room temperature for 0.5 h. The reagent was removed and
the gel pieces were dehydrated in 100 μL acetonitrile. The
acetonitrile was removed and the gel pieces were rehydrated
in 100 μL 0.1 M ammonium bicarbonate. The pieces were
dehydrated in 100 μL acetonitrile, the acetonitrile was removed and the pieces were completely dried by vacuum
centrifugation. The gel pieces were rehydrated in 20 ng/μL
trypsin in 50 mM ammonium bicarbonate on ice for 30 min.
Any excess enzyme solution was removed and 20 μL 50 mM
ammonium bicarbonate added. The sample was digested
overnight at 37 °C and the peptides formed were extracted
from the polyacrylamide in a 100 μL aliquot of 50% acetonitrile/5% formic acid. This extract was evaporated to 15 μL for
MS analysis.
The LC–MS system consisted of a Thermo Electron Velos
Orbitrap ETD mass spectrometer system with a Protana
nanospray ion source interfaced to a self-packed 8 cm × 75 μm
id Phenomenex Jupiter 10 μm C18 reversed-phase capillary
column. 7 μL of the extract was injected and the peptides were
eluted from the column by an acetonitrile/0.1 M acetic acid
gradient at a flow rate of 0.5 μL/min over 1.2 h. The nanospray
ion source was operated at 2.5 kV. The digest was analyzed
using the rapid switching capability of the instrument acquiring
a full scan mass spectrum to determine peptide molecular
weights followed by product ion spectra [20] to determine amino
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X
acid sequence in sequential scans. This mode of analysis
produces approximately 30,000 MS/MS spectra of ions ranging
in abundance over several orders of magnitude. Not all MS/MS
spectra are derived from peptides.
Trans-Proteomic Pipeline 4.7.0 on MASSyPup was used for
protein identification and validation, using Comet as search
engine. A concatenated target-decoy database was constructed,
using the NCBI protein entries for E. histolytica (version 12 June
2014) (ProteomeXchange accession: PXD001080). Results were
validated by PeptideProphet/ProteinProphet, using decoy hits to
pin down the negative distribution. The identification of the
protein was considered significant when at least two nonoverlapping peptides of a protein were detected with the
probability score of .95 and .99%. Additional protein identification and annotations were carried out with Scaffold 4 (starting
with 2110 HA-IP proteins) using AmoebaDB, version 1.7 (http://
amoebadb.org/amoeba/). Individual predicted protein sequences were manually analyzed by BLAST search (http://
www.ncbi.nlm.nih.gov/BLAST/) against the non-redundant database at NCBI.
3. Results
To search for pre-mRNA processing components that could be
also part of the splicing machinery the following experimental
approach was used. First we HA-tag cloned the putative
Entamoeba U1 snRNP component, splicing factor U1A. U1A can
also be found in free form, as such interacts with the auxiliary
splicing factor PSF (PTB-associated splicing factor), a component
of the polypyrimidine-tract binding protein complex, involved
in the later stages of pre-mRNA processing [42]. Therefore CLIP
of HA-tagged U1A would increase the chances of detecting E
complex components as well as B complex and C complex
factors. To circumvent mRNA degradation by the numerous
Entamoeba nucleases [43] during nuclear fractionation, and to
ensure detection of RNA processing proteins bound to the RNA
for MS/MS analysis, CLIP assays were carried out from nuclear
extracts obtained from UV cross-linked amoebae.
Using HA antibodies, HA-U1A expression and intracellular
localization in amoeba transfectants were analyzed by western
blot (Fig. 1A) and confocal microscopy (Fig. 1B). Whereas the
31-kDa HA-U1A fusion proteins were expressed as well as the
cytoplasmic positive control HA-AIG1 proteins of 34 kDa (lanes
1 and 3, respectively), no signals were observed in the extracts
from empty vector (mock) transfectants (lane 4). Correspondingly, HA signals (green channel) were detected within the
amoebae nuclei of HA-U1A and HA-nNS1 transfectants (lanes 5
and 6, respectively), colocalizing with the DAPI-stained DNA
signals (blue channel). Since the N-terminus of NS1 interacts
with U2–U6 snRNA dimers inhibiting pre-mRNA splicing [44],
HA-nNS1 nuclear localization was expected, although HA-nNS1
was expressed in the cytoplasm as well. Only blue signals were
detected in the mock controls (lane 4).
