Trypanosoma cruzi RNA-binding protein DRBD3: perinuclear foci formation during benznidazole exposure

CHAME, DANIELA F.; LAET-SOUZA, DANIELA DE; VIEIRA, HELAINE G. S.; TAHARA, ERICH B.; MACEDO, ANDREA MARA; MACHADO, CARLOS RENATO; FRANCO, GLÓRIA REGINA

doi:10.1590/0001-3765202420240321

Abstract

Benznidazole (BZ) is the trypanocidal compound of choice for Chagas disease, a neglected tropical disease in the Americas. However, this drug often fails to cure the infection. The regulation of gene expression in Trypanosoma cruzi, the causative agent of Chagas disease, is based on post-transcriptional mechanisms. When environmental changes cause translational arrest, RNA-binding proteins, and their target mRNAs assemble into cytoplasmic bodies, known as RNA granules, which act as RNA sorting centers. We have characterized the T. cruzi RNA-binding protein DRBD3, which has two RRMs domains, and a C-terminal low-complexity sequence rich in proline and glutamines. Using a tagged form of TcDRBD3 (rTcDRBD3), we showed that this protein resides in the cytoplasm, but localizes into perinuclear cytoplasmic foci after BZ exposure. RNA staining after BZ also showed that this molecule accumulates into perinuclear cytoplasmic foci. Moreover, BZ and puromycin treatment enhanced the colocalization of rTcDRBD3 and RNA, suggesting that TcDRBD3 granules repertoire harbors RNAs released from polysomes. Under starvation, rTcDRBD3 granules localized throughout the cytoplasm and also increased in number in the presence of puromycin. Our results suggest that TcDRBD3 accumulates into perinuclear granules that harbor RNA and also that its localization varies according to the type of stress.

Key words
Benznidazole stress; DRBD3; perinuclear granules; RNA-binding proteins

INTRODUCTION

The complex life cycle of trypanosomes requires rapid metabolic and morphological changes, and the adaptation to different hosts and stress conditions. The regulation of Trypanosoma cruzi gene expression relies on several layers of post-transcriptional events (Schwede et al. 2012). Besides the trans-splicing reaction, the fate of mRNAs depends on elements present in their primary structure, such as cis-regulatory elements in their untranslated regions, which in turn interact with trans-acting factors such as RNA-binding proteins (RBPs) (Kramer & Carrington 2011, Clayton 2013). Moreover, RBPs and their target RNAs may assemble in non-membranous cytoplasmic compartments, known as RNA granules, with an increase in assembly when translation is stalled (Eulalio et al. 2007, Anderson & Kedersha 2008, Saarikangas & Barral 2016). The formation of membraneless bodies allows the spatiotemporal activation or inactivation of cellular processes and thereby, prompting cellular adaptation to a particular stress condition (Courchaine et al. 2016).

T. cruzi has gained attention for its capacity to overcome several stress conditions, including prolonged periods of nutritional stress (Kollien & Schaub 1998). The fact that RNA granules are naturally present in epimastigotes under starvation conditions in the insect intestine suggests that their assembly protects mRNA during the stress response (Cassola et al. 2007). Although the function of RNA granules remains elusive in trypanosomes, several studies have focused on the identification of their components and their differences compared with the human host in order to identify potential targets for therapeutic applications (Holetz et al. 2007, Kramer 2014, Fritz et al. 2015).

The RNA binding-protein DRBD3 (also known as PTB1) is one of the most well-characterized stress granules (SGs) components in starved Trypanosoma brucei (Fernández-Moya et al. 2012). Additionally, high throughput sequencing analysis of T. brucei DRBD3-bound mRNAs showed that this protein acts on an RNA regulon basis and is crucial for the maintenance of cellular homeostasis, considering that the most overrepresented biological processes from isolated mRNAs are energy production and protein translation (Das et al. 2015). Likewise, T. cruzi has a DRBD3 homolog. Therefore, this is the first study aimed at characterizing T. cruzi DRBD3 (TcDRBD3).

Our results showed that TcDRBD3 harbors two RNA-binding domains and a C-terminal low-complexity sequence, suggesting that it functions as an RBP in T. cruzi. In parasites expressing a recombinant form of TcDRBD3, treatment with BZ elicited an accumulation of the protein as perinuclear granules, which were colocalized with RNA. Moreover, we observed that the localization of DRBD3 granules were dependent on the type of stress, since starvation caused the formation of DRBD3 foci throughout the parasite cytoplasm.

MATERIALS AND METHODS

T. cruzi DRBD3 gene characterization

T. cruzi CL Brener DRBD3 gene characterization was performed using the information retrieved from the TriTrypDB Kinetoplastid Genomics Resource database (Aslett et al. 2009). Characterization of gene features was performed based on the following parameters: chromosome location, genome location, gene copy number, and sequence similarity analysis for both Esmeraldo-like and non-Esmeraldo-like haplotypes, and also with the T. brucei DRBD3 gene, (Tb927.9.8740) from the TREU927 strain, previously published (De Gaudenzi et al. 2005).

