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Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi Fabı ´ ola B. Holetz 1 , Lysangela R. Alves 1 , Christian M. Pr obst 1 , Bruno Dallagiovanna 1 ,FabricioK.Marchini 1 , Patricio Manque 2,3 , Gregory Buck 2 ,MarcoA.Krieger 1 , Alejandro Correa 1 and Samuel Goldenberg 1 1 Instituto Carlos Chagas ⁄ FIOCRUZ, Curitiba, Brazil 2 Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA, USA 3 Instituto de Biotecnologia, Universidad Mayor, Santiago, Chile Keywords differentiation; P-bodies; stress granules; translation control; Trypanosoma Correspondence S. Goldenberg, Instituto Carlos Chagas ⁄ FIOCRUZ, Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010 PR, Brazil Fax: (55) 41 3316 3230 Tel: (55) 41 3316 3230 E-mail: sgoldenb@fiocruz.br (Received 29 January 2010, revised 3 June 2010, accepted 23 June 2010) doi:10.1111/j.1742-4658.2010.07747.x In trypanosomatids, the regulation of gene expression occurs mainly at the post-transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA-related processes in vari- ous eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunopre- cipitation assays with anti-TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabo- lism, regulating the expression of at least epimastigote-specific genes. Structured digital abstract l MINT-7909478: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0915) with PABP2 (uni- protkb: Q27335)byanti bait coimmunoprecipitation (MI:0006) l MINT-7909338: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0914) with ATP-depen- dent DEAD ⁄ H RNA helicase, putative (uniprotkb: Q4DIE1), Actin, putative (uniprotkb: Q4D7A6), Actin, putative (uniprotkb:Q4CLA9), Chaperonin HSP60, mitochondrial (uni- protkb: Q4DYP6), ATP-dependent Clp protease subunit, heat shock protein 100 (HSP100), putative (uniprotkb: Q4CNM5), Elongation factor 2, putative (uniprotkb:Q4D5X0), Elonga- tion factor 1-alpha (EF-1-alpha), putative (uniprotkb: Q4CU73), Heat shock protein 85, putative (uniprotkb: Q4CQS6), Glutamate dehydrogenase, putative (uniprotkb:Q4DWV8), Putative uncharacterized protein (uniprotkb: Q4CNI8), 40S ribosomal protein S11, putative (uniprotkb: Q4CRH9), Sterol 24-c-methyltransferase, putative (uniprotkb:Q4CMB7), Heat shock protein 70 (HSP70), putative (uniprotkb: Q4DTM9), Glutamate dehydrogenase, puta- tive (uniprotkb: Q4D5C2) and Calpain-like cysteine peptidase, putative (uniprotkb:Q4CYU3) by anti bait coimmunoprecipitation ( MI:0006) l MINT-7909469: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0915) with PABP1 (uniprotkb: Q4E4I9)byanti bait coimmunoprecipitation (MI:0006) Abbreviations eIF, eukaryotic initiation factor; IP, immunoprecipitated; MASP, mucin-associated surface protein; PABP, poly(A)-binding protein; P-bodies, processing bodies; SG, stress granule; SP, supernatant. FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS 3415 Introduction The life cycle of the three trypanosomatid parasites pathogenic to humans involves the alternation between a mammal and an arthropod host. The two main developmental stages of Trypanosoma brucei are extracellular, in both mammalian host blood and the tsetse fly intestine [1]. Trypanosoma cruzi exists in its amastigote form in the cytoplasm of the mammal infected cell, or as an epimastigote in the kissing bug intestine [2]. Protozoans of the Leishmania genus, in turn, multiply in the form of amastigotes within mac- rophages and as extracellular promastigotes in the insect vector [3]. The regulation of gene expression thus plays a major role in determining the adaptation and differentiation of these parasites throughout their life cycle. Trypanosomatids diverge from other eukaryotes in several aspects, including the editing of mitochondrial RNA molecules, trans-splicing, genes almost never interrupted by introns and a lack of typical promoter sequences for the transcription of protein-encoding genes. In addition, transcription by RNA polymer- ase II is constitutive and genes in the same polycis- tronic unit display different levels of processed mRNA [4–6]. Consequently, the regulation of gene expression in trypanosomatids seems to occurs mainly post-trans- criptionally [6,7]. When mRNAs enter the cytoplasm, they can be translated, stored for later translation or degradation, degraded or subjected to a combination of these processes. In mammalian cells, mRNAs that are not translated or destined for degradation are compart- mentalized in distinct cytoplasmic structures, known as ‘processing bodies’ (‘P-bodies’) and stress granules [8– 10]. These RNA granules are key structures in the reg- ulation of gene expression at the post-transcriptional level [11–13]. Recently, we have identified the protein TcDHH1, a putative DEAD box RNA helicase, in T. cruzi, homologous to Dhh1 (yeast) and Rck ⁄ p54 (mammals) proteins [14]. It has been reported that Dhh1 is involved in multiple RNA-related processes in various eukaryotes, and accumulates in stress granules and P-bodies of yeast, animal cells and T. brucei (reviewed in [15–21]). Trypanosoma cruzi DHH1 is pres- ent in polysome-independent complexes and is located diffusely in the cytoplasm and in cytoplasmic