To validate our experimental approach, nuclei enrichment/
purification efficiency and HA-U1A cross-reactivity and exposure/availability after UV cross-linking were monitored. Whereas the cytosolic protein cysteine synthase 1 (CS1) was detected
in the total, cytosolic and first wash fractions (Fig. 2A, lanes 1–3,
respectively), Histone 3 was detected in the nuclear fraction
only (lane 5), indicating that no significant cross-contamination
occurred during nuclear enrichment/purification. In addition,
HA-U1A signals shifted upwards after UV treatment (Fig. 2B,
lanes 3 and 5). This suggested HA-U1A–RNA complex formation, RNA-mediated HA-U1A protein–protein association and
HA-U1A epitope exposure and availability during CLIP (UV
cross-linking and HA-IP) procedures. Longer exposures showed
enhanced HA-U1A cross-linked and input signals without
significant increase of artifact signals (compare lanes 3 and 4
with lanes 5 and 6).
Next we verified that splicing RNA components and
intron-containing pre-mRNAs were associated with CLIP
products. To this end, RNA was purified from CLIP eluates of
HA-nNS1, HA-U1A and mock-amoeba transfectants. These
RNAs were used as templates to amplify by RT-PCR the two
components of the spliceosome catalytic core U6 snRNA and
U2 snRNA. To explore HA-U1A association with the mRNA
processing apparatus, the intron containing (CdcB) and
intron-less (actin) mRNAs were amplified also. As expected,
the HA-U1A and HA-nNS1 precipitates contained U2 snRNA,
U6 snRNA as well as Cdc2 mRNA (Fig. 3, top panels of lanes 1
and 2), whereas no actin mRNA was detected in these eluates
(lanes 1 and 2, bottom panel). As expected, none of the tested
RNA molecules were found in the mock CLIP (Fig. 3, lane 3)
and no amplifications were observed in the minus RT controls
(Supplemental Fig. 1). Thus far, our results suggest that the
Entamoeba splicing factor HA-U1A localizes in vivo in the
nucleus and is able to interact with RNA molecules from the
splicing machinery and with intron containing pre-mRNAs.
Parallel anti-HA CLIPs were analyzed by silver-stained
SDS-PGE. The same protein pattern between HA-nNS1 and
mock controls showed the light and heavy chains of the
antibodies and two bands of 90 and 45 kDa (Fig. 4, lanes 1 and
3). However, in the HA-U1A CLIP seven distinct bands of 280,
200, 125, 48, 40, 35, and 28 kDa were observed above the
background (Fig. 4, lane 2). Mock and HA-U1A lanes were
analyzed by MS/MS and the resulting protein dataset (2110
proteins) was filtered using the minimal threshold indexes of
the least represented high (U5-200K/Prp8) and low (SmD1)
molecular weight splicing factors (number of peptides/molecular mass), and eliminating the proteins present in the mock
controls (Supplemental Fig. 2). We confirmed and complemented our data with the proteins probed from MS2–
sepharose–IP eluates of Entamoeba MS2 aptamer-tagged
RabX13 intron transformants, compared to untagged gRabX13
and the intron-less RabC1 transformants, both with or without
UV cross-linking. Thus 36 splicing proteins corresponding to 32
cognate splicing factors (Table 1) and 50 out of 100 hypothetical
mRNP proteins (Table 2) were identified and categorized.
3.1 Entamoeba pre-mRNA processing
associated factors
Apart from the HA-tagged U1A, the HA-U1A-exclusive selection
included 36 Entamoeba splicing components, including 13 DExH/
D-box RNA helicases required for splicing, 11 splicing-related
helicases (Table 1), and 50 hypothetical proteins (Table 2). In
keeping with their CLIP recovery, categories were: MS2-specific
factors, intron-bound/unspecific (i.e. bound to untagged intron
as well) factors, pre-mRNA binding/cotranscriptional (all RNAs
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
5
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
mock
HA-U1A
HA-nNS1
DAPI
HA-AIG1
mock
kDa
250
B
HA-U1A
A
150
anti-HA
100
75
50
5 µm
PC
37
25
10 µm
20
1
2
3
5 µm
4
5
6
Fig. 1 – HA-U1A expression and nuclear localization in amoeba transformants. (A) Whole cell lysates of HA-U1A, mock and
HA-AIG1 (positive control) Entamoeba transformants were resolved by 4–20% SDS-PAGE and blotted onto nitrocellulose. Tagged
proteins were detected with anti-HA antibodies. The 31 and 34 kDa signals correspond to HA-U1A and HA-AIG1 fusion
proteins, respectively. (B) Mock (lane 4), HA-U1A (lane 5), and HA-nNS1 (lanes 6) amoeba transformants were treated for
immunofluorescence and observed under a confocal microscope. Green signals correspond to anti-HA Alexa antibodies and
blue signals correspond to DAPI-contrasted nuclear DNA. PC, phase contrast images merged with fluorescent signals.
tested), pre-mRNA binding (except tagged intron), and intronless RNA-binding; and according to attributed functions,
subcategories were: transcription/translation factors, kinases,
membrane/trafficking, and unassigned factors.
