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RESEARCH ARTICLE
Transcriptional profile of processing machinery of 30end
of mRNA in Trichomonas vaginalis
Miguel A
´ngel Del-Moral-Stevenel •Alma Villalobos-Osnaya •Mavil Lo
´pez-Casamichana •
Laura Itzel Quintas-Granados •Ce
´sar Lo
´pez-Camarillo •Jose
´Manuel Ferna
´ndez Sa
´nchez •
Selene Zarate-Guerra •Marı
´a Elizbeth Alvarez-Sa
´nchez
Received: 30 June 2014 / Accepted: 18 January 2015
ÓThe Genetics Society of Korea and Springer-Science and Media 2015
Abstract Trichomonas vaginalis is the causative agent of
trichomonosis, a sexually transmitted disease (STD) that
affects over 180 million people worldwide. This parasite is
capable to infect the urogenital tract of women and men,
both microenvironments might affect the expression of key
genes that may be involved in the parasite pathogenesis.
The processing of 30end of mRNA promotes mRNA sta-
bility in many eukaryotes, however in T. vaginalis this
molecular machinery is under research. By means of an in
silico analysis we identified putative proteins of the 30end
mRNA processing machinery of T. vaginalis, and by RT-
PCR assays we evaluated the expression of eight of these
genes in a female and male T. vaginalis isolates. According
to the in silico analysis, the T. vaginalis 30end mRNA
processing machinery, comprises a similar complex and
protein factors that those described in Homo sapiens,
Arabidopsis thaliana,Saccharomyces cerevisiae and Ent-
amoeba histolytica. The complex contains several sub-
complexes, including cleavage and polyadenylation speci-
ficity factor (CPSF), cleavage stimulation factor (CstF),
cleavage factor I (CFIm) and cleavage factor II (CFIIm).
We demonstrated that genes tvpsf2p, tvcfi25, tvcpsf160,
tvcpsf73, tvfip1, tvpap1, tvpc4 and tvpabp are expressed in
male or female T. vaginalis isolates. Besides we identify
two different isoforms of TvPC4. T. vaginalis genome
contains most of genes encoding for 30end mRNA pro-
cessing, which may be transcriptionally active and could be
involved in the capping, splicing, cleavage and polyade-
nylation of mRNAs in this parasite. Further studies are
necessary to elucidate the biological meaning of our
findings.
Keywords Trichomonas vaginalis 30End mRNA
processing machinery Transcriptional profile,
polyadenylation, genome
Abbreviations
2DE Double dimensional gel electrophoresis
WB Western blot
2DE-WB Double dimensional gel electrophoresis
Western blot assay
Introduction
The trichomonosis is caused by the parasitic protozoan
Trichomonas vaginalis. Since the draft of T. vaginalis
genome was released in 2007 (Carlton et al. 2007), there
have been huge advances in the in silico characterization of
the molecules governing gene transcription by upstream
regulatory sequences. Repeats and transposable elements
are highly abundance in this genome and hampered mea-
surement of genome size, estimate in *160 Mb. A core set
of *60,000 protein-coding genes had been identified in T.
vaginalis, leading this parasite one of the highest coding
capacities among eukaryotes (Carlton et al. 2007). Inter-
estingly, the Inr promoter element was found in *75 % of
50of untranslated region (UTR) of its genes (Liston and
Miguel A
´ngel Del-Moral-Stevenel and Alma Villalobos-Osnaya had
equal contribution to this manuscript.
M. A
´. Del-Moral-Stevenel A. Villalobos-Osnaya
M. Lo
´pez-Casamichana L. I. Quintas-Granados
C. Lo
´pez-Camarillo J. M. F. Sa
´nchez S. Zarate-Guerra
M. E. Alvarez-Sa
´nchez (&)
Posgrado en Ciencias Geno
´micas, Universidad Auto
´noma de la
Ciudad de Me
´xico (UACM), San Lorenzo # 290, Col., Del Valle,
CP 03100 Me
´xico, Distrito Federal, Mexico
e-mail: elizbethalvarezsanchez@yahoo.com.mx
123
Genes Genom
DOI 10.1007/s13258-015-0268-3
Johnson 1999), supporting its central role in gene expres-
sion (Schumacher et al. 2003). In T. vaginalis, the protein
IBP39 involved in the promoter recognition, has also been
described (Liston et al. 2001). However, the eukaryotic
transcription machinery of this parasite metazoan seems
that protist (Carlton et al. 2007). About 65 genes in T.
vaginalis genome appear to contain an intron (Carlton et al.