granules, which vary in number when the parasite is subjected to nutritional stress or conditions interfering with the translation process, e.g. treatment with cycloheximide or puromycin [14]. In this article, we show that TcDHH1-containing foci are present throughout the T. cruzi life cycle; how- ever, they are less well defined in the infective metacy- clic trypomastigote form. We also show that proteins directly or indirectly associated with TcDHH1 include those of diverse function, e.g. translation-related fac- tors, cytoskeleton proteins, heat shock proteins and metabolic proteins. We use a ribonomic approach to identify the mRNA content of the TcDHH1-contain- ing complexes. Microarray analyses reveal that the mRNAs associated with TcDHH1 in the epimastigote stage are some of those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of, at least, epimastigote-specific genes. Results TcDHH1-containing granules are present in all developmental forms of T. cruzi We have shown previously that, in epimastigotes, TcDHH1 proteins are localized in discrete cytoplasmic foci during the logarithmic growth phase and increase in number under nutritional stress [14]. In this study, we confirmed our previous data and also showed that TcDHH1 is localized to cytoplasmic foci in adherent parasites differentiating into metacyclic trypomastig- otes and amastigotes. TcDHH1-containing granules were not readily observed in trypomastigotes. This is evident in microscopic fields in which terminally differ- entiated metacyclic trypomastigotes and intermediate forms are present (Fig. 1D). However, when the expo- sure time for the photograph was at least doubled, a few TcDHH1-containing granules were observed in metacyclic trypomastigotes (data not shown). To a les- ser extent, TcDHH1 was also observed diffusely in the cytoplasm in all forms analyzed (Fig. 1). At least 10 random fields of each developmental form were ana- lyzed, and more than 95% of the parasites had the appearance described above. Protein composition of TcDHH1-containing complexes Cytoplasmic complexes containing the protein TcDHH1 in logarithmic growth phase epimastigotes were immunoprecipitated with specific antiserum. As seen by western blot analysis, the antiserum recognizes a unique band of the expected mass size in a cyto- plasmic extract of T. cruzi (Fig. 2). Pre-immune serum was used as an experimental control. Three different DHH1-mRNPs in Trypanosoma cruzi F. B. Holetz et al. 3416 FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS immunoprecipitation experiments were performed. Proteins associated with immunoprecipitated (IP) com- plexes and proteins not associated with TcDHH1 from the supernatant (SP) were resolved by SDS ⁄ PAGE and visualized by silver staining (Fig. 3A). The IP and SP samples obtained with pre-immune serum were pro- cessed in the same way and are shown in Fig. 3C. To provide a measure of the efficiency of TcDHH1 immu- noprecipitation, IP and SP samples were loaded onto an SDS ⁄ PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the TcDHH1 antibody (Fig. 3B). The results showed that different protein profiles were obtained for the IP and SP silver- stained gels (Fig. 3A, C). Western blot analysis using the TcDHH1 antiserum allowed the identification of the band corresponding to TcDHH1 (Fig. 3B), which was not detected in the IP fraction obtained with the pre-immune serum (Fig. 3D). The fact that TcDHH1 could also be detected in the SP fraction is probably a A B Fig. 2. Western blot analysis of T. cruzi protein extracts using anti-TcDHH1. (A) Western blot analysis of protein extracts from epimastigotes (Epi), epimastigotes under nutritional stress (Stress), differentiating epimastigotes (cells adherent after 24 h) (Ad24h), metacyclic trypomastigotes (Meta) and amastigotes (Ama), probed with antiserum against TcDHH1 protein (1 : 100 dilution). The extracts were standardized and all lanes were loaded with 10 lgof protein. (B) SDS ⁄ PAGE stained with Coomassie brilliant blue to control protein load. TcDHH1 has an expected molecular mass of 46.7 kDa. The molecular mass marker (in kDa) is the Benchmark Protein Ladder (Gibco, Grand Island, NY, USA). A B C D E Fig. 1. Localization of TcDHH1 during the life cycle of T. cruzi. (A) Epimastigotes in logarithmic growth phase. (B) Epimastigotes under nutritional stress. (C) Differentiating epimastigotes (24 h adherent cells). (D) Metacyclic trypomastigotes. (E) Amastigotes. Cells were incubated with antiserum against TcDHH1 and the immune com- plexes were reacted with Alexa-labeled goat anti-mouse antibodies. Kinetoplasts and nuclei were stained with propidium iodide. Open arrowheads indicate an epimastigote form in the metacyclic trypo- mastigote population. Filled arrowheads point to a trypomastigote among the amastigotes. Bar, 10 lm. F. B. Holetz et al. DHH1-mRNPs in Trypanosoma cruzi FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS 3417 result of the abundance of TcDHH1, as inferred from recent data from T. brucei [22]. IP proteins were digested with trypsin and subjected to analysis by two-dimensional nano LC-MS ⁄ MS. The mass spectra obtained were compared with spectra