Conserved domains and secondary structure predictions

Conserved domains were identified by sequence searches using the software InterProScan (Jones et al. 2014) and the NCBI Conserved Domains Database (Marchler-Bauer & Bryant 2004). Queries were performed using the TcDRBD3 sequences of both CL Brener Esmeraldo-like (TcCLB.506649.80) and non-Esmeraldo-like (TcCLB.508349.39) haplotypes. Secondary structure was predicted using the software PSSpred (Yan et al. 2013). Results were compared to the T. brucei ortholog, Tb927.9.8740, and a graphical representation was constructed using the software Illustration for Biological Sequences (Liu et al. 2015). Protein sequences were aligned using the T-coffee aligner (Notredame et al. 2000), and a graphical representation of the alignment was prepared using the Boxshade server. The identity between sequences was obtained using the UniProt BLAST tool (The UniProt Consortium 2017).

Parasite growth, cloning, and transfection

T. cruzi clone CL Brener epimastigotes were cultured at 28˚C in liver infusion tryptose medium, pH 7.4, supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies), streptomycin sulfate (100 μg mL-1), and penicillin (100 units mL-1) (Life Technologies) (Camargo 1964). For cloning procedures, a 173-bp DNA fragment was synthesized harboring flanking 5’-NheI and 3’-SalI and internal XbaI and XhoI restriction sites. An SBP tag and a C-terminal 6-His tail were also included (Epoch Life Science, Inc). The pROCK_SBP/6HIS vector was constructed by cloning this 173-bp sequence into the pROCK_GFP_Neo vector, as previously described (DaRocha et al. 2004). After digestion, the flanking 5’-NheI and 3’-SalI sequences formed compatible cohesive ends with the XbaI and XhoI restriction sites in pROCK_GFP_Neo. The T. cruzi DRBD3 gene (TcCLB.506649.80) was amplified by PCR using primers 5’-TCT AGA GAT GTA CGG TCA GCA GTT TCC-3’ and 5’-CTC GAG GCT CCA CTG CTG TGG GAT AA-3’, which contain XbaI and XhoI restrictions sites, respectively. As a negative control, a GFP-coding sequence harboring XbaI and XhoI restrictions sites at its ends was amplified (Teixeira et al. 1999). The amplified products were cloned into the new XbaI and XhoI restriction sites introduced in the pROCK_Neo_SBP/His vector to create the pROCK_Neo_SBP/His_DRBD3 and pROCK_Neo_SPB/His_GFP. Diagrams of these vectors are shown in Figure S1 (Supplementary Material - Figure S1). The vectors were amplified using eletrocompetent E. coli DH5α cells and were isolated using a NucleoSpin Plasmid NoLid kit (Macherey-Nagel). The sequences of the purified plasmids were confirmed by Sanger sequencing (Sanger et al. 1980), and they were then digested with the restriction enzyme NotI (New England Biolabs Inc). CL Brener epimastigotes transfections were performed using an Amaxa Nucleofector Electroporator (Lonza), according to a previously published method (Burkard et al. 2011). Parasites containing the transfected plasmids were selected after four weeks of culturing in the presence of 200 mg mL-1 of the Geneticin™ Selective Antibiotic (G418 Sulfate) (Thermo Fisher Scientific).

Western blot analysis

CL Brener epimastigotes were washed in PBS, pH 7.4, and boiled in Laemmli sample buffer. The lysate was quantified and 30 mg of the total protein was subjected to 12% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (PVDF), blocked for 2 h with PBS supplemented with 5% bovine serum albumin (BSA), and then incubated overnight with a primary anti-His-probe antibody (AD1.1.10, Santa Cruz Biotechnology) diluted (1/1,000) in PBS supplemented with 3% BSA and 0.05% Tween 20. The membranes were washed three times with 0.05% Tween 20 in PBS and then incubated for 1.5 h with the secondary antibody (Amersham™ ECL™ Anti-Mouse IgG, horseradish peroxidase-linked whole antibody, GE healthcare life sciences) at a dilution of 1/10,000 in PBS supplemented with 3% BSA and 0.05% Tween 20. PVDF membranes were rewashed and signal detection was performed using an Immobilon Western Chemiluminescent HRP Substrate (Merck-Millipore). Immunostaining was imaged using a Gel Logic 1500 imaging system (Kodak).

BZ treatment and nutritional stress

BZ (LAFEPE) treatment, and doses used were performed as described previously (Rajão et al. 2014). Briefly, epimastigote cultures of 1×107 cells/mL were incubated with 120 or 240 μM BZ for 24 and 48 h. Cells were counted daily in a Neubauer chamber after vital staining with erythrosine. Results are presented as growth curves compared to the untreated cells. Samples were collected at 24 and 48 h for immunofluorescence assays. Nutritional stress was induced according to a previously published method (Cassola et al. 2007). Briefly, the cells were cultured for 48h in PBS to induce starvation. Puromycin (Sigma-Aldrich) was added at 200 μg mL-1, 1 h before sample collection. All the experiments were performed in triplicate.

