An mRNA-Binding Protein of The Ancient Protist Trichomonas Vaginalis

An in vivo UV-crosslinking assay using lysates of Trichomonas vaginalis was used to identify a 21.5-kDa protein called RNA-binding protein 1 (RBP1) that interacts with mRNA. A single transcript was detected using Northern blots that showed RBP1 mRNA was expressed in parasites. Nitrocellulose with RBP1 blotted after electrophoresis of total proteins from high versus low-iron parasites and probed with MAb CF130C3 to RBP1 were identical. A cDNA expression library screened with CF130C3 yielded a cDNA encoding for RBP1 of 189 amino acids with Mr 21.5-kDa and pI 11.01. The nucleotide sequence of the 3’-end revealed the presence of the ATTTA (AUUUA for mRNA) putative transcript destabilizing element and poly(A)-tail. Importantly, the amino acid sequence of RBP1 disclosed the presence of ribonucleoprotein (RNP) elements found within the functional domains of known RNP RNA-binding proteins. Furthermore, RBP1 was found to have two distinct RNP RNA-binding domains (RNP-1 and RNP- 2) found among eukaryotic RNA-binding proteins. Southern analysis indicated a single copy gene. In vitro RNA-binding assays using the mRNA subclones without 5’- and 3’-untranslated regions (UTRs) of the ap51-1 adhesin of T. vaginalis showed the binding of RBP1 to the 3’-UTR. Finally, in addition to T. vaginalis , RBP1 was detected among three different members of the family Trichomonidae . This is the first demonstration of a protein that binds to the 3’-UTR of transcripts identified for this ancient protist. 21.5-kDa RNA-binding protein (RBP1) was isolated, and RBP1 was shown to bind mRNA In vivo using UV-crosslinking experiments. RBP1 was present in equal amounts regardless of the iron status of parasites and was constitutively expressed. The sequence of recombinant cDNA clones indicated that RBP1 contains both primary domains and secondary structural elements characteristic of members of the RNA-binding protein family [14-16]. We show that RBP1 binds to motifs at the 3’-untranslated region (3’-UTR) of the mRNA of adhesin protein AP51-1. Thus, this work established an initial framework into the regulation of T. vaginalis gene expression.


Introduction
Trichomonas vaginalis is responsible for the most common, nonviral sexually transmitted infection (STI) worldwide [1]. A hallmark of this STI is the asymptomatic nature of infection among both women and men. In addition, there is an association of infection in women with the development of other genitourinary complications, including adverse pregnancy outcome [2], increased susceptibility to HIV infection [3], and cervical neoplasia [4].
Interestingly, the transcripts of the adhesin proteins differ in the 3'-untranslated regions (UTR) [5,9]. Further, several adhesin mRNA transcripts possess AU-rich elements (AREs) [5,9] known to regulate RNA instability in other systems [10]. One of the adhesins, AP51-2, was found not to respond to iron in up-regulation of expression [5]. Therefore, members within these multigene families may be differentially regulated. To date, however, no regulatory proteins have been identified that may play a role in any of these parasite responses to the extracellular environment.
With the goal of understanding the mechanism(s) of regulation of gene expression in T. vaginalis and given the knowledge base available on post-transcriptional regulation in other systems [11-21.5-kDa RNA-binding protein (RBP1) was isolated, and RBP1 was shown to bind mRNA In vivo using UV-crosslinking experiments. RBP1 was present in equal amounts regardless of the iron status of parasites and was constitutively expressed. The sequence of recombinant cDNA clones indicated that RBP1 contains both primary domains and secondary structural elements characteristic of members of the RNA-binding protein family [14][15][16]. We show that RBP1 binds to motifs at the 3'-untranslated region (3'-UTR) of the mRNA of adhesin protein AP51-1. Thus, this work established an initial framework into the regulation of T. vaginalis gene expression.
The vector pCDNAII was used to generate the cDNA library [17], and all PCR products were cloned into pCR2.1. Induction of RBP1 expression in recombinant E. coli was done with 1 mM isopropyl-β-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, MO, USA).