deduced from protein sequences in the T. cruzi data bank. We used the following criteria to reliably select proteins from the database for further analysis: (a) proteins with probability values equal to or < 0.001; (b) proteins present in at least two experiments using TcDHH1 antiserum; and (c) pro- teins that did not appear in any of the three control samples. Proteins that fulfilled the above criteria are listed in Table 1 (for a complete list with all proteins reliably identified in the immunoprecipitation assays, but not meeting the selection criteria, see Table S1). These complexes contain various proteins in addition to TcDHH1, including heat shock proteins, mRNA- binding proteins, initiation and elongation translation factors, ribosomal proteins and metabolic proteins. Abundant proteins, such as heat shock proteins and elongation factor 1a, were present in the TcDHH1 immunoprecipitates, whereas other highly abundant proteins, such as the cysteine proteinase cruzipain, epimastigote-specific mucins and paraflagellar rod proteins, were not detected in any of the experi- ments. TcDHH1 granules contain poly(A)-binding proteins (PABPs) As described previously, PABP1 is a core component of stress granules in mammals [23]. Two PABPs with a high similarity to other eukaryotic PABPs have been identified previously in T. cruzi, TcPABP1 and TcPABP2, with molecular masses of 63.8 and 61.4 kDa, respectively [24,25]. To test for the presence of these proteins in TcDHH1 granules, epimastigotes in the exponentially growing phase or under nutri- tional stress were used for colocalization analyses. PABP2 antiserum showed a punctate distribution in the cytoplasm of epimastigotes and stressed epimastig- otes. Most granules in these cells seemed to colocalize with granules containing TcDHH1; those that did not colocalize were grouped in defined regions of the cyto- plasm lying next to each other (Fig. 4). Colocalization assays with PABP1 antiserum showed a slightly differ- ent staining pattern, with diffuse fluorescence and some more intense fluorescent foci throughout the cytoplasm. These foci partially colocalized with TcDHH1 in both epimastigotes and epimastigotes under nutritional stress (Fig. 4). In parallel with the colocalization experiments, we carried out immunopre- cipitation assays using mouse TcDHH1 antiserum, and the presence of TcPABP1 and TcPABP2 proteins in the precipitated complex was determined using western blot analysis with rabbit LmPABP1 and LmPABP2 antisera (Fig. 5). Both antibodies specifically recog- nized a single band with the expected molecular mass in western blots, confirming that TcPABPs are part of the TcDHH1 protein complex. mRNAs present in TcDHH1-containing complexes are regulated in a stage-specific manner We used a ribonomic approach to identify the mRNAs associated with TcDHH1, and thus to infer the role CD AB Fig. 3. Analysis of TcDHH1 immunoprecipitation. (A) Epimastigote proteins immunoprecipitated with TcDHH1 antibody (IP) and proteins from the supernatant fraction (SP) were resolved by SDS ⁄ PAGE and visualized by silver staining. (B) Western blot analy- sis of IP and SP samples probed with antiserum against TcDHH1 protein (1 : 100 dilution), showing the efficiency of TcDHH1 immu- noprecipitation. (C) Epimastigote proteins immunoprecipitated with pre-immune serum (IP) and proteins from the supernatant fraction (SP) were resolved by SDS ⁄ PAGE and visualized by silver staining. (D) Western blot analysis of IP and SP samples, obtained with pre-immune serum, and probed with serum against TcDHH1 pro- tein (1 : 100 dilution). It should be noted that tracks in (C) and (D) have a higher IgG background when compared with those in (A) and (B) as pre-immune serum was used at a lower dilution in order to discard the spurious recognition of TcDHH1. The molecular mass marker (in kDa) is the Benchmark Protein Ladder (Gibco). DHH1-mRNPs in Trypanosoma cruzi F. B. Holetz et al. 3418 FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS played by this protein in the regulation of gene expres- sion in T. cruzi. The mRNAs associated with IP com- plexes from epimastigotes were compared with the mRNAs not associated with TcDHH1 from SP. IP and SP samples were obtained from six experiments, and were compared using competitive hybridization assays in oligo-DNA microarrays. The amount of RNA extracted from IP samples was generally smaller than that from SP samples. This was expected, as the mRNA population associated directly or indirectly with TcDHH1 is likely to represent only a fraction of the total RNA. We identified mRNAs that were present in IP and SP samples in different amounts, based on a two-fold difference in mRNA levels and a 5% false discovery rate. For epimastigote forms in the logarithmic growth phase, 203 distinct mRNAs displayed higher levels in the IP than SP fraction, with 265 mRNAs present at lower levels in the SP sample. Most of the mRNAs associated with TcDHH1 were from mucin-associated surface protein (MASP) and mucin protein families, with others corresponding to several hypothetical and hypothetical conserved proteins. We also observed mRNAs corresponding to metabolic