2007), and the ones recognized are short and contains a 12
nucleotides conserved sequence (Van
ˇa
´c
ˇova
´et al. 2005). T.
vaginalis also has a potential upstream control element
(UCE) that was located 80 bp upstream of the transcription
start point (TSP). An element of transcription termination
was identified within 34 bp region set immediately down-
stream of the 28S coding sequence. The function of this
element depends upon polarity and the presence of both a
stretch of uridine residues (U’s) and a hairpin structure in
the transcript.
After the synthesis of mRNA in T. vaginalis, the tran-
scripts are polyadenylated, but this event is still unknown
in this parasite. Reports provide evidence that the 30poly
(A) tail is involved in a regulatory mechanism that provides
stability to mRNA. Moreover, the processing factors and
RNA sequence elements (cis-elements) located up- and
downstream, are involved in the recognition of the poly-
adenylation site (Proudfoot et al. 2002). Previous reports
proposed that the tetranucleotide UAAA functions as a
polyadenylation signal (PAS) in T. vaginalis (Espinosa
et al. 2002). Previously we reported that the mRNA
expression profile is different between a female or male T.
vaginalis isolates, for instance mp50 is differentially
expressed in both isolates (Quintas-Granados et al. 2013).
In order to determinate if the differences in the transcript
expression in both isolates might be due to the mRNA
processing machinery, we identify and analyze the tran-
scriptional profile of putative genes encoding for proteins
involved in the 30end processing of mRNAs, in female and
male T. vaginalis isolates.
Materials and methods
In silico analysis for the identification of the processing
machinery
The proteins involved in the mRNA processing in Homo
sapiens,Saccharomyces cerevisiae,Arabidopsis thaliana
and Entamoeba histolytica were used to perform an
alignment (BLASTP) by mean of the TrichDB (http://
trichdb.org/trichdb/), Swiss Prot (http://web.expasy.org/
docs/swiss-prot_guideline.html), and AmoebaDB (http://
amoebadb.org/amoeba/). Putative T. vaginalis proteins
were selected from BLASTP analysis according to the
following criteria: (i) at least 20 % identity and 35 %
homology to the query sequence; (ii) E value lower than
0.002; and (iii) absence of stop codons in the coding
sequence (Frith et al. 2010).
Model building
In order to propose a model for the 30UTR processing
machinery, we performed a functional protein association
network analysis using the STRING database (http://string-
db.org/). In this analysis, we include only the sequences,
which mRNA were observed by RT-PCR analysis. In order
to propose a model, these interactions were corroborated by
reference searching. The model includes the proteins which
expression was corroborate by RT-PCR analysis, such as
160 and 73 CPSF subunits, CFI25, PAP1, PAPB, FIP1, and
PC4. The model also includes the protein–protein interac-
tions results.
Primers design
The mRNA sequences of proteins involved in the mRNA
processing machinery from different species were used to
perform a BLASTN analysis in the TrichDB. The specific
primers were designed using the most divergent sequences
from each candidate gene (Table 1).
Trichomonas vaginalis culture
T. vaginalis isolates from a female and a male trichomo-
nosis patients (CNCD147 and HGMN01, respectively)
were axenically grown for 24 h in trypticase-yeast extract-
maltose (TYM) medium at pH 6.2 using 10 % heat-inac-
tivated horse serum (Gibco). Samples were taken at 24 h
for assessing the amount of parasites using a Neubauer-
counting chamber, and viability was measured by the try-
pan blue dye exclusion test (Alvarez-Sanchez et al. 2000).
RNA isolation
A sample containing 2 910
7
parasites was collected by
centrifugation at 9009gfor 5 min at 4 °C (AllegraTM X-22
Centrifuge, Beckman Coulter). The pellet was suspended in
1 ml of TRIzolR reagent (Invitrogen, Life Technologies,
Carlsbad, CA, USA), and the total RNA was extracted as
recommended by the manufacturer. RNA concentration was
determined spectrophotometrically using a NanoDrop 2000
(Thermo Scientific, Villebon Sur Yvette, France). All
260/280 ratios were between 1.8 and 2.1.