Altogether, probed proteins included pre-mRNA processing
factors and components of all stages of the splicing reactions:
complex A, complex B, complex B* (activated), complex C,
splicing step II factors, disassemble components, and factors of
the exon junction complex/messenger ribonucleoprotein particles (EJC/mRNP); Fig. 5 compares the splicing factors found here
with the previously reported human and yeast spliceosomes.
As previously reported for in vitro and in vivo intron-labeled
recruited spliceosomes [17–30], our combined approach with
MS2-CLIP allowed us to probe 11 additional splicing factors,
not detected with the HA-U1A CLIP (Table 1), which include
components of the U2 snRNP (U2A′, SF3a120, SF3a60/Prp9, and
Prp43), or of the U6 snRNP, U4/U6 · U5 tri-snRNP (LSm2, LSm5,
CPR6, 65K/SAD1, and Prp38), or components involved in
complex C formation (p68/DDX5, and Abstrakt). In addition,
two splicing factors (CPR6, and the Prp19/CDC5L complex core,
Prp19), and two splicing-involved (EhDExH13 and EhDEAD3),
and one splicing-related (Chain A of Mtr4) DExH/D RNA
helicases were recovered with this approach.
Taking advantage of their conservation to human splicing
factors, antibodies against Prp8, TIAR and U2AF35 were used
to probe cytosolic and nuclear fractions of E. histolytica (Fig. 6).
Despite Prp8 signal being somewhat lower than expected
(250 kDa), this factor was detected in the nuclear faction only.
Both TIAR and U2AF35 were detected in the cytosol, but
the latter is more abundant in the nuclear fractions. Their
apparent molecular weight corresponded to those expected
for Entamoeba, 35 and 29 kDa, respectively. These data
partially validate these E. histolytica previously predicted
splicing factors. Functional assays are being conducted for
thorough characterization of some of these splicing and
mRNP factors.
4 Discussion
Consistent with their nuclear localization, our HA-CLIP
assays showed that in vivo, the E. histolytica nuclear U1A
splicing factor associates with the catalytic core snRNA
components U2 snRNA and U6 snRNA, as much as the
transfected nNS1 influenza virus protein, used here as a
control. Possibly, the C-terminus of NS1 is also required for its
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X
C
W1
W2
N
50
CS1
37
IP
mock
T
HA-nNS1
kDa
HA-U1A
A
U6
120
20
80
150
+
-
+
-
+
*
Actin
B
RT-PCR
U2
Cdc2
H3
15
kDa
250
100
75
50
37
100
HA-U1A
25
20
Fig. 2 – Cellular fractionation assessment and UV
cross-linking of HA-U1A in enriched nuclear fractions. (A)
Protein extracts from total (T, lanes 1 and 6), cytoplasmic
(C, lanes 2 and 7), first and second wash (W1, lanes 3 and 8;
W2, lanes 4 and 9, respectively) and enriched nuclei (N, lanes
5 and 10) fractions were blotted onto nitrocellulose and
probed against the cysteine synthase 1 (CS1) cytoplasmic
protein (lanes 1–5) or Histone 3 (lanes 6–10). (B) CLIP (+) or no
UV treated (−) nuclear enriched protein extracts of mock
(lanes 1 and 2) and HA-U1A (lanes 3 and 4) amoeba
transformants were probed with anti-HA antibodies. After
development, blots were exposed for 10 s or 1 min (lanes 5
and 6) to discriminate free HA-U1A signals after UV
cross-linking, and artifact signals (asterisk).
nuclear-exclusive localization. U1A interacts with introncontaining mRNA molecules, suggesting that the methods
used here allowed us to detect mRNP particles. This view is
supported by the fact that additional protein signals were
revealed in the HA-U1A CLIP compared with those observed
with nNS1 or the mock background. Furthermore, additional
bands above the background were obtained from MS2-CLIP
assays also, with no difference between treatments (not
shown). Both cDNA and genomic RabX13 constructs rendered
a single 25 kDa protein signal, indicating that the gRabX13 clone
product is properly spliced (not shown), and suggesting that the
220
200
180
200
200
160
140
120
100
80
bp
bp
Fig. 3 – HA-U1A interacts with components of the splicing
machinery and mRNP particles in vivo. CLIP assays were
carried out with HA-nNS1 (lane 1), HA-U1A (lane 2) and mock
(lane 3) amoeba transformants. Total RNA was isolated form
the immunoprecipitates and was used as template to
amplify by RT-PCR the U6 snRNA, the U2 snRNA, and the
Cdc2 (intron containing), and Actin (intronless) mRNAs.
same stands for the gRabx13-MS2 clone products and that its
splicing signals were sufficient to recruit components of the
splicing machinery.