proteins, mRNA- binding proteins, amastin and cyclin, among others (Fig. 6A, Table 2) (for a detailed list of mRNAs, see Table S2). A semi-quantitative approach, RT-PCR and densitometry analysis of gel bands for five mRNAs with different distribution patterns between IP and SP samples confirmed these results (Fig. 6B). Surface proteins (MASP and mucins) are encoded by multigene families. Although these gene families were the largest in the T. cruzi genome, we did not identify any of the corresponding mRNAs in the SP sample, demonstrating the specific presence of these mRNAs in the IP sample. Moreover, mRNAs from other large gene families in T. cruzi, e.g. those encoding trans-siali- dase and cysteine protease, were not found in the IP sample. Discussion Recently, we have identified a putative RNA helicase (TcDHH1), which is similar to its eukaryotic orthologs [14]. Members of this protein family are involved in several aspects of mRNA metabolism and localize to distinct foci in the cytoplasm [26]. We compared the replicative, nutritionally stressed, differentiating and infective forms of T. cruzi, and showed that these T. cruzi life cycle stages display distinct granular pat- terns of cytoplasmic TcDHH1-containing foci (Fig. 1). These granules were not as clearly visible in trypom- astigotes as in the other forms, but TcDHH1 protein levels remained similar for all T. cruzi developmental stages [14]. Thus, the variation in the number of TcDHH1-containing granules does not seem to be related to changes in gene expression levels, but is probably related to diffuse or foci-like distributions. In a recent study, Cassola et al. [20] showed that epim- astigotes in culture and parasites at different time points during in vitro differentiation did not display mRNA granules. Although, at first, these findings seem to disagree with our work, it is not possible to compare these studies because different experimental approaches were used to investigate the presence of mRNA granules in developmental forms of T. cruzi. Table 1. Protein composition of TcDHH1-containing complexes. IP, complexes immunoprecipitated with anti-TcDHH1 antibody; C, complexes immunoprecipitated with pre-immune serum; 1, 2, 3, biological replicates; •, indicates the presence of a protein in that fraction. Protein description IP1 IP2 IP3 C1 C2 C3 Tc00.1047053510127.79 actin, putative •• Tc00.1047053510573.10 actin, putative •• Tc00.1047053507641.280 Cnp60 chaperonin HSP60, mitochondrial precursor; groELprotein; heats •• Tc00.1047053508665.14 ATP-dependent Clp protease subunit, heat shock protein 100,putative •• Tc00.1047053508169.20 elongation factor 2, putative ••• Tc00.1047053508949.4 elongation factor 1a (EF-1a), putative ••• Tc00.1047053507713.30 heat shock protein 85, putative ••• Tc00.1047053507875.20 glutamate dehydrogenase, putative •• Tc00.1047053508111.30 glutamate dehydrogenase, putative •• Tc00.1047053509139.10 hypothetical protein, conserved •• Tc00.1047053511139.20 40S ribosomal protein S11, putative •• Tc00.1047053506983.39 calpain-like cysteine peptidase, putative •• Tc00.1047053504191.10 sterol 24-c-methyltransferase, putative •• Tc00.1047053511211.160 heat shock protein 70 (HSP70), putative •• Tc00.1047053506959.30 ATP-dependent DEAD ⁄ H RNA helicase, putative •• F. B. Holetz et al. DHH1-mRNPs in Trypanosoma cruzi FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS 3419 In yeast, the Dhh1 protein interacts with decapping and deadenylation complexes, stimulating mRNA decapping [15–18]. Dhh1p homologs from Caenorhabd- itis elegans (Cgh1), Drosophila (Me31b) and Xenopus (Xp54) are involved in the storage of translationally repressed maternal mRNAs [16,17]. In addition, the RNA helicase DOZI, involved in the storage and silencing of certain mRNA species, has been identified recently in Plasmodium berghei and is localized to cyto- plasmic granules in female gametocytes [19]. Dhh1 ⁄ Rckp54 is common to both P-bodies and stress granules [10–13]; both of these types of granule may therefore exist in T. cruzi. Therefore, we studied mRNPs containing TcDHH1 and investigated their potential similarity with mRNPs in other organisms. We have shown previously that TcDHH1 is present in cytoplasmic complexes containing mRNA and pro- teins. TcDHH1-containing complexes have been puri- fied previously from polysome and polysome-free fractions [27]. TcDHH1 must therefore be, at least partly, associated with mRNAs that are independent of the translation machinery. Our analysis of IP complexes showed that TcDHH1 interacts with proteins that are described as stress granule components (SGs). We identified TcDHH1, as expected, and proteins previously identified in stress granules, such as heat shock proteins and 40S ribo- somal subunit proteins. Translation initiation factors (eukaryotic initiation factors 3 and 4, eIF3 and eIF4), also typical of SGs, were observed in one of the three A B Fig. 4. Colocalization assays of PABP proteins with TcDHH1. (A) Logarithmic growth phase epimastigotes. (B) Epimastig- otes under nutritional stress. Antibodies against PABP1 and PABP2 were produced in rabbit and tested at 1 : 100 dilutions. Antibodies against TcDHH1 