In vivo UV-irradiation and purification of the mRNA complex
Parasites (3 × 10 8 ) washed three times with cold PBS were suspended in 1 ml PBS containing 100 μM Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma). Organisms were placed in individual wells of 24-well culture plates and then exposed for 5 minutes (min) at 4°C to a wavelength of 254 nm at 4.5cm.
Complete lysis of trichomonads was achieved by passaging three times through a sterile 18-gauge needle and examining by darkfield microscopy. The lysate was incubated at 90°C for 5 min followed by ice for 2 min, and then 0.5 M LiCl and 70 mg oligo(dT)-cellulose (Thermo Fisher Scientific, Waltham, MA USA) were added. The mixture was incubated overnight at 4°C with constant agitation followed by washing the oligo(dT)-cellulose with 70 ml of 10 mM Tris-HCl, pH 7.4, containing 500 mM LiCl prior to transfer to an RNA spin column (Thermo Fisher) for elution of mRNA-protein complexes with 10 mM Tris-HCl, pH 7.4. RNA was digested with 10 U RNase cocktail (Stratagene, La Jolla, CA USA) for 1 hour (h) at 37°C. Proteins were precipitated overnight with 10% trichloroacetic acid. The proteins were pelleted by centrifugation and washed three times with cold PBS. The final pellet was solubilized in 20 µl electrophoresis dissolving buffer [5,7,18]. After boiling for 3 min, solubilized proteins were electrophoresed as described below.

Isolation of C23-1 cDNA encoding recombinant RBP1 and sequencing rbp1
The MAb CF130C3 to RBP1 was generated as has been extensively described [18,19]. The cDNA expression library screened using MAb CF130C3 isolated a recombinant E. coli with a cDNA insert (Figure 1 Part A), and colonies of E. coli colonies were subjected to insert analysis [5,17]. The recombinant cDNA E. coli clone labeled C23-1 encoded RBP1.

Figure 1:
Complete nucleotide and amino acid sequences of RBP1. Part A is the 605-bp nucleotide sequence of C23-1 cDNA within which is the 566-coding sequence of RBP1 from start codon (ATG) to stop codon (TAA). The underlined and italics in addition to the dashed sequence were missing in the original cDNA clone and were acquired as described in Methods. The 3'-UTR nucleotide sequence shows a putative AU-rich element (ATTTA) (double underlined, AUUUA for mRNA) and the corresponding poly(A) tail. Part B is the 189 amino acid sequence with the corresponding ribonucleotide-binding sequences labeled RNP 1a, RNP 1b, RNP-2a, and RNP-2b. Part C is the predicted secondary structure with the βαββαβ organization consistent with that reported for other RNA-binding proteins [14].

Figure 2:
Northern and Southern analyses of RBP1. Part A Northern hybridization using 10 μg of RNA electrophoresed in 1% denaturing agarose get followed by blotting onto Zeta-probe membrane. Hybridization was under high stringency conditions with nick-translated, 32 P-labeled C23-1 cDNA. The single band of ~1-kbp in size encodes for RBP1. Part B is of Southern analysis of genomic DNA digested to completion with restriction enzymes, as indicated, and electrophoresed in 0.8% agarose gel followed by transferring to Zeta-probe membrane. Hybridization under high stringency with 32 P-labeled C23-1 cDNA revealed single bands indicative of the single copy nature of rbp1.
Methods for Northern analysis was performed as before [5,17,20]. Purified RNA obtained by FastTrack ® 2.0 as specified by the manufacturer (Thermo Fisher) was from trichomonads grown in normal TYM-serum medium. RNA (10μg) was electrophoresed in 1% denaturing agarose and then transferred onto Zeta probe membrane for hybridizing at high stringency conditions with nicktranslated 32 P-labeled C23-1 cDNA.

Determination of mRNA-binding location by RBP1 using competitors in UV-crosslinking assays using RBP1 in lysates and recombinant RBP1 in recombinant E. coli extracts
To show the location of RBP1 binding to mRNA in competition experiments, the T. vaginalis adhesin protein ap51-1 gene [5] was used to generate the full-length ap51-1 transcript and ap51-1 subclones (Table 1) (Table 1), 50 μCi 32 P-UTP (NEN) was included in the reaction. RNAs were ultimately passed through MicroSpin TM G-25 columns (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).
All in vitro RNA competition experiments used either lysates of parasites or French press extracts of E. coli expressing the cDNA C23-1 that encodes for recombinant RBP1 [3]. Competitor, unlabeled RNA (2 pmol) was added to either lysate or extract and incubated for 10 min at RT followed by addition of 20 fmol radiolabeled RNA probe in 10 mM Tris-HCl (pH 8.0), 15 mM KCl, 1 mM MgCl 2 , 5 U RNase Block, 0.2 μM DTT, 5 % glycerol, 0.2 μg tRNA, and 100 μg/ ml bovine serum albumin [20]. The reactions were incubated for 20 min, and RNA was crosslinked to RBP1 by exposure to UV for 5 min on ice. The mixture was then digested with 10 U RNase Cocktail for 1h at 37°C. The proteins were subjected to SDS-PAGE for autoradiography. The intensity of bands in X-ray films were scanned for quantitation.  b. Primers corresponding to the 5'-end of each gene (S) include ATG start codon (bold). Primers for the 5'-ends of clones SC5.2 and SC5.3 also contain the Inr-like promoter element (underlined) [27]. c.
Sense (S) and antisense (A) primers are relative to the ap51-1 gene as described before [5].