4.1. Splicing/mRNP factors
To gain insights into the E. histolytica spliceosome, the MS/MS
analysis of the HA- and MS2-CLIP eluates allowed us to probe
100 splicing related proteins. Out of these 100 proteins, only 36
were cognate splicing factors from all stages of the splicing
process including components of the A-, B- (U1 snRNP
associated factors), and B*-complexes, U2 snRNP associated
factors (17S particle), U4/U6 core and associated components,
and U4/U6 · U5 specific factors. The HA-CLIP was designed
to identify E complexes and perhaps unravel some splicing
later-stage factors. Surprisingly, such strategy rendered also
core and associated constituents of the Prp19/CDC5L complex,
and of the Transcription–Export (TREX) complex, which are
major components of the catalytic core of the spliceosome
[45–47]. Most of these factors were also detected in the
MS2-CLIP, designed to rescue introns engaged in the splicing
and post-splicing stages. Although to a limited extent, our
results coincide with a number of the human, yeast and chicken
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
k
1A
oc
m
-U
HA
HA
-n
NS
1
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
kDa
250
150
100
75
50
37
25
20
15
Fig. 4 – HA-U1A immunoprecipitated proteins. CLIP assays
were carried out as in Fig. 3. Samples of the
immunoprecipitates were loaded onto a 4–20% SDS-PAGE gel
and silver-stained. Bands of 280, 200, 125, 48, 40 35, and
28 kDa appearing in the HA-U1A immunoprecipitates
(lane 2), above the HA-nNS1 (lane 1) and mock (lane 3)
background are indicated by arrowheads.
spliceosomal factors probed in vivo [18,26,27,30,48,49], and
resemble the pattern obtained with trypanosome tagged-SMN
copurifications [29,50–52].
Except U6 snRNP, the snRNP particles contain a very stable
core formed by seven Sm proteins (Sm E, F, G, D1, D2, D3, and
B/B′) [49], and the LSm 2–7 proteins that belong to the U6
snRNP dissociate during spliceosome activation during
the formation of the B*-complexes [53]. The active particle
catalyzes the first step of splicing. In our experiments, three
Sm proteins were detected with the HA-CLIP, and LSm
proteins were probed with MS2-tagged introns.
Consistently, SF1 was not detected in our assays but the
uridine-rich binding factor TIA-1/TIA L-1 (TIAR) was identified. TIAR reinforces U1 snRNP binding to 5′ss in front of
polypyrimidine (PPy) tracts [54,55] resembling most Entamoeba
5′ss [8]. Intriguingly, although U1 snRNA has not been predicted
in Entamoeba [56], we probed the U1 snRNP components U1A
and U1-70K.
7
With our combined approach we detected components of
the B*, U4/U6 and U4/U6 · U5 spliceosomal particles. Moreover,
we probed components of the A- and B-complexes, proteins
important for the first (Prp2), and second (Prp16, Prp22, and
Abstrakt) catalytic reactions, and for spliceosome disassembly,
Prp43 [57].
Sub2p/UAP56 was the sole TREX component identified.
TREX was probed in yeast spliceosomal complexes assembled
in vivo by using UV cross-linking combined with affinity
purification of exon-tagged and U2 snRNP-tagged proteins
[58,59]. TREX binds introns with functional ss concentrated in
speckles where splicing occurs [47], as well as to poly(A) + and
intron-less mRNAs [60], and it may be associated with the
cleavage stimulation factor [61]. It is possible that the
Entamoeba Mtr4 and Cca1p homologs found here may be also
part of the TREX complex.
As expected and in agreement with intron retention, the
preferred route of alternative splicing in Entamoeba [31], no
SRSF proteins (positive splicing modifiers) [62], or hnRNP
proteins (negative splicing modifiers) [63] were detected here.
Eight RNA helicases, conserved between human and yeast,
are required at each of the spliceosome assembly steps,
including the catalytic processes [12,57], and they can be
isolated in complexes and purified by using molecular and
biochemical tools [26,30]. The best characterized are Sub2p/
UAP56, U5-100K (DDX3X)/Prp28, U5-200K/Brr2, and SF3b3/
DDX42. From the factors here identified, UAP56 associates
with U2AF65 and, together with Prp5, facilitates the interchange between SF1 and U2 snRNP during A-complex
formation. In vitro, it helps U2 snRNP recruiting to the BP
[57]. The U5-100K helicase is involved in the switch of U1 for
U6 at the 5′ss, which also requires U5-220K/Prp8 [12,30].