protein were produced in mouse and used at 1 : 100 dilution. Immune complexes reacted with Alexa-labeled 546 goat anti-rabbit and Alexa-labeled 488 goat anti-mouse antibod- ies (1 : 400). Bar, 10 lm. DHH1-mRNPs in Trypanosoma cruzi F. B. Holetz et al. 3420 FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS biological replicates (Table S1). Although abundant proteins, such as heat shock proteins and eIF1a, are present in these complexes, other proteins highly abun- dant in epimastigotes, such as the cysteine proteinase cruzipain, the family of GP63 metalloproteases, many ribosomal proteins, epimastigote-specific mucins and the epimastigote-specific metabolic protein histidine ammonia-lyase, were not detected in any of the experi- ments [28–30]. Nonetheless, it remains to be elucidated whether these proteins indeed interact with TcDHH1 or are fraction contaminants. Interestingly, Pare et al. [31] have revealed recently that a heat shock protein (Hsp90) is important for recruiting the argonaute protein ( hAgo2) to SGs and for efficient biogenesis and ⁄ or stability of P-bodies in mammals. Thus, it seems probable that the heat shock proteins identified in our study might interact with TcDHH1. We also identified proteins that are not characteristic of these structures, such as translation elongation factors, metabolic proteins and actin (Table 1). Although actin is not a AB Fig. 6. mRNAs present in TcDHH1-containing complexes in epimastigotes. (A) Pie chart diagram displaying the percentage representation of the most abundant mRNAs present in TcDhh1-containing complexes. (B) RT-PCR analysis of five mRNAs with different distribution patterns between IP and SP fractions. Putative glucose-regulated protein 78, more represented in the SP fraction, showed an IP ⁄ SP ratio of 0.51. Putative (H+)-ATPase G subunit, equally represented in both fractions, resulted in an IP ⁄ SP ratio of 0.98. Putative cyclin, putative mucin TcMUCII and hypothetical protein were more represented in the IP fraction, with IP ⁄ SP ratios of 2.0, 6.7 and 1.8, respectively. AB Fig. 5. Co-immunoprecipitation assays of TcDHH1 with PABP pro- teins. Epimastigote proteins were immunoprecipitated with mouse TcDHH1 antibody (I) or pre-immune serum (PI), resolved by SDS ⁄ PAGE, electrotransferred onto Hybond-C membranes and probed with rabbit anti-LmPABP1 (A) and anti-LmPABP2 (B) anti- sera (1 : 100 dilution), showing that PABPs are part of the TcDHH1 protein complex. The molecular mass marker (in kDa) is the Bench- mark Protein Ladder (Gibco). Table 2. mRNAs present in TcDHH1-containing complexes in epimastigotes. Gene description No. of genes Mucin-associated surface protein (MASP), putative 61 Mucin-associated surface protein (MASP, pseudogene), putative 4 Mucin TcMUCII, putative 52 Mucin TcMUCII (pseudogene), putative 3 Mucin TcSMUGS, putative 1 Hypothetical protein, conserved 31 Hypothetical protein 34 ADP-ribosylation factor family, putative 1 Amastin, putative 1 Cyclin, putative 1 Expression site-associated gene (ESAG-like) protein, putative 1 Meiotic recombination protein SPO11, putative 1 Mitochondrial carrier protein, putative 1 Nucleoside transporter 1, putative 1 Phosphatidic acid phosphatase, putative 1 Protein kinase, putative 1 Ras-related GTP-binding protein, putative 1 RNA-binding protein 5, putative 1 RNA-binding protein, putative 1 Serine acetyltransferase, putative 1 Serine ⁄ threonine protein phosphatase, putative 1 Serine ⁄ threonine protein phosphatase 2A, catalytic subunit, putative 1 Serine-, alanine- and proline-rich protein, putative 1 Trypanothione synthetase, putative 1 F. B. Holetz et al. DHH1-mRNPs in Trypanosoma cruzi FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS 3421 typical SG, there seems to be a close interaction between cytoplasmic mRNA granules and the cytoskel- eton. Indeed, granules remaining motionless are associ- ated with actin filaments, whereas those that move in the cytoplasm remain associated with microtubules [32]. Thus, it is possible that actin is indeed a compo- nent of TcDHH1-containing complexes and not a putative contaminant. The fact that other abundant proteins were not detected in this fraction would be consistent with this. Interestingly, we did not identify P-body core proteins, such as LSMs and exonuclease 5¢–3¢ XRN1. These findings suggest that TcDHH1- containing complexes are more likely to be compo- nents of SGs than of P-bodies. We cannot rule out the possibility that the identified proteins of TcDHH1-con- taining complexes also correspond to the cytoplasmic granule-free fraction of TcDHH1. We tested for the colocalization of TcDHH1 with PABP present in stress granules to extend our findings from the immunoprecipitation experiments and to gain further insight into the function of TcDHH1-contain- ing granules. Our results suggest that TcDHH1- containing granules contain PABPs. Indeed, PABP2 seemed to colocalize with most granules containing TcDHH1 in both unstressed epimastigotes and epi- mastigotes subjected to nutritional stress. Granules appearing to contain only PABPs remained adjacent to these TcDHH1-containing granules (Fig. 4). Our findings are in agreement with