Semi-quantitative RT-PCR for analysis gene expression
1lg of total RNA was treated with DNase I, (Invitrogen)
according to manufacturer
´s instructions and then the
Genes Genom
123
treated-RNA was reverse-transcribed using the Superscript
Reverse Transcriptase Kit (Invitrogen) and the oligo-dT
(dT18) (10 pmol/ll) primer. PCR was performed in 12 ll
reactions containing 1 lg of cDNA, 10 pmol of each pri-
mer pair (Table 1), and 0.25 U of Taq DNA polymerase
(Invitrogen). Amplification was carried out in a GeneA-
mpPCR System 9700 thermal cycler (Applied Biosystems)
using the following steps 95 °C/30 s, 52 °C/30 s and
72 °C/1 min for 35 cycles.
As a positive control we amplified the112 bp of the b-
tubulin gene (Leon-Sicairos et al. 2004). The products were
analyzed on 3 % agarose gels and visualized by Red Gel
staining in triplicate assays. Steady-state mRNA levels
were semi-quantified by densitometric analysis using the
Quantity One Software (Bio-Rad, Hercules CA). Data are
the mean of three independent assays. Records from den-
sitometry of the housekeeping gene (b-tubulin) were used
to normalize the data.
qRT PCR for tvpc4
Transcript levels from tvpc4 were analyzed by qRT-PCR
using the QuantiTect SYBR Green PCR kit (Qiagen)
according to manufacturer
´s instructions. The relative
quantification of tvpc4 expression was calculated after the
threshold cycle (Ct) and was normalized with the Ct of b-
tubulin gene. All reactions including non template and RT
minus controls for each mRNA were run in duplicate. All
experimental data were expressed as means standard
deviation from three separate biological experiments. The
significance of the difference between means was deter-
mined by ANOVA with SigmaPlot software.
Double dimensional gel electrophoresis (2DE)
For proteomic maps, we used a previously reported pro-
tocol (Vazquez-Carrillo et al. 2011) with minimal modifi-
cations. Briefly T. vaginalis (20 910
6
) were collected by
centrifugation at 9009gfor 5 min at 4 °C and washed
three times with PBS pH 7.0. For the first dimension,
parasites were lysed in a final volume of 200 ll rehydration
solution (7 M urea, 4 % CHAPS, 70 mM DTT, 2 % IPG
buffer pH 4–7, trace bromophenol blue; Bio-Rad). The
supernatant was centrifuged at 13,0009gfor 10 min at
4°C to remove insoluble material, and samples of 200 lg
were applied to an IPG strip (17 cm, pH 4–7 linear; Bio-
Rad) for passive rehydration for 12 h. All isoelectric
focusing took place on a Protean IEF system (Bio-Rad) as
follows: step 1—gradient from 1 to 500 V over 1 h; step
2—1,000 V for 1 h; step 3—6,000 V for 2.5 h, and step
4—gradient until 20,000 V/h, Before the second dimen-
sion, proteins were reduced (10 mg/ml DTT) and alkylated
(25 mg/ml iodoacetamide) step-wise, 15 min for each step,
in equilibration buffer (6 M urea, 2 % SDS, 300 mM Tris–
Table 1 Primers used to evaluate the expression of T. vaginalis mRNA processing machinery genes
Gene/Locus Primer sequence Amplicon length (bp)
psf2p/TVAG_121040 F 50-AGGTGACACTATAGAATAGTTCCACATTCCAGCTTC-30186
R5
0-GTACGACTCACTATAGGGAGCTTCTCAACGTTGTTGTAG-30
cfi25/TVAG_495750 F 50-AGGTGACACTATAGAATACGTATTCCCTCGATCGG-30209
R5
0-GTACGACTCACTATAGGGATACCCATGTCCCAAAGC-30
cpsf160/TVAG_077030 F 50-AGGTGACACTATAGAATACAATCGAGGATTGCTTGG-30399
R5
0-GTACGACTCACTATAGGGACTCAGTCCGCAAAGAGAT-30
cpsf73/TVAG_437970 F 50-AGGTGACACTATAGAATAGACGCAATTGATCCTGC-30338
R5
0-GTACGACTCACTATAGGGACAGGGTAGCATGTCATCT-30
fip1/TVAG_463950 F 50-AGGTGACACTATAGAATAAGCCAAAGGATGAGAATCC-30229
R5
0-GTACGACTCACTATAGGGAGTTGAATTAATGCGCGTGT-30
pap1/TVAG_388620 F 50-AGGTGACACTATAGAATACCGAATTGCAGGCTAGTA-30262
R5
0-GTACGACTCACTATAGGGATTACGACACCTAAACGGTAAG-30