Immunofluorescence assays

Parasites were fixed for 20 min in a 4% formaldehyde solution in PBS, washed twice in PBS, spread directly on poly-L-lysine–coated slides, air dried for 20 min, and finally washed with PBS. Samples were blocked and permeabilized for 0.5 h with 2% BSA and 0.5% Triton X-100 (Sigma-Aldrich) in PBS and then incubated overnight with primary anti-His-probe antibody (AD1.1.10, Santa Cruz Biotechnology) diluted 1/100 in PBS supplemented with 2% BSA. Parasites were washed three times with PBS and incubated for 1.5 h with Alexa Fluor® 488 conjugated goat anti-muse IgG antibody (H&L) (ab150113, Abcam) diluted 1/600 with PBS supplemented with 5% of normal goat serum (Thermo Fisher Scientific, MA, US). DNA in the nuclei and kinetoplasts was stained for 20 min using Hoechst 3342 diluted 1/1,000 in PBS (Thermo Fisher Scientific) and RNA was stained using the Click-iT™ RNA Alexa Fluor™ 594 Imaging Kit (Thermo Fisher Scientific), following the manufacturer instructions. The 5-ethynyl-uridine reagent (EU) was incubated immediately after BZ and nutritional stress treatments. Slides were then mounted with ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific) and observed using an Apotome.2 (Zeiss) microscope at the Centro de Aquisição e Processamento de Imagens (CAPI) at the Universidade Federal de Minas Gerais (UFMG).

Statistical analysis

Data are presented as mean ± standard deviation. All growth curves were generated using Prism 5.0 software (GraphPad).

RESULTS

T. cruzi DRBD3 gene characterization

Using the TriTrypDB Kinetoplastid informatics Resource database, we identified two syntenic DRBD3 sequences in the T. cruzi CL Brener strain genome: the Esmeraldo-like allele (TcCLB.506649.80) on chromosome 8-S, and the non-Esmeraldo-like allele (TcCLB.508349.39) on chromosome 8-P. The non-Esmeraldo-like sequence is annotated as a gene fragment. The Esmeraldo-like allele is intronless, consisting of 978 bp, while the non-Esmeraldo-like gene fragment is 495 bp. Sequence alignment showed 79% identity between the Esmeraldo-like allele and T. brucei DRBD3 (TbDRBD3), and 83% identity for the non-Esmeraldo-like gene fragment. Due to the non-Esmeraldo-like partial nature, further analyses focused on the Esmeraldo-like allele.

The Esmeraldo-like allele predicts a product of 325 amino acids. Alignment of TcDRBD3 Esmeraldo-like and TbDRBD3 revealed 84.7% identity (Figure 1a). A domain prediction using InterProScan, identified two RNA recognition motifs (RRM) in TcDRBD3, flanked by disordered regions and a C-terminal region rich in proline and glutamine (Figure 1b). BLAST searches indicated 94.7% identity between T. cruzi and T. brucei RRM1 and 92.6% identity for RRM2. NCBI conserved domain analysis classified both RRM domains in the polypyrimidine tract-binding protein (PTB) subfamily (Figure 1b). TcDRBD3 and human PTB1 alignment showed 28.6% identity overall, with 32.9% and 34.2% identity for RRM1 and RRM2, respectively. Secondary structure prediction indicated that TcDRBD3 contains five α-helices and nine β-sheets within a coil region, mirroring the secondary structure arrangement found in TbDRBD3 and human PTB1 (Figure 1c).

Figure 1
Sequence alignments and structural comparison. a) Alignment of DRBD3 protein sequences of T. brucei Tb927.9.8740 and T. cruzi TcCLB.506649.80 (Esmeraldo-like). Identical residues are marked with black, while conservative amino acid substitutions are shaded with gray. b) Schematic diagrams of conserved domains and motifs predicted for TbDRBD3 (Tb927.9.8740) and TcDRBD3 (TcCLB.506649.80), compared with human PTB1 (NP_002810.1). RRM: RNA recognition motif, P/Q-rich: glutamine and proline-rich region. c) Secondary structure predictions for TbDRBD3 and TcDRBD3, compared with human PTB1.

T. cruzi epimastigotes expressing rTcDRBD3 exhibited similar growth rates to wild-type cells under normal and stress conditions

We generated a T. cruzi population transfected with the Esmeraldo-like haplotype, TcCLB.506649.80, carrying a streptavidin-binding peptide (SBP) tag and a histidine (6-His) tail to facilitate further detection (rTcDRBD3). A diagram of the recombinant plasmid is shown in Figure S1. All transfection procedures were approved by the National Technical Commission of Biosafety for genetically modified organisms (process number 01250.026820/2017-07). rTcDRBD3 was detected in whole cell extracts by western blotting assays. A green fluorescent protein (GFP) control was also detected by western blotting using an anti-His-probe antibody (Figure S2). Transfected epimastigotes under normal conditions and after BZ exposure, were counted daily using erythrosine staining to assess whether rTcDRBD3 expression affected cell growth. The observed growth rates for transfected (rTcDRBD3) and untransfected (wild-type) parasites were similar under normal conditions (Figure 2). BZ treatment was performed using two doses, 120 µM and 240 µM. After 120 µM BZ treatment, we observed cell growth arrest until 96 h (Figure 2a), whereas 240 µM BZ treatment caused cell death after 48 h (Figure 2b). There were no statistical differences between cells transfected with rTcDRBD3 and control cells (wild-type and GFP-transfected cells) after treatment with BZ.

Figure 2
Growth curves of DRBD3 and GFP transfected epimastigotes. Growth curves of rTcDRBD3, GFP-transfected parasites exposed or not to a) 120 μM or b) 240 μM BZ. The number of viable cells was determined daily by erythrosine staining. Values are expressed as the means of three replicates and error bars represent the standard deviation.