the data pub- lished by Cassola et al. [20], who demonstrated that, in starved parasites, TcPABP1 and TcPABP2 showed strong accumulation in mRNA granules. However, in contrast with our study, these authors showed that TcPABP1 and TcPABP2 were not recruited to mRNA granules in parasites not subjected to starvation. One possible explanation for this difference is the fact that these authors used parasites overexpressing green fluo- rescent protein fusions, in contrast with the native pro- teins evaluated here. PABP1 is used as a marker for stress granules and is absent from P-bodies in mam- mals. However, Brengues and Parker [33] showed that PABP1 is present in the P-bodies of Saccharomyces cerevisiae, and that mRNPs containing poly(A) + mRNA, PABP1, eIF4E and eIF4G enter these struc- tures, possibly representing a transitional state during mRNA exchange between P-bodies and the translation machinery. These authors also demonstrated that PABP1 may be present in P-bodies even in the absence of stress. Another study demonstrated that granules containing PABP1 in S. cerevisiae were distinct from the P-bodies formed specifically under stress caused by glucose deprivation, and that these two types of gran- ule partially colocalize. These granules may function as mRNA storage compartments, called EGP-bodies, allowing mRNA translation to resume when cell growth conditions are restored, and are analogous to the stress granules observed in mammals [34]. The characterization of mRNAs from mRNP com- plexes immunoprecipitated with TcDHH1 antiserum revealed the presence of several mRNA species, with overrepresentation of those encoding MASP, mucins, hypothetical proteins and amastin. In general, mRNAs associated with TcDHH1 are not translated into pro- teins in epimastigotes, but are predominantly trans- lated during other stages of the parasitic life cycle. For example, TcMUCII proteins are specific to, and MASP proteins are mostly produced in, the trypomas- tigote forms [35,36]. The mRNA encoding amastin, a protein predominantly produced in amastigotes [37], was also present in the TcDHH1-containing complexes from epimastigotes. These findings suggest that epi- mastigote TcDHH1-associated mRNAs are either stored for later use or are present in the initial steps to degradation, given that their poly(A) tails are mostly intact. Accordingly, recent work using an ATPase-defi- cient dhh1 mutant provided evidence that a pathway including Dhh1 has a selective role in the destabiliza- tion of many regulated mRNAs in procyclic forms of T. brucei [22]. It should be noted that the ribonomic approach used in this study favors the identification of polyadenylated mRNAs present in IP complexes; deadenylated mRNAs destined for, or in the initial stages of, degradation would not be detected by these microarray analyses. Materials and methods Parasites The T. cruzi clone Dm28c [38] was maintained at 28 °Cin liver infusion tryptose medium supplemented with 10% heat-inactivated fetal bovine serum. Epimastigotes under nutritional stress, metacyclic trypomastigotes and amastig- otes were obtained in vitro as described previously [39,40]. Immunofluorescence and imaging Immunofluorescence assays were carried out using a proto- col described previously [14], which was modified slightly to ensure that parasites were resuspended and washed in NaCl ⁄ P i for only 5 min before being fixed. Mouse polyclonal anti-TcDHH1 antibody was produced as described previously [14]; the antiserum was affinity puri- fied and stored in aliquots at )20 °C prior to use. Rabbit polyclonal antibodies against PABP1 and PABP2 were kindly provided by Dr Osvaldo P. de Melo Neto (Centro DHH1-mRNPs in Trypanosoma cruzi F. B. Holetz et al. 3422 FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS de Pesquisas Aggeu Magalha ˜ es, Fiocruz, Recife, Brazil). Serum dilutions of antibodies were as follows: rabbit anti-LmPABP1 and anti-LmPABP2, 1 : 100; mouse anti- TcDHH1, 1 : 100. Alexa Fluor-488 and Alexa Fluor-546 were used as conjugated secondary antibodies (1 : 400; Molecular Probes, Invitrogen, Eugene, OR, USA). Images showing subcellular localization were acquired using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan) coupled to a Cool SNAP-PRO color camera (Media Cybernetics, Bethesda, MD, USA). Merged images were obtained by superimposing image files with image-pro plus software (Media Cybernetics). DHH1 immunoprecipitation assays: protein content Immunoprecipitation assays with the anti-TcDHH1 anti- body were carried out in cytoplasmic extracts from loga- rithmic growth phase epimastigotes. Mouse anti-TcDHH1 (50 lL) was incubated with 150 lL of resin containing pro- tein G Sepharose (Sigma) for 8 h at 4 °C, with moderate stirring. Pre-immune serum was incubated with resin under the same conditions and used as a control for immunopre- cipitation reaction specificity. After incubation, the resin was collected by centrifugation at 600 g for 2 min, the supernatant was discarded and the resin was incubated with 5% nonfat milk in NaCl ⁄ P i for 30 min. The resin was then washed twice with NaCl ⁄ P i . To obtain T. cruzi cytoplasmic extracts, 2 · 10 9 parasites