pc4/TVAG_255210 F 50-AGGTGACACTATAGAATAGACATCCTACGTCTTTCCTT-30177
R5
0-GTACGACTCACTATAGGGATTCCTTTCTTGCCAGGAA-30
pabp/TVAG_385330 F 50-AGGTGACACTATAGAATACAGTTCAGGAACCCAGA-30168
R5
0-GTACGACTCACTATAGGGAAAGTGGTTTGATGTAGAGT-30
b-tub F5
0-AGGTGACACTATAGAATACATTGATAACGAAGCTCCTTTACGAT-
30
159
R5
0-GTACGACTCACTATAGGGAGCATGTTGTGCCGGACATAACCAT-30
Fforward primer, Rreverse primer
Genes Genom
123
Cl pH 8.8, 20 % glycerol, and 0.002 % bromophenol blue)
at room temperature. Equilibrated IPG strips were sepa-
rated on 12 % SDS-PAGE gels and were run at 35 mA/gel
at room temperature until the tracking dye left the gel and
stained with Sypro Ruby following procedures described
by the manufacturer. Finally, gels were documented using
Gel Doc EQ (Bio-Rad). Image analysis was performed
using the pDQuest software (Bio-Rad).
Western Blot (WB) and 2DE-WB
Total protein extract from parasites (2 910
7
) were
obtained by TCA-precipitation as previously described
(Alvarez-Sanchez et al. 2000). Solubilized proteins were
resuspended in Laemmli buffer boiled, and loaded onto a
15 % polyacrylamide gel with an equivalent of 4 910
5
parasites/lane. Protein extracts separated by one or two
dimensional electrophoresis were blotted onto nitrocellu-
lose membranes and blocked with 5 % skim milk in PBS
(pH 7.0) for 18 h at 4 °C. Membranes were incubated for
18 h at 4 °C with anti-EhPC4 from Entamoeba histolytica
(1:100 dilution), a heterologous primary antibody as pre-
viously reported (Lo
´pez-Camarillo et al. 2010). Then the
blotted membrane was washed five times with a PBS pH
7.0–0.1 % Tween 20-buffer. The primary antibody was
detected with a secondary goat anti-mouse-IgG (H ?L)
horseradish peroxidase conjugate (goat source) (1:3,000
dilution, Invitrogen). The membrane was washed with PBS
pH 7.0–0.1 % Tween 20 and visualized using an enhanced
chemiluminescence ECL Plus Western Blotting Detection
System (GE Healthcare) according to the manufacturer’s
instructions.
Results and discussion
Most eukaryotic mRNA precursors (pre-mRNAs) under-
goes extensive processing, including capping, splicing,
cleavage and polyadenylation at its 30end, which is con-
trolled by sequence elements in the mRNA precursors (cis
elements) and by protein factors.
Pre-mRNA processing of the eukaryotic cell has a cru-
cial function. The 30end processing promotes the transport
from the nucleus to the cytoplasm (Vinciguerra and Stutz
2004), and the stability of mRNAs (Wickens et al. 1997;
Wilusz et al. 2001). In addition, the mRNAs are degraded
from the 50end, indicating the importance of capping
(Wickens et al. 1997). Moreover, the addition of the
poly(A) tail, which is the binding sequence of the
poly(A) binding protein (PABP), prevents degradation in
mammalians cells (Ford et al. 1997). The 30end processing
enhances the translation of mRNA into protein (Mandel
et al. 2008). Interestingly, this machinery is coupled to the
transcription and splicing events, since the 50end pro-
cessing complex interacts with transcriptional factors and
with the C-terminal domain (CTD) of Pol II to help to
control transcriptional initiation, and a proper poly(A) sig-
nal, which is essential for transcriptional termination
(Mandel et al. 2008). Since distinct T. vaginalis isolates
differentially expresses certain genes such as mp50
(Quintas-Granados et al. 2013), we investigated if this
difference might be generates by differences in the mRNA
processing machinery.
Since the mRNA processing machinery in Homo sapi-
ens,Saccharomyces cerevisiae,Arabidopsis thaliana and
Entamoeba histolytica had been highly described, we
selected these organisms for identification of putative
proteins involved in this processing in T. vaginalis.