BZ induced the assembly of DRBD3 into perinuclear foci

To determine whether rTcDRBD3 assembles into cytoplasmic granules, we examined its localization by immunofluorescence analysis of transfected parasites under normal conditions and after BZ treatment. rTcDRBD3 was detected using an antibody against the 6-His tag, since anti-TcDRBD3 antibodies were not available. Under normal conditions rTcDRBD3 showed a cytoplasmic localization (Figure 3). The recruitment of rTcDRBD3 into cytoplasmic foci was observed after 24 h of treatment with 120 μM BZ (Figure 3). The foci induced by BZ were characterized by granules in close contact with the nucleus (Figure 3). We did not observe the accumulation of GFP foci after BZ exposure (Figure 3).

Figure 3
rTcDRBD3 perinuclear foci formation after BZ exposure. Untreated cultures of rTcDRBD3-expressing epimastigotes, and cultures of rTcDRBD3 and GFP-expressing epimastigotes after 24 h of BZ exposure to 120 µM. Transfected rTcDRBD3 and GFP were detected via the 6-His probe and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Nuclear and kinetoplast DNA were stained with Hoechst 33342. Magnification bars: 10 µm.

BZ induced RNA recruitment into perinuclear granules

We also detected RNA synthesis using a Click-iT Imaging Kit, which is based on the detection of the uridine analog, 5-ethynyluridine (EU), incorporated into RNA. Samples were incubated with EU and collected after 24 and 48 h of treatment. Untreated parasites showed strong nuclear staining of newly synthesized RNA (Figure 4a). However, in parasites treated with BZ, RNA was recruited into cytoplasmic granules in close contact with the nucleus, after both 24 and 48 h of treatment. We also noted perinuclear foci of rTcDRBD3 that, in some cases, colocalized with the RNA granules (Figure 4b). In GFP-transfected cells, we did not observe GFP recruitment into perinuclear foci. However, we observed RNA granules located in the perinuclear region after 24 and 48 h of BZ treatment (Figure 4c). We also used puromycin, which promotes premature translational termination by disassembling polysomes and causing the release of untranslated mRNAs, thus increasing the assembly of SGs (Kedersha et al. 2000). Puromycin alone was not capable of inducing granules assembly (Figure 5a). However, puromycin treatment increased the localization of rTcDRBD3 foci and RNA, both of which were recruited into perinuclear granules (Figure 5b). In GFP-transfected cells, puromycin did not alter the protein localization of GFP, compared to BZ treatment alone (Figure 5c).

Figure 4
Perinuclear localization of RNA after BZ exposure. Immunofluorescence images of rTcDRBD3 and GFP-transfected epimastigotes. Transfected rTcDRBD3 and GFP were detected via the 6-His probe and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Nuclear and kinetoplast DNA were stained with Hoechst 33342. RNA staining was performed using the Click-iT RNA Alexa Fluor 594 Imaging Kit. Magnification bars: 10 µM. a) untreated cultures of rTcDRBD3-transfected parasites. b) rTcDRBD3 and c) GFP-transfected epimastigotes 24 and 48 h of treatment with 120 µM BZ. White arrows indicate colocalization of rTcDRBD3 and RNA.

Figure 5
Cellular localization of RNA under normal and after BZ with puromycin treatment. Immunofluorescence images of rTcDRBD3- and GFP-transfected cell lines. Transfected rTcDRBD3 and GFP were detected via the 6-His probe and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Nuclear and kinetoplast DNA were stained with Hoechst 33342. Magnification bars: 10 µM. a) rTcDRBD3- and GFP-transfected cells incubated with puromycin (200 µg mL-1). b) rTcDRBD3 and c) GFP-transfected epimastigotes after 24 and 48 h of treatment with 120 µM BZ and puromycin. White arrows indicate RNA and TcDRBD3 colocalization.

Nutritional stress and puromycin promoted the assembly of cytoplasmic rTcDRBD3 foci

Based on the characterization of TbDRBD3 under nutritional stress (Fernández-Moya et al. 2012), we also induced starvation by culturing rTcDRBD3-transfected epimastigotes in phosphate-buffered saline (PBS) for 48 h. To further characterize rTcDRBD3 recruitment into cytoplasmic foci, we used puromycin, which has been shown to increase the formation of SGs (Kedersha et al. 2000). As observed previously, puromycin treatment alone did not alter rTcDRBD3 localization in the cytoplasm (Figure 6). However, in starved parasites, we observed rTcDRBD3 recruitment into cytoplasmic foci and this was increased by puromycin treatment (Figure 6). Cytoplasmic foci formation was not observed in GFP-transfected cells (Figure 6).

Figure 6
Cellular localization of rTcDRBD3 under nutritional stress, with or without puromycin treatment. Immunofluorescence images of rTcDRBD3- and GFP-transfected cell lines. Transfected rTcDRBD3 and GFP were detected via the 6-His probe and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Nuclear and kinetoplast DNA were stained with Hoechst 33342. Magnification bars: 10 µM.

DISCUSSION

RNA binding proteins are essential modulators of gene expression, especially in trypanosomes, in which transcription is polycistronic, and the regulation of gene expression relies on post-transcriptional mechanisms (Clayton 2013). Here, we characterized the gene coding for the T. cruzi RNA-binding protein DRBD3 (TcDRBD3) and conducted a motif identification based on its primary structure. We also obtained parasites expressing a recombinant form of TcDRBD3 and measured their growth rate under normal conditions and after BZ exposure. Additionally, we assessed the cellular localization of recombinant TcDRBD3 and RNA synthesis in untreated and BZ-treated parasites.