During spliceosome activation (B to B* complex transition),
Prp2 participates with U5-200K/Brr2 for unwinding U4/U6
[57,64], rearranging the spliceosome and repositioning the
substrate [65], probably involving the Brr2 modulator
U5-116K/Snu114 GTPase. As expected for splicing superfamily 2 helicases [66], the C-terminus of most Entamoeba DExH/
D-box RNA helicases involved in splicing is conserved
(Supplemental Fig. 3A). Furthermore, EhDEAD18 maintains
the amino methionine terminal (NMT) domain of Sub2p/
UAP56 (Supplemental Fig. 3B) involved in mRNA export and
genomic stability in yeast independently of the helicase
activity in vitro [67]. Surprisingly, even though E. histolytica
expresses and recruits in spliceosomal complexes the RNA
helicases/ATPases that proofread incorrectly selected 5′ss
(Prp28), BS (Prp5, Prp16) or exon-ligation events (Prp16, Prp22,
hPrp43) [15,16,68–71], numerous splicing products lack BS
and 3′ss selection fidelity [72], suggesting either that additional splicing checkpoint factors might be absent in this
parasite, or that splicing defects are somehow buffered by the
amoebae.
Another twelve DNA/RNA helicases were identified
(Rad3p, Ssl2p, Ssl2p-like, Rvb1p-like, Mtr4, EhDExH12,
EhDEAD6, EhDEAD9, and two gene products each of Sgs1p
recQ and isw2p). However, their function during spliceosome
assembly or catalysis remains to be investigated. Some DNA
helicases have dual functions as RNA or DNA helicases [73].
Therefore their participation in spliceosome assembly cannot
be ruled out, since other DNA helicases, such as DNJC-8, have
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
8
Particle/complex/class
Protein name
Gene ID
Protein
access
MW
Number or peptides a
Splicing
factor
U1A
Sm proteins
U1 snRNP
Accessory
U2 snRNP
U2-related
U5 snRNP
LSm proteins
U4/U6 snRNP
Small nuclear ribonucleoprotein Sm D1
Small nuclear ribonucleoprotein
Small nuclear ribonucleoprotein F
U1snRNP-specific protein
U1 small nuclear ribonucleoprotein subunit
RNA-binding protein TIA-1
Leucine rich repeat
Splicing factor
Splicing factor 3A subunit 3
Splicing factor 3B subunit 1
Splicing factor 3b subunit 3
U2 snRNP auxiliary factor large subunit
U2 snRNP auxiliary factor
EhDExH9
EhDExH13
EhDExH7
EhDEAD3
Splicing factor Prp8
EhDExH10/U5 snRNP-specific 200kd
EhDExH1
U5 small nuclear ribonucleoprotein subunit
Pre-mRNA splicing factor
EhDEAD4
EHI_052090
EHI_163710
EHI_060400
EHI_050780
EHI_153670
EHI_056660
EHI_167290
EHI_058680
EHI_038600
EHI_049170
EHI_048160
EHI_098300
EHI_192500
EHI_184530
EHI_090040
EHI_096230
EHI_013960
EHI_060350
EHI_045170
EHI_131080
EHI_021380
EHI_093960
EHI_021440
183234262
67476256
67475017
67482465
67482015
67465872
67468662
67468502
67483146
183235217
67473926
67475980
67476636
183233848
67465050
67483238
183231239
67475030
67484208
67480341
183231926
67484108
67469545
12
13
22
27
63
35
96
28
54
103
125
84
29
110
140
77
82
266
206
206
110
101
66
U6 snRNA-associated Sm-like protein LSm2
LSM domain containing protein
Peptidyl–prolyl cis–trans isomerase
EHI_068580
EHI_076840
EHI_020340
183230265
183232658
67478366
7
10
20
SmD1
SmD3
SmF
U1A, HA-tagged
U1-70K
TIA-1/TIAR
U2A′
SF3a120
SF3a60/Prp9
SF3b1
SF3b3/DDX42
U2AF65
U2AF35
Prp43
Prp43
Prp43
Prp5
220K/Prp8
200K/Brr2
200K/Brr2
116K/Snu 114
102K/Prp6
U5-100K
(DDX3X)/Prp28
LSm2
LSm5
CPR6
2
19
5
C1
C1 +
1
2
1
22
2
5
X
X+
Max %
coverage b
M
M+
12
8
7
15
3
20
3
1
1
2
1
HA
2
1
3
2
1
2
2
2
1
1
2
7
2
1
7
3
2
2
3
1
3
1
1
3
2
3
1
1
1
1
1
1
1
1
2
2
3
1
1
1
1
1
1
1
1
4
1
1
2
1
1
3
1
1
1
1
2
1
2
6
2
1
6
2
6
2
1
2
4
1
7
MS2
31
7
5
3
2
6
3
2
10
4
1
5
3
2
2
2
2
3
37
27
14
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Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
Table 1 – Components of Entamoeba histolytica pre-mRNA splicing complexes.