According to in silico analysis and the identity, similarity
and E values, this amitochondriate parasitic protozoon
contains twenty nine genes encoding putative proteins
involved in the 30end mRNA processing machinery
(Table 2). Most of putative proteins were identified closer
to H. sapiens and S. cerevisiae orthologs. Sixteen of these
proteins were considered members of the complex. Com-
ponents of sub-complexes, including cleavage and poly-
adenylation specificity factor (CPSF), cleavage stimulation
factor (CstF), cleavage factor I (CFIm), and cleavage factor
II (CFIIm) were properly recognized. The others thirteen
were included into de group of proteins not associated with
complexes, which comprises others enzymes necessary for
the 30end mRNA processing such as symplekin and RNA
polymerase II C-terminal elements (CTD-RNApolII), and
protein factors as poly(A) polymerase, poly(A)-binding
protein and symplekin subunit (Mandel et al. 2008; Shi
et al. 2009). Furthermore, we found two versions for
CstF64 (TvCstF64-I and TvCstF64-II), CstF50 (TvCstF50-
I and TvCstF50-II), RNA15p (TvRna15p-I and TvRna15p-
II), PAP (TvPAP-I and TvPAP-II), and Glc7p (TvGlc7p-I
and TvGlc7p-II) (Table 2). Duplication is evident for many
other genes in T. vaginalis, and reflexes the massive gene
expansion inside the large genome of this pathogen
(Bethke et al. 2006).
The close relationship between proteins involved in the
30end processing machinery from H. sapiens, A. thaliana,
E. histolytica and S. cerevisiae reveals that this process has
been highly conserved throughout evolution (Hunt 1994;
Mandel et al. 2008; Zhao et al. 1999).
As a first step towards establishing the role of this
machinery in T. vaginalis, we evaluated the mRNA
expression of nine representative genes of this machinery
by semi-quantitative RT-PCR using specific primers
(Table 1), and two T. vaginalis isolates, obtained from a
male patient (HGMN01) (Fig. 1a) or a female patient
(CNCD147) with trichomonosis (Fig. 1b). The tvpsf2p,
tvcpsf160, tvcpsf73, tvfip1, tvpap1, tvpabp, tvcfi25, and
Genes Genom
123
tvpc4 transcripts were observed in both isolates and no
significant differences were found in their mRNA expres-
sion levels (Fig. 1c) (p [0.02). We found that tvcpsf160,
tvpabp, and tvpc4 transcripts had higher expression levels
in both isolates. The b-tubulin (btub) mRNA levels were
very similar in both isolates. According to qRT-PCR data
there is not a statistically significant difference (P=0.815)
between the tvpc4 transcript levels from female
(CNCD147) and male (HGMN01) isolates (Fig. 1d).
Interestingly, in spite of processing of 30end of mRNA is a
crucial event for handling of any transcript in the cellular
dynamic, our findings suggest that the genetic variability of
male and female isolates could differentially control the
mRNA steady state levels of some of these genes. How-
ever, as the main regulation might be occurring at trans-
lational and/or posttranslational level, a further evaluation
Table 2 Putative proteins involved in the mRNA processing machinery in Trichomonas vaginalis identified from orthologs
T. vaginalis
predicted protein
Locus name in
T. vaginalis genome
a
Orthologous protein
in H. sapiens, A. thaliana,
S. cerevisiae, E. histolytica
s
Accesion
number
c
I (%) S (%) E value
CPSF complex
TvCPSF160 TVAG_077030 HsCPSF160 Q10570 23 42 2.7 e-25
TvCPSF100 TVAG_363680 AtCPSF100 Q9LKF9 33 54 6.6 e-80