We characterized two alleles of the TcDRBD3 gene in the CL Brener strain: the Esmeraldo-like allele and the non-Esmeraldo-like allele, since CL Brener is a hybrid strain from discrete typing units II and III (Zingales et al. 2009). Although the non-Esmeraldo-like allele was annotated as a gene fragment, sequence alignment showed that both alleles were highly similar. The analysis of DRBD3 protein sequences from several trypanosomatids species showed that this protein was highly conserved, especially in regions predicted as RRMs. Proline and glutamine (PQ) tracts were commonly observed at the C-terminal region of the proteins from species of the genus Trypanosoma.

RNA-binding proteins from RNA granules harbor two components that selectively target mRNAs to these structures: N-terminal RRMs and C-terminal low-complexity sequences, such as the PQ tract (Molliex et al. 2015). TcDRBD3 structure analysis showed that this protein had both RRMs and C-terminal low-complexity sequences, suggesting that it can assemble into RNA granules. Although the molecular mechanisms of RNA granules assembly are still elusive, recent reports have suggested that low-complexity domains drive their formation through a process of liquid-liquid phase separation and these membraneless foci are referred to as droplets organelles (Molliex et al. 2015).

Both T. cruzi and T. brucei DRBD3 RRMs were predicted as members of the PTB1 subfamily. PTB1 is a multifunctional RNA-binding protein that shuttles between the nucleus and the cytoplasm and was first described as a repressor of alternative splicing (Sawicka et al. 2008, Romanelli et al. 2013). The translocation of this protein to the cytoplasm usually occurs under stress conditions, such as viral infections or exposure to genotoxic agents (Florez et al. 2005, Dobbyn et al. 2008). Moreover, PTB1 is involved in all mRNA metabolic processes, such as polyadenylation, mRNA stability, and translation initiation (Sawicka et al. 2008, Romanelli et al. 2013). T. brucei DRBD3 is also known as TbPTB1, due to its similarity to human PTB1 (Stern et al. 2009). High throughput sequence analysis of TbDRBD3-bound mRNAs showed that, like human PTB1, this protein was also involved in mRNA metabolism and translation (Das et al. 2015).

Studies of RNA-binding proteins and granules assembly during BZ-induced stress in T. cruzi have not been reported. Our approach to studying TcDRBD3 protein was through transfection of epimastigotes with a recombinant TcDRBD3 protein harboring an SBP and a 6-His C-terminal tag (rTcDRBD3). We assessed rTcDRBD3 localization by detection of the 6-His tag. The SBP tail allows further screenings, such as immunoprecipitations assays, to investigate TcDRBD3 targets mRNAs. Transfected epimastigotes had similar growth under normal conditions and after BZ treatment compared to the non-transfected controls.

In the present study, we observed rTcDRBD3 recruitment into cytoplasmic perinuclear foci in parasites exposed to BZ. Interestingly, previous electron microscopy studies of epimastigotes under BZ stress showed the accumulation of granular electron-dense bodies in the parasite cytoplasm (Rajão et al. 2014). We also found that BZ triggered RNA recruitment into granules located in the nuclear periphery. RNA granules located in the nuclear periphery have previously been observed in trypanosomatids after exposure to drugs capable of inhibiting mRNA maturation (Kramer et al. 2012). The inhibition of RNA maturation, through an incomplete trans-splicing process, can lead to the formation of transcripts containing more than one open reading frame, that are potentially toxic after export to the cytoplasm (Kramer et al. 2012). Furthermore, the increased colocalization of rTcDRBD3 and RNA foci after the combined treatment of puromycin and BZ, suggested that TcDRBD3 granules harbor RNAs recruited from the translating pool, since puromycin causes premature release of RNAs present in polysomes, thus increasing SG formation (Kedersha et al. 2000).

Trypanosomes have a variety of different RNA granules and their composition is dependent on the type of stimulus, such as starvation (Kramer 2014). TbDRBD3 is described as a component of nutritional SGs (Fernández-Moya et al. 2012). Also, upon arsenite-induced stress, which like BZ treatment, generates reactive oxygen species, TbDRBD3 localizes to the nucleus. This translocation has been attributed to the shuttle function of TbDRBD3, which transports target mRNAs from the nucleus to the cytoplasm (Fernández-Moya et al. 2012). Since nutritional stress is associated with RNA granules assembly in trypanosomatids, we assessed rTcDRBD3 localization in starved epimastigotes. It has been shown that starvation-induced stress, combined with puromycin treatment, promotes increased SG assembly (Kedersha et al. 2000, Holetz et al. 2007, Cassola et al. 2007). Our results showed that puromycin alone was unable to induce cytoplasmic granular accumulation of rTcDRBD3, which was consistent with the results of previous studies (Kedersha et al. 2000, Cassola et al. 2007, Bounedjah et al. 2014). However, under conditions of starvation, we observed rTcDRBD3 granular assembly and this was enhanced by puromycin treatment. The increase in SGs assembly after puromycin treatment is attributed not only to the excess mRNA resulting from polysome dissociation, but also to the presence of pro-aggregating stimuli triggered under conditions of stress (Bounedjah et al. 2014).