Prp19/CDC5L complex
Prp19/CDC5L complex-related
Complex B/B a
Complex C
Step II factors
EJC/mRNP
Peptidyl–prolyl cis–trans isomerase
Ubiquitin carboxyl-terminal hydrolase domain
containing protein
PRP38 family protein
WD domain containing protein
Regulator of nonsense transcripts
EhDEAD20
EhDExH4
EhDExH8
EhDEAD1
EhDExH5
EhDEAD18
Helicases with other splicing-related functions
MS2-specific/splicing related
Rad3p DNA repair helicase
EHI_125840
EHI_152110
67482289
67484272
18
52
CPR6
65K/SAD1
EHI_000490
EHI_130870
EHI_193520
EHI_096390
EHI_033720
EHI_077640
EHI_175030
EHI_122790
EHI_151600
67474026
67478341
67472499
67483276
67469329
67466830
67475258
67477533
67480889
23
52
108
62
87
105
66
98
47
Prp38
Prp19
KIAA0560 (fSAP164)
p68 (DDX5)
Prp2
Prp22
Abstrakt
Prp16
Sub2p/UAP56
EHI_132410
67466685
90
Transcription/
translation
Intron-binding, unspecific
Intron-binding, unspecific
Ssl2p-like DNA repair helicase
Ssl2p DNA repair helicase
Rvb1p-like DNA helicase
EHI_077260
EHI_088430
EHI_040360
67479133
67467062
67471882
69
75
48
Intron-binding, unspecific
Sgs1p recQ family DNA helicase
Sgs1p recQ family helicase
isw2p helicase
isw2p helicase
Chain A structure of Mtr4
EHI_023090
EHI_028890
EHI_012470
EHI_044890
EHI_125170
67475629
67469885
67479899
67483974
67464927
59
138
98
112
123
EhDExH12
EhDEAD6
EHI_134610
EHI_145050
67472639
67463088
111
72
EhDEAD9
EHI_151190
183229616
58
Intronless RNA-binding
Nonsense-mediated mRNA decay
and rRNA processing / biogenesis
2
5
3
1
3
1
1
1
2
2
1
2
6
2
1
4
4
2
1
1
1
1
2
2
1
3
1
2
1
2
3
1
1
1
1
1
1
1
1
1
1
1
2
8
5
15
6
3
4
6
2
3
5
8
15
2
5
4
5
7
4
2
4
3
1
2
3
2
3
2
1
2
3
3
1
1
7
5
5
3
2
1
1
2
1
1
1
1
2
Intronless-binding
5
4
1
1
1
1
4
Pre-mRNA-binding/
cotranscriptional
8
1
1
3
Chromatin
remodeling
Poly-adenylaton/
nuclear exosome
complexes
1
2
a
Number of peptides are listed for each sample: HA-IP (HA) using nuclear extracts from mock and HA-U1A (U1A) amoeba transformants, and MS2-IP (MS2) using nuclear extracts from untreated or UV
crosslinked (+) HA-RabC1 (C1), HA-gRabX13 (X) and HA-gRabX13-MS2 (M) amoeba transformants.
b
Percent of maximal coverage obtained for each protein in the different IP assays.