TvCPSF73 TVAG_437970 HsCPSF73 Q9UKF6 40 61 8.5 e-137
TvFIP1 TVAG_463950 HsFIP1 Q92797 42 59 1.9 e-06
TvFIPS5 TVAG_210560 AtFipS5 AED96990 20 42 6.7 e-16
CstF complex
TvCstF77 TVAG_343030 AtCstF77 Q8GUP1 29 50 1.1 e-51
TvCstF64-I TVAG_021720 HsCstF64 P33240 45 60 1.0 e-13
TvCstF64-II TVAG_211580 EhCstF64 C4LSN8 32 52 4.3 e-07
TvCstF50-I TVAG_374220 HsCstF50 Q05048 28 42 2.3 e-19
TvCstF50-II TVAG_493960 EhCstF50 C4LSW0 24 40 3.6 e-14
TvRna15p-I TVAG_267990 ScRNA15 P25299
P25299
32 65 1.1 e-09
TvRna15p-II TVAG_023270 ScRNA15 28 62 2.7 e-09
CFI complex
TvCFI25 TVAG_495750 EhCFIm25 C4M2T1 31 56 1.8 e-22
CFII complex
TvCLPS3 TVAG_162880 AtCLPS3 Q9SR06 27 47 6.0 e-22
TvCLP1 TVAG_317830 ScClp1 Q08685 25 43 2.0 e-06
TvPCF11 TVAG_100880 HsPCF11 O94913 36 47 8.0 e-05
Proteins not associated with complexes
TvPAP-I TVAG_388620 HsPAP-b Q9NRJ5 38 59 5.5 e-78
TvPAP-II TVAG_107950 AtPAP F4KE92 23 42 5.7 e-06
TvPAB1 TVAG_385330 ScPab1 P04147 37 56 6.9 e-55
TvPC4 TVAG_255210 HsPC4 P53999 43 66 1.3 e-07
Tv Symplekin TVAG_494090 Hs Symplekin Q92797 21 40 0.00036
TvFY TVAG_121040 AtFY F4K3Y7 35 52 1.4 e-63
TvSsu72 TVAG_229140 ScSsu72p P53538 35 50 3.1 e-29
TvHrp1 TVAG_245890 ScHrp1p Q99383 30 50 6.1 e-15
TvMpe1 TVAG_265310 ScMpe1 P35728 32 53 2.7 e-05
TvSw2p TVAG_016940 ScSwd2p P36104 24 43 2.0 e-11
TvGlc7p-I TVAG_101240 ScGlc7p P32598 56 75 1.6 e-96
TvGlc7p-II TVAG_379450 ScGlc7p P32598 57 74 2.0 e-96
TvhRrbp6 TVAG_480160 HsRrbp6 Q7Z6E9 20 41 2.0 e-24
a
TrichDB database
b
Hs Homo sapiens,ScSaccharomyces cerevisiae,AtArabidopsis thaliana,EhEntamoeba histolytica
c
Swiss-Prot/TrEMBL databases
Iidentity, Ssimilarity
Genes Genom
123
of protein expression will help us to better understating of
these processes.
In summary, these results reveal that T. vaginalis has at
least eight active genes that probably encode proteins
responsible for 30end mRNA processing. The results from
protein–protein interaction analysis (Fig. 2) help us to
build a model (Fig. 3). Since the mRNA from TvCPSF160,
TvCPSF73, TvCFI25, TvPAP1, TvPAPB, TvFIP1, and
TvPC4 were expressed, we include these proteins in the
model. Previous reports showed that CPSF interacts with
PAP, and according to our protein–protein interaction
analysis, TvPAP interacts with CPSF, for this reason,
TvPAP was included in the model. Moreover, according to
our protein–protein interaction analysis, TvCstF77, and
TvCstF64 interact with TvCPSF160 and TvPC4, respec-
tively; for this reason these proteins were included in the
model. It important to mention that these interactions were
previously reported (Calvo and Manley 2001; Zhao et al.
1999). Since Clp1 interacts with PAP and CFI25 (de Vries
et al. 2000), so TvClp1 was included in the model.
According to our protein–protein interaction analysis
CPSF30 and CPSF100 interacts with TvCPSF160 and
TvFIP1, for this reason, we include TvCPSF30 and
TvCPSF100. Kaufman and colleagues reported the inter-
action CPSF160 and FIP1 (Kaufmann et al. 2004).
TvCPSF160, TvCPSF73, and TvFIP1 could be consid-
ered as factors of CPSF complex, involved in cleavage and
polyadenylation of RNA in this pathogen. The 160 kDa
CPSF subunit is highly conserved in the eukaryotic king-
dom and participates with PAS in the polyadenylation
reaction. This subunit is involved in several biological
processes, such as transcription due its association with
transcriptional initiation factors (TFIID) (Dantonel et al.