In summary, this is the first reported study of RNA-binding proteins and granule assembly in T. cruzi during BZ-induced stress. Our results indicated that TcDRBD3 differentially assembled into discrete perinuclear granules after BZ exposure. Furthermore, RNA recruitment into these perinuclear foci suggested that TcDRBD3 foci harbor RNA. The increased colocalization of rTcDRBD3 and RNA after the combination of BZ and puromycin treatment, which dismantles polysomes and releases translating transcripts, suggested that TcDRBD3 granules also harbor RNAs from the translating pool. Moreover, TcDRBD3 foci under conditions of starvation and puromycin treatment, resemble SGs described in previous studies (Kedersha et al. 2000, Holetz et al. 2007, Cassola et al. 2007, Bounedjah et al. 2014). The study of these cytoplasmic foci components is crucial to understanding the response of T. cruzi to stress conditions, such as BZ exposure, since trypanosomes repertoire of RNA granules is dependent on the type of stress.

SUPPLEMENTARY MATERIAL

Figures S1 and S2.

ACKNOWLEDGMENTS

The immunofluorescence data presented in this paper were obtained using the microscopes and equipment at Centro de Aquisição e Processamento de Imagens (CAPI - ICB/UFMG) (www2.icb.ufmg.br/capi/). This work was supported by the following funding agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, Grant IDs 479623/2013-9 and 310741/2013-0 to G.R.F. and M.Sc fellowship to D.L.S., Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil, Ph.D fellowship to D.F.C., Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil, Grant ID CBB - APQ-02566-16 to G.R.F. and Pró-reitoria de Pesquisa – UFMG, Brazil.DFC designed and performed the majority of the experimental procedures and analyses, generated the figures, and wrote the manuscript. DLS assisted with the experimental procedures and manuscript writing. HGSV performed vector construction and cloning experiments. EBT, AMM, and CRM were involved in discussions on the study and contributed their expert insights. GRF conceived the study, participated in its design and coordination, and provided financial support for all experiments. All authors were involved in the discussion of results, reviewed the manuscript and agreed with its submission.