Nucleotide sequence accession number
The GenBank accession number for the nucleotide sequence of rbp1 obtained in this study is AF084572. The accession number for the ap51-1 adhesin gene of T. vaginalis is U87093.

Identification of a T. vaginalis RNA-binding protein
We identified the RNA-binding protein RBP1 from T. vaginalis by performing In vivo UV crosslinking experiments using organisms grown in normal TYM-serum medium. As can be seen in Figure   3, The sequences of the RBD1 and RBD2 do not possess homology at the amino acid level either to each other or to other RNA-binding proteins [14]. Finally, the amino acid sequence indicates a lack of a transmembrane domain.
Furthermore, Part C illustrates the secondary structure predicted for each RBD and consists of a βαββαβ organization, as has been reported for other RNA-binding proteins [14]. These data indicate that upon protein folding, the structure is expected to have a four-stranded anti-parallel β sheet on which lie two perpendicular α-helices [16], an arrangement that may occur twice in the RBP1 relative to the four RNP sequences.

The mRNA transcript and single copy of the rbp1 gene
We next performed Northern analysis of 10 μg purified RNA derived from trichomonads grown in normal TYM-serum medium.
The RNA was electrophoresed in 1% denaturing agarose followed by blotting onto Zeta probe membrane and probing with nicktranslated 32 P-labeled C23-1 cDNA. Figure 2 (part A) shows the radioactive probe detected a single band of ~1-kb in size and sufficient to encode the transcript for RBP1.
Next, purified genomic DNA was digested to completion with AvaI, EcoRI and EcoRV followed by electrophoresis in 0.8% agarose gel. The gel was then transferred onto Zeta-probe membrane and hybridized with nick-translated 32 P-labeled C23-1 cDNA. Only single bands were obtained that show RBP1 is single copy. Genomic DNA was also digested to completion with HincII, BamHI, and HindIII, which gave similar single band patterns (data not shown). These data suggest that rbp1 is a single copy gene.

RBP1 associates with the 3'-UTR
In Figure 6 data are presented from UV-crosslinking experiments using lysates of T. vaginalis organisms (part A) and extracts of recombinant E. coli expressing RPB1(part B) to examine the region of a transcript recognized by RBP1. RNA-binding assays were performed using in vitro-transcribed ap51-1 adhesin RNA and subclones [5]. Table 1   coli extracts with subclone C23-1 encoding RBP1. For these experiments RNA-binding assays were performed using in vitro-transcribed ap51-1 adhesin RNA reported before for the trichomonad AP51 adhesin protein [5]. Unlabeled AP51 subclones for competition experiments (Table 1)   to both natural RBP1 and recombinant RBP1 by 65% and 40%, respectively. Therefore, these data indicate that RBP1 recognizes motifs found within the 3'-UTR.

rbp1 is not unique to T. vaginalis
Finally, we determined rbp1 was conserved within the family Trichomonidae. PCR was performed using genomic DNA from Pentatrichomonas hominis (intestinal), Tritrichomonas foetus (bovine), T. tenax (oral) and T. vaginalis. Primers P1 and P3 were as described in Methods. As can be seen in Figure 7, PCR products of identical size were amplified from all four genomes. Each of these products was subsequently cloned and sequenced. The sequence data showed the PCR products were identical to the sequence obtained for T. vaginalis (Figure 1 Part A). Genomic DNA from the different trichomonads was subjected to PCR using the C23-1-derived primers P1 and P3 (Methods). PCR products were electrophoresed in 1% agarose and stained with ethydium bromide. Reactions were performed with DNA from T. suis, T. foetus, P. hominis, and T. vaginalis. Lane 1 represents to 100-bp DNA ladder. The DNA sequence of the PCR product for all species were identical (not shown).