1997), elongation (McCracken et al. 1997), and transcrip-
tional termination (Proudfoot 2004). We proposed that the
putative TvCPSF160 bounds to UAAA sequence located in
Fig. 1 mRNA expression profiles of genes involved in the 30end
mRNA processing of T. vaginalis. Parasites obtained from male
(HGMN01, a) and female (CNCD147, b) trichomonosis patients were
used to carry out RT-PCR assays to amplify of tvpsf2p (186 bp, lane
1), tvcfi25 (209 bp, lane 2), tvcpsf160 (399 bp, lane 3), tvcpsf73
(338 bp, lane 4), tvfip1 (229 bp, lane 5), tvpap1 (262 bp, lane 6),
tvpc4 (177 bp, lane 7), tvpabp (168 bp, lane 8), and btubulin
(159 bp, lane 9). The RT reaction (-) was included as a negative
control (lane 10). Arrow heads refer amplicon length. cDensitometric
analysis of RT-PCR products (gray bars for CNCD147 and black
bars for HGMN01 isolate). The data were normalized using btubulin
as loading control and represent the mean of three independent assays.
dtvpc4 mRNA levels. cDNA samples from female (CNCD147) and
male (HGMN01) isolates were analyzed by qRT-PCR. Bars indicate
the normalized Ct levels of tvpc4 mRNA in trichomonads from both
isolates (P=0.815)
Genes Genom
123
the T vaginalis mRNAs, as it has been reported in plants,
mammalians, yeast and amoeba (Bernstein and Toth 2012;
Lo
´pez-Camarillo et al. 2005; Shi et al. 2009). In this
context, TvCPSF160 could interact with other subunits of
this complex, such as TvCPSF100 and TvCPSF30 that also
were found in the T. vaginalis genome (Table 2). A. tha-
liana has two paralogous sequences for the 73 kDa subunit
of CPSF complex. The second sequence is called CPSF73-
II and is related to the flowering of this plant (Xu et al.
2004). Interestingly, CPSF73 was observed by RT-PCR
analysis.
In mammals, FIP1 functions as a polymerase-regulating
component of CPSF complex, which cooperates with
cleavage factors including the cleavage factor I (CFIm) and
that directly interacts with poly(A) polymerase (Helmling
et al. 2001). Accordingly, we suggest that TvFIP1 could be
working in cooperation with TvCFI25 and TvPAP1, both
expressed in this parasite, as has been reported in E. his-
tolytica (Lo
´pez-Camarillo et al. 2010; Pezet-Valdez et al.
2013).
T. vaginalis only has the 25 kDa subunit of the CFIm
complex (TvCFI25), although four factors have been
described in mammals (Zhao et al. 1999), and no one for
yeast. Moreover, T vaginalis also expressed genes for two
central proteins in polyadenilation event, the polyA poly-
merase (TvPAP1) and its binding protein (TvPAPB). In
mammals, it has been shown that nuclear PAP catalyzes
the poly(A) tail synthesis, after pre-mRNA 30end cleavage
(Martin et al. 2000) and then, PABP binds to the nascent
poly(A) tract increasing the efficiency of polyadenylation
(Kerwitz et al. 2003). Another two putative genes, here
evaluated: tvpsf2p and tvpc4, were transcriptionally active
in this parasite. Since tvpc4 mRNA was highly expressed,
we evaluate the TvPC4 expression using polyclonal anti-
bodies against E. hystolytica PC4 (EhPC4) (Fig. 4a).
Interestingly, a single band was immunorecognized by the
Fig. 2 T. vaginalis protein–protein interaction network using String
database. The proteins which mRNA was expressed were used to
analyze their interactions using the String database (http://string-db.
org/). The codes indicate the following T. vaginalis proteins:
TvPSF2P (A2D7P9), TvCFI25 (A2DVL4), TvCPSF160 (A2D9V0),
TvCPSF73 (A2FCF8), TvFIP1 (A2E247), TvPAP1 (A2DYJ5),
TvPC4 (A2FPY1), TvPAPB (A2FXM5). The protein interaction
between the protein hits is shown with lines that indicate different
predicted functional parameters (binding, expression, catalysis and
reaction)
Fig. 3 Working model for the 30end mRNA processing machinery of
T. vaginalis. Taking advantage of described machineries for H.
sapiens,S. cerevisiae, and E. histolytica, our bioinformatic analysis
and the transcriptional profile of several orthologous genes here
evaluated for T. vaginalis, we propose the referred diagram. Gray
elements refer the proteins, whose expression was corroborated at
transcriptional level in this work, and white elements denote other
proteins whose homologous genes have been found in the T. vaginalis
genome and their interactions were predicted by the protein–protein
interaction analysis
Fig. 4 TvPC4 is expressed in T. vaginalis.aTotal protein extract
from CNCD147 (lane 1) was transferred into a nitrocellulose
membrane to perform a Western blot using the preimmune sera (lane
2) or the heterologous a-EhPC4 antibody (lane 3). The arrowhead
shows the immune-recognized band of approximately 11.5 kDa that
correspond to TvPC4. bThe purified recombinant EhPC4 from E.