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
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complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
U4/U6.U5 tri-snRNP
9
10
Number of peptides a
IP/function
Protein
Gene ID
Prot
access
MW
U1A
EHI_048270
EHI_134790
EHI_129490
EHI_098010
EHI_180940
67473904
67472676
183235170
67470175
67472461
196
59
51
84
78
EHI_023310
EHI_114110
EHI_107540
EHI_059040
EHI_062090
EHI_180150
67480583
67462946
67463446
183235228
67468296
183232515
71
151
94
154
156
135
EHI_064610 183231977
EHI_067070 67465327
EHI_009890 67483780
119
134
120
3
2
1
2
1
1
EHI_065240 183233793
EHI_118100 67481393
219
98
4
1
1
1
EHI_198900
EHI_061870
EHI_042880
EHI_177440
EHI_035460
67479193
183235096
67473767
183231390
183231337
47
235
95
108
17
8 12 3
7 11 2
1 1
1 1 2
1
EHI_092300
EHI_175920
EHI_152420
EHI_138750
183230507
67465319
67483395
67468156
150
125
200
124
11 10
1 2
14 10 1
7 6 3
Max %
coverage b
C1 C1 + X X+ M M +
HA
BLAST scores
MS2 Total Query
score cover
E value
Ident
Accession
MS2-specific
EhSec7 domain
Ehzinc finger protein
EhCXXC-rich protein
H
KIAA0216, myosin XVIIIA
H
Transmembrane protein, putative
Intron-binding/unspecific
EhRING zinc finger protein
H
Similar to zinc finger protein 650
H
Uncharacterized protein LOC101882025
EhProtein kinase domain containing
EhProtein kinase domain containing
EhProtein tyrosine kinase
domain-containing
EhProtein kinase domain containing
EhProtein kinase
EhPhosphatidylinositol-4,5bisphosphate 3-kinase
H
Protein tyrosine kinase
H
WD40 repeat-containing protein
Pre-mRNA-binding/co-transcriptional
Transcription/translation factors
EhCXXC-rich protein
H
Zinc finger domain, LSD1 subclass family
EhDNA topoisomerase III
H
cca1p
Eh40S ribosomal protein S13
Kinases
EhProtein kinase
EhProtein kinase
EhTyrosine kinase
EhTyrosine kinase
3
2
1
2
2
1
1
3
2
1
2
1
1
2
2
1
4 2
1 2
1 2
11 10
10 7
5 3
1
1
1
2
1
2
1
2
5
2
2
1
2
1
2
1
2
1
6
7
3
5
14
3
3
4
3
6
7
2
3
4
3
10
6
1
2
4
2
1
1
2
1
2
7
2
2
1
4
3
1
2
2
2
1
1
5
2
1
5
2
5
36
9
2
2
9
16
5
5
2
29
3
3
2
1
1
1
1
4
12
4
6
5
2
1
1
4
2
1
1
6
3
1
3
4
1
7
3
5
4
182
66%
70.5 25%
6.0E −18
2.0E −09
11%
30%
BAA13206.2
XP_002908461.1
81.6 36%
304
56%
2.0E −12
3.0E −88
22%
36%
XP_969420.2
XP_005164424.1
1911
451
25%
36%
5.0E −31
2.0E −08
29%
WP_006508829.1
750
83%
3.0E −48
24%
XP_001015310.1
0.67
25%
EJS43939.1
43.1 26%
5
3
4
6
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Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
Table 2 – High ranked proteins selected from MS/MS analysis of HA-U1A and gRabX13-MS2 IPP. Data are presented as in Table 1. For hypothetical proteins (H labels on the
first column) or for proteins identified in other organisms, BLAST scores and accession identifiers are also presented.
H
Unassigned
H
H
H
Pre-mRNA-binding
EHI_127440 67473072
EHI_030120 67476356
EHI_101280 183234347
187
155
134
3
2
1
1
3 1
EHI_064500 67476998
133
3
1
EHI_037140 67465629
EHI_128800 183236164
EHI_055710 67472469
129
145
129
2
1
1
2
2 2
EhCysteine surface protein
EhCysteine surface protein
EhGal/GalNAc lectin heavy subunit
Ras-like GTPase
EHI_116260
EHI_160750
EHI_046650
EHI_090940
183235261
183235324
67469085
183234136
103
64
135
93
Predicted protein
Hypothetical protein
EMIHUDRAFT_240854
T17H7.18
EHI_064100 183235702
EHI_094080 67465773
155
97
EHI_159840 183231093
110
EHI_098370 183231153
EHI_047750 67469203
EHI_015280 67478161
65
36
150
3
2
EHI_078270 67472246
86
1
67482879
67479391
67479617
183232838
183232826
13
42
18
113
25
10
7
1
1
1
EHI_154180 67468884
EHI_169070 183232471
117
69
EhCleavage stimulation factor
EhNucleotide-binding protein
EhUDP-glucose:glycoprotein
glucosyltransferase
H
Piso0_004841
Intronless RNA-binding
EhActin
EhGTP-binding protein
Eh40S ribosomal protein S19
Ehtyrosine kinase
DNA-directed RNA polymerase II
largest subunit
H
Ehzinc finger domain containing protein
H
NF-X1 finger transcription factor
EHI_107290
EHI_014370
EHI_198740
EHI_017760
EHI_017570
11 10 6
4 1
8 14 2
3 2 2
2
1
1
2
1
3
5
5
6
5
5
3
5
3
3
2
2
3
5
4
5
4
3
187
272
21%
36%
4.0E −46
4.0E −76
39%
36%
XP_004338582.1
AFW67835.1
3
11
13
11
6
11
6
6
3
336
56%
1.0E −103
39%
XP_005164424.1
2
3
1
3
114
467
35%
19%
2.0E −22
3.0E −21
21%
40%
XP_001772062.1
EOD21833.1
3
3
49.3 13%
0.008
28%
AAD32943.1
1
2
8
2
6
8
2
1
1
5
54.3 33%
2.0E −04
27%
XP_004194353.1
36.6 46%
8.9
24%
XP_003072524.1
5.0E −43
36%
EFY87775.1
3
2
1
3
1
2
1
1
3
2
2
3
1
1
1
1 1
1
1
1
1
2
1
2 1
1 1
1
1
1
8
1
1
2
2
1
2
2
1
2
3
2
1
1
32
7
7
5
14
2
1
3
1
2
1
1
1
2
3
3
3
341
48%
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
H: BLAST results are shown for the annotated hypothetical proteins.