Discussion
In this report the T. vaginalis RBP1 was the only protein detected by the In vivo UV-crosslinking assay from T. vaginalis regardless of the iron status or parasites (Figure 4), indicating that RBP1 is constitutively expressed. This single copy gene is 566 nucleotides and encodes for a protein of 189 amino acids in length.
Consistent with other known ribonucleotide-binding proteins (RNPs), RBP1 also had consensus sequences to other RNA-binding domains (RBDs) [14], and the two RNA-binding domains (RBD1 and RBD2) had the secondary structure predictions of the βαββαβ organization [14,16]. Although extremely diverse in function, RNPs have conservation of primary, secondary, and tertiary structure within each RNP domain [14]. Each of the two RBDs of 1 to 85 and 90 to 167 amino acid sequences contains two RNP elements and conserved hydrophobic residues required for proper folding of the peptide and RNA-binding activity [14,15]. Although requiring experimental verification, the initial characterization of RBP1 appears to have the features predicted for these proteins. The sequence data may indicate that this RNA-binding protein also has a four-stranded anti-parallel β sheet on which lie two perpendicular α-helices ( Figure 1, part C) as reported for other RBPs [14,15].
The data using full-length subclones of the T. vaginalis adhesin protein AP51 (Table 1 and Figure 6) indicates that RBP1 interacts with the 3'-UTR of mRNA transcripts. It is noteworthy that the transcripts of the adhesin proteins of T. vaginalis differ in the 3'-untranslated regions (3'-UTR) [5,9]. Thus, it will be important to examine the interaction of RBP1 with the distinct transcripts to delineate at the molecular level the exact sequences recognized by this RNA-binding protein. Interestingly, the 3'-UTR nucleotide sequence had a putative AU-rich element (ARE) (ATTTA) reported to confer instability to mRNA species in other eukaryotes [10] and had the corresponding poly(A) tail. AREs have repeatedly been shown to be bound by RBPs, resulting in altered transcript stability [10,22]. Located within 3'-UTRs, this motif has also been found in several T. vaginalis transcripts [5,17,20], although its function remains unknown. For example, it has been reported that transcripts for at least three adhesins and the phenotypically varying immunogen P270 possess either the AUUUA pentamer or UUAUUUAUU nonamer ARE in their 3'-UTRs [3,9,20]. This may suggest that the rbp1 3'-UTR with this motif may have functional significance (Figure 1).
It is known that one of the four RNA-binding domains of yeast RBP associates with poly(A) sequences In vivo [23] and interacts with eukaryotic translation initiation factor 4G (eIF4G) [24]. That RBP1 interacts with poly (A) cannot be excluded, and future work is needed to further define the exact binding sites by which RBP1 associates with mRNAs of T. vaginalis. Although speculative, the basic nature and high pI of 11.01 of RBP1 may promote associations

American Journal of Biomedical Science & Research
Copy@ JF Alderete of this protein with RNA. In addition, many characterized RBPs interact with other proteins [24][25][26][27]. Therefore, identification of proteins binding to RBP1in trichomonads is needed to further understand the function of RBP1 In vivo. It should be noted that proteins unrelated to RBP1 that interact with RNA stem loop structures and that may be involved in regulatory functions have been identified in T. vaginalis [28,29].
Finally, that RBP1 has been identified in members of the family Trichomonidae, all primitive amitochondriate protists, may suggest that this is a product early in evolution. Further, this identical conserved rbp1 gene among the Trichomonidae (Figure 7) may indicate that this gene was acquired in an ancestor common to all members of the family.

Conclusion
This report shows the identity of RBP1, a 21.5-kDa RNAbinding protein that interacts with the 3'-UTR of the ap51-1 adhesin of T. vaginalis. Synthesis of RBP1 was unaffected by the levels of iron during growth and is constitutively expressed. Two distinct ribonucleotide-binding domains (RBDs) were identified in RBP1. RBD1 from amino acid 1 to 85 and RBD2 from 90 to 167 had no homology at the amino acid sequence level within the protein and with other RNA-binding proteins [14]. The secondary structure predicted for each RBD consisted of a βαββαβ organization, as has been previously reported [14]. RBP1 is without a transmembrane domain. The single copy rbp1 gene is present among different members of the family Trichomonidae. This is the first RNA-binding protein identified for this ancient protist.

Conflict of Interest
I declare that there are no conflicts of interest. I alone designed the study and was responsible in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.