were washed with NaCl ⁄ P i and incubated in 2 mL of IMP1 buffer (KCl, 100 mm; MgCl 2 ,5mm; Hepes, 10 mm, pH 7.0; protease inhibitor, 1 : 100; RNase OUT, 200 UÆmL )1 ; Nonidet P40, 0.5%) for 2 h on ice, with mod- erate agitation. Parasite lysis was monitored with a light microscope. Cytoplasmic extracts were obtained by centri- fugation at 7000 g for 20 min at 4 °C; 1 mL of this extract, corresponding to the lysis of 1 · 10 9 parasites, was incu- bated with resin previously coupled to anti-TcDHH1 anti- body or to the pre-immune serum, as described above, for 16 h at 4 °C, with moderate agitation. The IP complexes were collected by centrifugation at 600 g for 2 min and SPs were saved. The resin was washed three times with IMP2 buffer (KCl, 100 mm; MgCl 2 ,5mm; Hepes, 10 mm, pH 7.0; protease inhibitor, 1 : 100; RNase OUT, 200 UÆmL )1 ; Nonidet P40, 1%), followed by the same centrifugation step. Proteins linked to the resin were eluted with 150 lL of glycine (0.1 m, pH 2.0) and the pH was adjusted to 7.5–8.0. Next, 150 lL of buffer (urea, 7 m; thiourea, 2 m; Chaps, 2%; Triton, 2%; dithiothreitol, 1%; nuclease, 1 : 100; protease inhibitor, 1 : 100) was added to the sample and proteins were analyzed by two-dimensional nano LC-MS ⁄ MS. Fifty micrograms of protein were puri- fied before analysis using a two-dimensional clean-up kit, following the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). Proteins were reduced with dith- iothreitol, alkylated with iodoacetamide and digested over- night with trypsin. The resulting tryptic peptides were desalted on C8 cartridges (Michrom BioResources, Auburn, CA, USA) and subjected to two-dimensional nano LC ⁄ MS ⁄ MS analyses on a Michrom BioResources Paradigm MS4 Multi-Dimensional Separations Module, a Michrom NanoTrap Platform and an LCQ Deca XP plus ion trap mass spectrometer. The mass spectrometer was used in data-depen- dent mode, and the four most abundant ions in each mass spectrum were selected and fragmented to produce tandem mass spectra. The MS ⁄ MS spectra were recorded in the pro- file mode. Proteins were identified by comparing the MS ⁄ MS spectra obtained with our T. cruzi database and its reversed complement using Bioworks v3.2. Peptide and protein hits were scored and ranked using the probability-based scoring algorithm incorporated in Bioworks v3.2 and adjusted to a false positive rate of less than 1%. Only peptides showing fully tryptic termini, with cross-correlation scores (X corr ) greater than 1.9 for single-charged peptides, 2.3 for double- charged peptides and 3.75 for triple-charged peptides, were used for peptide identification. In addition, delta correlation scores (DC n ) were set to be > 0.1 and, for increased strin- gency, proteins were accepted only if their probability score was less than 0.001. The following search parameters were used: taxonomy, eukaryota; monoisotopic mass tolerance, 0.1 Da; partial methionine oxidation; and one missed tryptic cleavage allowed. Criteria for positive protein identification included Mascot scores and sequence coverage. Parallel to MS, IP and SP samples were analyzed by SDS ⁄ PAGE. Gels were silver stained according to the following protocol: incubation in fixing solution (50% ethanol, 12% acetic acid, 0.02% formaldehyde) for 1 h, three washes in 50% ethanol for 15 min and sensibilization in 0.02% sodium thiosulfate for 1 min, followed by an extensive wash in distilled water. Staining was performed by incubating the gel for 30 min in silver nitrate solution (0.2% silver nitrate, 0.02% formaldehyde), followed by washing three times in distilled water for 1 min. The gel was developed in 3% sodium carbonate and 0.05% formal- dehyde. Staining was stopped in 50% ethanol and 12% ace- tic acid for 5 min. For western blot analysis, IP and SP samples were separated on a 13% SDS ⁄ PAGE gel and transferred to a nitrocellulose membrane. Nonspecific bind- ing sites were blocked by incubating the membrane with 5% nonfat milk powder and 0.1% Tween-20 in NaCl ⁄ P i for 30 min. For analysis of the efficiency of TcDHH1 immunoprecipitation, membranes were incubated for 1 h with anti-TcDHH1 antibody (1 : 100 dilution) or with pre- immune serum. For co-immunoprecipitation analysis of TcDHH1 and TcPABPs, membranes were incubated for 1 h with rabbit anti-LmPABP1 and anti-LmPABP2 (1 : 100 dilution). The membranes were then extensively washed in NaCl ⁄ P i and incubated with goat phosphatase-conjugated anti-rabbit IgG (Sigma) diluted 1 : 10 000. The color reaction was developed with 5-bromo-4-chloro-3-indolyl F. B. Holetz et al. DHH1-mRNPs in Trypanosoma cruzi FEBS Journal 277 (2010) 3415–3426 ª 2010 The Authors Journal compilation ª 2010 FEBS 3423 phosphate and nitroblue tetrazolium (Promega, Fitchburg, WI, USA). DHH1 immunoprecipitation assays: ribonomics To determine which mRNAs were associated with the TcDHH1 protein ⁄ complexes, anti-TcDHH1 was incubated with resin containing protein A Sepharose (Sigma) for 16 h at 4 °C with agitation. Antiserum (150 lL) was mixed with 150 lL of resin and 1 lL of RNAse OUT (Invitrogen). After incubation, the resin was collected by centrifugation and processed as described above. Cyto- plasmic