IP/function: Functions attributed according to the MS2-IP sample(s) where proteins were found (tagged intron, intron-containing and intron-less mRNA).
a
Number of peptides are listed for each sample: HA-IP (HA) using nuclear extracts from mock and HA-U1A (U1A) amoeba transformants, and MS2-IP (MS2) using nuclear extracts from untreated or UV
crosslinked (+) HA-RabC1 (C1), HA-gRabX13 (X) and HA-gRabX13-MS2 (M) amoeba transformants.
b
Percent of maximal coverage obtained for each protein in the different IP assays.
11
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complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
H
H
Membrane/traffic
EhTyrosine kinase
EhTyrosine kinase
EhProtein tyrosine kinase
domain-containing
EhProtein tyrosine kinase
domain-containing
EhReceptor protein kinase
Protein kinase domain containing protein
Putative AGC protein kinase family protein
12
JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
13
Fig. 5 – Splicing factors of Entamoeba histolytica. The compositional dynamics of the yeast and human spliceosomes (Refs.
[29,74]) was used as template to compare the Entamoeba splicing factors here identified (in bold). Yeast protein factors appear
first, followed by the human orthologs, separated by a slash. The Entamoeba snRNAs, previously identified are also shown.
been identified previously in human and chicken spliceosome
preparations [30].
Actin, myosin, Sec7-, Zn finger-, and CXXC-rich transcription
factors, DNA directed polymerase II, and the 40S ribosomal
proteins S13 and S19, the cleavage stimulating factors, and
nucleotide binding and GTP binding proteins have been
reported to belong to spliceosome particles. The remaining 39
factors listed in Table 2 include: 16 protein kinases (seven of
them tyrosine-kinases), one phosphatidylinositol kinase, DNA
topoisomerase III, two cysteine surface proteins, one Gal/
GalNac lectin, one vacuolar targeting protein, and 17 hypothetical Entamoeba proteins (labeled H). Only 3 or the 17 hypothetical
proteins could not be assigned by BLAST analysis.
As reported for humans and yeasts, the spliceosome has a
complex and dynamic composition of proteins, which changes
during assembly of each of its functional particles [29,74]. Fig. 5
summarizes the spliceosomal components previously reported
along with the factors identified here. The list of spliceosomal
components is still growing, and new factors are likely to be
identified in the future. Whether all of these newly identified
factors are components of the spliceosomal particle, or are just
part of the mRNP are questions that will be addressed in the
future.
Our work is the first to report the identification of the
snRNP and non-snRNP splicing and mRNP factors immunoprecipitated with one Entamoeba Early complex splicing factor
and one aptamer-tagged functional intron. All of the data
suggested that the profiles of the factors identified in this
work include factors involved in all steps of the splicing
process as well as mRNP factors.
Please cite this article as: Valdés J, et al, Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing
complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027
14
JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X
kDa
C
250
N
Prp8
150
37
25
TIAR
U2AF35
Fig. 6 – Subcellular localization of the Entamoeba histolytica
splicing factors Prp8, TIAR and U2AF35. Entamoeba histolytica
proteins isolated from cytoplasmic (C) and nuclear enriched
(N) fractions were resolved by 10% SDS-PAGE, blotted onto
nitrocellulose and probed with human antibodies to Prp8,
TIAR, and U2AF35 (top, middle and bottom panels,
respectively).
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2014.07.027.
Conflict of interest
I hereby state that none of the authors have any conflict of
interest with any researcher in the field.
Acknowledgments
This work was supported by CONACyT grants 49355M and
127557M and ICYT grant 71/2012 to J.V. and a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT) of Japan (23390099), a
Grant-in-Aid for Scientific Research on Innovative Areas from
MEXT of Japan (3308, “Matryoshka-type evolution”, 23117001,
23117005), a grant for research on emerging and re-emerging
infectious diseases from the Ministry of Health, Labour and
Welfare (MHLW) of Japan (H23-Shinkosaiko-ippan-014) to T.N.
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complexes, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.07.027