REFERENCES

ANDERSON P & KEDERSHA N. 2008. Stress granules: the Tao of RNA triage. Trends Biochem Sci 33: 141-150.
ASLETT M ET AL. 2009. TriTrypDB: A functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38: 457-462.
BOUNEDJAH O, DESFORGES B, WU T-D, PIOCHE-DURIEU C, MARCO S, HAMON L, CURMI PA, GUERQUIN-KERN J-L, PIÉTREMENT O & PASTRÉ D. 2014. Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules. Nucleic Acids Res 42: 8678-8691.
BURKARD GS, JUTZI P & RODITI I. 2011. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol Biochem Parasitol 175: 91-94.
CAMARGO EP. 1964. Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop Sao Paulo 6: 93-100.
CASSOLA A, DE GAUDENZI JG & FRASCH AC. 2007. Recruitment of mRNAs to cytoplasmic ribonucleoprotein granules in trypanosomes. Mol Microbiol 65: 655-670.
CLAYTON C. 2013. The regulation of trypanosome gene expression by RNA-binding proteins. PLoS Pathog 9: 9-12.
COURCHAINE EM, LU A & NEUGEBAUER KM. 2016. Droplet organelles? EMBO J 35: 1603-1612.
DAROCHA WD, SILVA RA, BARTHOLOMEU DC, PIRES SF, FREITAS JM, MACEDO AM, VAZQUEZ MP, LEVIN MJ & TEIXEIRA SMR. 2004. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92: 113-120.
DAS A, BELLOFATTO V, ROSENFELD J, CARRINGTON M, ROMERO-ZALIZ R, DEL VAL C & ESTÉVEZ AM. 2015. High throughput sequencing analysis of Trypanosoma brucei DRBD3/PTB1-bound mRNAs. Mol Biochem Parasitol 199: 1-4.
DE GAUDENZI J, FRASCH AC & CLAYTON C. 2005. RNA-binding domain proteins in Kinetoplastids: a comparative analysis. Eukaryot Cell 4: 2106-2114.
DOBBYN HC, HILL K, HAMILTON TL, SPRIGGS KA, PICKERING BM, COLDWELL MJ, DE MOOR CH, BUSHELL M & WILLIS AE. 2008. Regulation of BAG-1 IRES-mediated translation following chemotoxic stress. Oncogene 27: 1167-1174.
EULALIO A, BEHM-ANSMANT I & IZAURRALDE E. 2007. P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Biol 8: 9-22.
FERNÁNDEZ-MOYA SM, GARCÍA-PÉREZ A, KRAMER S, CARRINGTON M & ESTÉVEZ AM. 2012. Alterations in DRBD3 ribonucleoprotein complexes in response to stress in Trypanosoma brucei. PLoS ONE 7: e48870.
FLOREZ PM, SESSIONS OM, WAGNER EJ, GROMEIER M & GARCIA-BLANCO MA. 2005. The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation. J Virol 79: 6172-6179.
FRITZ M, VANSELOW J, SAUER N, LAMER S, GOOS C, SIEGEL TN, SUBOTA I, SCHLOSSER A, CARRINGTON M & KRAMER S. 2015. Novel insights into RNP granules by employing the trypanosome’s microtubule skeleton as a molecular sieve. Nucleic Acids Res 43: 8013-8032.
HOLETZ FB, CORREA A, AVILA AR, NAKAMURA CV, KRIEGER MA & GOLDENBERG S. 2007. Evidence of P-body-like structures in Trypanosoma cruzi. Biochem Biophys Res Commun 356: 1062-1067.
JONES P ET AL. 2014. InterProScan 5: genome-scale protein function classification. Bioinformatics 30: 1236-1240.
KEDERSHA N, CHO MR, LI W, YACONO PW, CHEN S, GILKS N, GOLAN DE & ANDERSON P. 2000. Dynamic shuttling of Tia-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol 151: 1257-1268.
KOLLIEN AH & SCHAUB GA. 1998. The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation. J Eukaryot Microbiol 45: 59-63.
KRAMER S. 2014. RNA in development: how ribonucleoprotein granules regulate the life cycles of pathogenic protozoa. Wiley Interdiscip Rev RNA 5: 263-284.
KRAMER S & CARRINGTON M. 2011. Trans-acting proteins regulating mRNA maturation, stability and translation in trypanosomatids. Trends Parasitol 27: 23-30.
KRAMER S, MARNEF A, STANDART N & CARRINGTON M. 2012. Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate. J Cell Sci 125: 2896-2909.
LIU W ET AL. 2015. IBS: An illustrator for the presentation and visualization of biological sequences. Bioinformatics 31: 3359-3361.
MARCHLER-BAUER A & BRYANT SH. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32: 327-331.
MOLLIEX A, TEMIROV J, LEE J, COUGHLIN M, KANAGARAJ AP, KIM HJ, MITTAG T & TAYLOR JP. 2015. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163: 123-133.
NOTREDAME C, HIGGINS DG & HERINGA J. 2000. T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302: 205-217.
RAJÃO MA ET AL. 2014. Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55: 309-321.
ROMANELLI MG, DIANI E & LIEVENS PMJ. 2013. New insights into functional roles of the polypyrimidine tract-binding protein. Int J Mol Sci 14: 22906-22932.
SAARIKANGAS J & BARRAL Y. 2016. Protein aggregation as a mechanism of adaptive cellular responses. Curr Genet 62: 711-724.
SANGER F, COULSON AR, BARRELL BG, SMITH AJH & ROE BA. 1980. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143: 161-178.
SAWICKA K, BUSHELL M, SPRIGGS KA & WILLIS AE. 2008. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein. Biochem Soc Trans 36: 641-647.
SCHWEDE A, KRAMER S & CARRINGTON M. 2012. How do trypanosomes change gene expression in response to the environment? Protoplasma 249: 223-238.
STERN MZ, GUPTA SK, SALMON-DIVON M, HAHAM T, BARDA O, LEVI S, WACHTEL C, NILSEN TW & MICHAELI S. 2009. Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism. RNA 15: 648-665.
TEIXEIRA SR, OTSU K, HILL K, KIRCHHOFF L & DONELSON J. 1999. Expression of a marker for intracellular Trypanosoma cruzi amastigotes in extracellular spheromastigotes. Mol Biochem Parasitol 98: 265-270.
THE UNIPROT CONSORTIUM. 2017. UniProt: the universal protein knowledgebase. Nucleic Acids Res 45: D158-D169.
YAN R, XU D, YANG J, WALKER S & ZHANG Y. 2013. A comparative assessment and analysis of 20 representative sequence alignment methods for protein structure prediction. Sci Rep 3: 2619.
ZINGALES B ET AL. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104: 1051-1054.

Publication Dates

Publication in this collection
22 Nov 2024
Date of issue
2024

History

Received
25 Mar 2024
Accepted
12 July 2024

This is an open-access article distributed under the terms of the Creative Commons Attribution License

[1] ANDERSON P & KEDERSHA N. 2008. Stress granules: the Tao of RNA triage. Trends Biochem Sci 33: 141-150.

[2] ASLETT M ET AL. 2009. TriTrypDB: A functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38: 457-462.

[3] BOUNEDJAH O, DESFORGES B, WU T-D, PIOCHE-DURIEU C, MARCO S, HAMON L, CURMI PA, GUERQUIN-KERN J-L, PIÉTREMENT O & PASTRÉ D. 2014. Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules. Nucleic Acids Res 42: 8678-8691.

[4] BURKARD GS, JUTZI P & RODITI I. 2011. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol Biochem Parasitol 175: 91-94.

[5] CAMARGO EP. 1964. Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop Sao Paulo 6: 93-100.

[6] CASSOLA A, DE GAUDENZI JG & FRASCH AC. 2007. Recruitment of mRNAs to cytoplasmic ribonucleoprotein granules in trypanosomes. Mol Microbiol 65: 655-670.

[7] CLAYTON C. 2013. The regulation of trypanosome gene expression by RNA-binding proteins. PLoS Pathog 9: 9-12.

[8] COURCHAINE EM, LU A & NEUGEBAUER KM. 2016. Droplet organelles? EMBO J 35: 1603-1612.

[9] DAROCHA WD, SILVA RA, BARTHOLOMEU DC, PIRES SF, FREITAS JM, MACEDO AM, VAZQUEZ MP, LEVIN MJ & TEIXEIRA SMR. 2004. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92: 113-120.