extract, corresponding to 2 · 10 9 cells, was incu- bated with resin previously linked to anti-TcDHH1 antibodies for 2 h in an ice bath, with agitation. The resin was then collected by centrifugation and SP was retained for a control. Resin was washed three times with IMP2 buffer. The RNAs from the SP or resin (IP) were purified with an RNeasy Ò (Qiagen, Hilden, Germany) kit using the ‘Animal Cells I’ protocol in the manufacturer’s manual, with the additional step of DNase treatment in a column. Linearly amplified RNA was generated from 1 lg of total RNA (single round) using a MessageAmp amplified RNA kit (Ambion, Austin, TX, USA), follow- ing the manufacturer’s instructions. cDNA was synthe- sized from 1 lg of total or affinity-purified RNA using an oligo(dT) primer (US Biochemical Corporation, Cleve- land, OH, USA) and reverse transcriptase (IMPROM II; Promega), as recommended. Oligonucleotide DNA microarrays The microarray was constructed with 70-mer oligonucleo- tides. As a result of the hybrid and repetitive nature of the sequenced T. cruzi strain (CL Brener), all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered using the blastclust program, with 40% coverage and 75% identity. For the probe design, array- oligoselector software (v. 3.8.1) was used, with 50% GC content; 10 359 probes were designed to the longest T. cruzi CDS of each cluster; 393 probes correspond to genes of an external group (Cryptosporidium hominis) and 64 spots con- tain only spotting solution (NaCl ⁄ Cit, 3 ·), totaling 10 816 spots. These oligonucleotides were spotted from a 50 lm solution onto poly-l-lysine-coated slides and cross-linked with 600 mJ UV. Each probe corresponding to T. cruzi genes was identified following the T. cruzi Genome Consor- tium annotation (http://www.genedb.org). The microarray slides were produced at Virginia Commonwealth Univer- sity, Richmond, VA, USA. Microarray hybridization and analysis Fluorescent cyanin (Cy) dyes, Cy3 or Cy5 as appropriate, were incorporated into second-strand cDNA synthesis using 2 lg of amplified RNA as the starting material for each sample. Labeled cDNA was purified with a Microcom 30 device (Millipore, Carrigtwohill, Ireland). Microarray hybridizations and washes were carried out in a GeneTac automated hybridization station (Genomic Solutions, Chelmsford, MA, USA). The Cy3- and Cy5-labeled cDNAs were mixed and added to 120 lL of hybridization solution and allowed to hybridize for 14–16 h at 42 °C. The micro- array slides were then washed in buffer of increasing strin- gency (0.5 · and 0.05 · NaCl ⁄ Cit) and dried by centrifugation at 280 g for 5 min. The dried slides were scanned in a 428 Array Scanner (Affymetrix, Santa Clara, CA, USA). The images were analyzed with spot software. The resulting data were corrected for background and nor- malized, using the normexp and PrintTip-Loess methods, respectively, within the Limma package [41]. A total of six individual IP and SP pairs was hybridized in a semi-balanced dye design; overrepresented genes from both fractions were selected using sam software [42]. Genes were thus selected on the basis of at least a two-fold differ- ence in mRNA levels and a 5% false discovery rate. Micro- array data were submitted to ArrayExpress accession number E-MEXP-2448. RT-PCR cDNA was synthesized from 1 lg of total RNA using 1 lL of 10 lm random primers (USB Corporation, Cleveland, OH, USA) and 1 lL of reverse transcriptase (IMPROM II; Promega), according to the manufacturer’s instructions. PCR was carried out with 20 ng of cDNA as template, 20 mm Tris-HCl (pH 8.4), 10 pmol of primers, 2.5 mm MgCl 2 , 0.0625 mm dNTPs and 1 U Taq polymerase (Invitrogen). The oligonucleotide primer sets used for PCR were as follows: putative cyclin (Tc00.1047053506945.270), F, 5¢-TGGGGAGGATTATAGCGATG-3¢;R,5¢-ACTTC GGCAGAGCACTTCAT-3¢; putative mucin TcMUCII (Tc00.1047053506131.20),F,5¢-GCGGAGAACAAGATG AGGA-3¢;R,5¢-TCGCTTTTGAAATAGGCACC-3¢; hypothetical protein (Tc00.1047053509891.40),F,5¢-GCCG TCATGCAAAAATATCC-3¢;R,5¢-CCTTTTCAGCCAA AAAGCTG-3¢; putative glucose-regulated protein 78 (Tc00.1047053506585.40),F,5¢-TGGCGGTAAGAAGAA GCAGT-3¢;R,5¢-CCGAGGTCAAACACAAGGAT-3¢; putative (H+)-ATPase G subunit (Tc00.1047053510993. 10),F,5¢-ACAACGTGCAAAGGCTTCTT-3 ¢;R,5¢-CTC GTGCCAACTCCAAGTTT-3¢. PCR, using a Bio-Cycler II thermocycler (Peltier Thermal Cycler; Bio-Rad, Hercules, CA, USA), included heating at 94 °C for 2 min, followed by 25 cycles of 94 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s, with a final extension of 72 °C for 3 min. Ten microliters of RT-PCR products were resolved by 2% agarose gel electrophoresis, visualized by ethidium bromide staining. Gel photographs were taken using a UVP Bioimaging System (UVP, Upland, CA, DHH1-mRNPs in Trypanosoma cruzi F. B. 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TcDHH1, including heat shock proteins, mRNA- binding proteins, initiation and elongation translation factors, ribosomal proteins and metabolic proteins. Abundant. putative 1 Protein kinase, putative 1 Ras-related GTP-binding protein, putative 1 RNA-binding protein 5, putative 1 RNA-binding protein, putative 1 Serine acetyltransferase,