hystolytica was immuno-recognized by the preimmune sera (lane 1)
or by the a-EhPC4 antibody (lane 2). Arrowhead recognized a band
of approximately 22 kDa that correspond to native EhPC4
Genes Genom
123
heterologous a-EhPC4 antibody (Fig. 4a, lane 3) in
trichomonad total protein extract (Fig. 4a, lane 1) and no
band were observed using the preimmune serum (PI)
(Fig. 4a, lane 2). As a control, the a-EhPC4 antibody
recognized the purified recombinant EhPC4 as a single
band of approximately 22 kDa that correspond to native
EhPC4 as obtained in previous reports (Lo
´pez-Camarillo
et al. 2010) (Fig. 4b, lane 2) and the PI did not recognized
any band (Fig. 4b, lane 1).
Furthermore, we evaluate the expression of TvPC4 by
double dimensional gel electrophoresis (2DE). The total
protein extract of T. vaginalis CNCD147 was separated by
2DE (Fig. 5a) and was transferred into a nitrocellulose
membrane to perform a Western blot assay using the het-
erologous a-EhPC4 antibody (Fig. 5c). In the CNCD147
proteome showed in the Fig. 5a, 470 spots were obtained.
Interestingly, the a-EhPC4 antibody immunorecognized
two spots from the T. vaginalis proteome (Fig. 5c, spots 1
and 2). According to PDQuest software, the spot 1 and 2
had similar pI value (5.1), but different molecular mass,
12.3 and 11.7 kDa respectively (Fig. 5b, d). In addition, the
amino acid sequence from TvPC4 was analyzed using the
YinOYang Software in order to find the putative post-
translational modifications that could explained the results
obtained by 2DE-WB analysis. According to this in silico
analysis, TvPC4 has to putative O-glycosylation sites in the
T17 and S95 amino acid residues. However, we cannot rule
out the possibility that other posttranslational modification
such as phosphorylation or N-glycosylation could be
affecting the protein shifting.
In S. cerevisiae, PFS2p plays an essential role in 30end
formation by bridging different processing factors and
thereby promoting the assembly of the processing complex
(Ohnacker et al. 2000). Likewise, PC4 has been charac-
terized as a transcriptional positive coactivator at different
levels. Human transcriptional coactivator PC4 is involved
in cellular processes such as transcription to chromatin
organization. PC4 as an activator of non-homologous end
joining (NHEJ) and double-strand break (DSB) repair
activities (Batta et al. 2009).
Our findings constitute the initial efforts to evidence the
presence the 30end processing machinery for mRNA in T.
Fig. 5 TvPC4 has two
isoforms. Total protein extracts
from T. vaginalis CNCD147
was isolate by 2DE using IPTG
strips pI 4–7 and stained with
Sypro Ruby (a) or transferred
into a nitrocellulose membrane
to perform a Western Blot assay
using the heterologous a-EhPC4
antibody (c). Close-up from the
spots 1and 2(b) or the immune-
recognized spots (d). The
numbers (1and 2) indicate the
spots that were immune-
recognized by the antibody
Genes Genom
123
vaginalis, and will contribute to the further elucidation of
events regulating gene expression in this early-branch
protozoan.
Conclusions
Bioinformatic analyses of fully sequenced genomes are
useful to identify molecular machineries. The 30end
mRNA processing machinery is highly conserved in
mammalians, plants, yeast, E. histolytica and also in T.
vaginalis. Future directions would include protein expres-
sion and characterization, as well as protein–protein
interactions analysis, and functional assays in order to
contribute to the further clarification of mechanisms regu-
lating gene expression in this ancient eukaryotic pathogen.
Acknowledgments This work was supported by the Universidad
Auto
´noma de la Ciudad de Me
´xico (UACM) and by grants from
ICyTDF 221/2011, 18/2012 and CONACyT 83808. MADMS was
scholarship recipient from CONACYT (230118) and ICyTDF (179/
2011) and AVO was supported by ICyTDF (ICyTDF/SRI/70/2011).
Conflict of interest The authors declare that they have no com-
peting interests.
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