[10] DAS A, BELLOFATTO V, ROSENFELD J, CARRINGTON M, ROMERO-ZALIZ R, DEL VAL C & ESTÉVEZ AM. 2015. High throughput sequencing analysis of Trypanosoma brucei DRBD3/PTB1-bound mRNAs. Mol Biochem Parasitol 199: 1-4.

[11] DE GAUDENZI J, FRASCH AC & CLAYTON C. 2005. RNA-binding domain proteins in Kinetoplastids: a comparative analysis. Eukaryot Cell 4: 2106-2114.

[12] DOBBYN HC, HILL K, HAMILTON TL, SPRIGGS KA, PICKERING BM, COLDWELL MJ, DE MOOR CH, BUSHELL M & WILLIS AE. 2008. Regulation of BAG-1 IRES-mediated translation following chemotoxic stress. Oncogene 27: 1167-1174.

[13] EULALIO A, BEHM-ANSMANT I & IZAURRALDE E. 2007. P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Biol 8: 9-22.

[14] FERNÁNDEZ-MOYA SM, GARCÍA-PÉREZ A, KRAMER S, CARRINGTON M & ESTÉVEZ AM. 2012. Alterations in DRBD3 ribonucleoprotein complexes in response to stress in Trypanosoma brucei. PLoS ONE 7: e48870.

[15] FLOREZ PM, SESSIONS OM, WAGNER EJ, GROMEIER M & GARCIA-BLANCO MA. 2005. The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation. J Virol 79: 6172-6179.

[16] FRITZ M, VANSELOW J, SAUER N, LAMER S, GOOS C, SIEGEL TN, SUBOTA I, SCHLOSSER A, CARRINGTON M & KRAMER S. 2015. Novel insights into RNP granules by employing the trypanosome’s microtubule skeleton as a molecular sieve. Nucleic Acids Res 43: 8013-8032.

[17] HOLETZ FB, CORREA A, AVILA AR, NAKAMURA CV, KRIEGER MA & GOLDENBERG S. 2007. Evidence of P-body-like structures in Trypanosoma cruzi. Biochem Biophys Res Commun 356: 1062-1067.

[18] JONES P ET AL. 2014. InterProScan 5: genome-scale protein function classification. Bioinformatics 30: 1236-1240.

[19] KEDERSHA N, CHO MR, LI W, YACONO PW, CHEN S, GILKS N, GOLAN DE & ANDERSON P. 2000. Dynamic shuttling of Tia-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol 151: 1257-1268.

[20] KOLLIEN AH & SCHAUB GA. 1998. The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation. J Eukaryot Microbiol 45: 59-63.

[21] KRAMER S. 2014. RNA in development: how ribonucleoprotein granules regulate the life cycles of pathogenic protozoa. Wiley Interdiscip Rev RNA 5: 263-284.

[22] KRAMER S & CARRINGTON M. 2011. Trans-acting proteins regulating mRNA maturation, stability and translation in trypanosomatids. Trends Parasitol 27: 23-30.

[23] KRAMER S, MARNEF A, STANDART N & CARRINGTON M. 2012. Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate. J Cell Sci 125: 2896-2909.

[24] LIU W ET AL. 2015. IBS: An illustrator for the presentation and visualization of biological sequences. Bioinformatics 31: 3359-3361.

[25] MARCHLER-BAUER A & BRYANT SH. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32: 327-331.

[26] MOLLIEX A, TEMIROV J, LEE J, COUGHLIN M, KANAGARAJ AP, KIM HJ, MITTAG T & TAYLOR JP. 2015. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163: 123-133.

[27] NOTREDAME C, HIGGINS DG & HERINGA J. 2000. T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302: 205-217.

[28] RAJÃO MA ET AL. 2014. Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55: 309-321.

[29] ROMANELLI MG, DIANI E & LIEVENS PMJ. 2013. New insights into functional roles of the polypyrimidine tract-binding protein. Int J Mol Sci 14: 22906-22932.

[30] SAARIKANGAS J & BARRAL Y. 2016. Protein aggregation as a mechanism of adaptive cellular responses. Curr Genet 62: 711-724.

[31] SANGER F, COULSON AR, BARRELL BG, SMITH AJH & ROE BA. 1980. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143: 161-178.

[32] SAWICKA K, BUSHELL M, SPRIGGS KA & WILLIS AE. 2008. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein. Biochem Soc Trans 36: 641-647.

[33] SCHWEDE A, KRAMER S & CARRINGTON M. 2012. How do trypanosomes change gene expression in response to the environment? Protoplasma 249: 223-238.

[34] STERN MZ, GUPTA SK, SALMON-DIVON M, HAHAM T, BARDA O, LEVI S, WACHTEL C, NILSEN TW & MICHAELI S. 2009. Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism. RNA 15: 648-665.

[35] TEIXEIRA SR, OTSU K, HILL K, KIRCHHOFF L & DONELSON J. 1999. Expression of a marker for intracellular Trypanosoma cruzi amastigotes in extracellular spheromastigotes. Mol Biochem Parasitol 98: 265-270.

[36] THE UNIPROT CONSORTIUM. 2017. UniProt: the universal protein knowledgebase. Nucleic Acids Res 45: D158-D169.

[37] YAN R, XU D, YANG J, WALKER S & ZHANG Y. 2013. A comparative assessment and analysis of 20 representative sequence alignment methods for protein structure prediction. Sci Rep 3: 2619.

[38] ZINGALES B ET AL. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104: 1051-1054.