The Trypanosoma cruzi RNA-binding protein RBP42 is expressed in the cytoplasm throughout the life cycle of the parasite

Abstract

Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans, has a complex life cycle that promotes survival in disparate environments. In each environment, the parasite must fine-tune its metabolic pathways to divide and multiply. In the absence of recognizable transcriptional gene regulation, it is apparent that protein levels are determined by post-transcriptional mechanisms. Post-transcriptional gene control is influenced by RNA-binding proteins that target mRNAs in the cell’s cytoplasm. To initiate the study of post-transcriptional activities in T. cruzi, we studied this organism’s ortholog of RBP42, a trypanosomal RNA-binding protein. RBP42 was originally detected in Trypanosoma brucei and was shown to target a subset of mRNAs that encode proteins governing central carbon metabolism. T. cruzi RBP42 structurally resembles T. brucei RBP42, sharing an NTF2 domain at its amino terminus and a single RNA-binding domain (specifically, the RNA recognition motif, or RRM), at its carboxy terminus. A phylogenetic analysis reveals that an NTF2 and a single RRM are distinguishing features of all RBP42 orthologs within the broad kinetoplastid grouping. T. cruzi RBP42 is expressed in all life cycle stages of the parasite as determined by immunoblot and immunofluorescence microscopy. In each case, the protein is localized to the cytoplasm, indicating a role for T. cruzi RBP42 in post-transcriptional activities in all stages of the parasite life cycle. We speculate that RBP42 influences the dynamic metabolic pathways responsible for parasite infection and transmission.

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References

  1. Aibara S, Valkov E, Lamers M, Stewart M (2015) Domain organization within the nuclear export factor Mex67:Mtr2 generates an extended mRNA binding surface. Nucleic Acids Res 43(3):1927–1936. https://doi.org/10.1093/nar/gkv030

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Araujo PR, Teixeira SM (2011) Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review. Mem Inst Oswaldo Cruz 106(3):257–266. https://doi.org/10.1590/S0074-02762011000300002

    CAS  Article  PubMed  Google Scholar 

  3. Archer SK, Luu VD, de Queiroz RA, Brems S, Clayton C (2009) Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle. PLoS Pathog 5(8):e1000565. https://doi.org/10.1371/journal.ppat.1000565

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bastin P, Bagherzadeh Z, Matthews KR, Gull K (1996) A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei. Mol Biochem Parasitol 77(2):235–239. https://doi.org/10.1016/0166-6851(96)02598-4

    CAS  Article  PubMed  Google Scholar 

  5. Bern C (2015) Chagas’ disease. N Engl J Med 373(5):456–466. https://doi.org/10.1056/NEJMra1410150

    CAS  Article  PubMed  Google Scholar 

  6. Blackinton JG, Keene JD (2014) Post-transcriptional RNA regulons affecting cell cycle and proliferation. Semin Cell Dev Biol 34:44–54. https://doi.org/10.1016/j.semcdb.2014.05.014

    CAS  Article  PubMed  Google Scholar 

  7. Bonney KM (2014) Chagas disease in the 21st century: a public health success or an emerging threat? Parasite 21:11. https://doi.org/10.1051/parasite/2014012

    Article  PubMed  PubMed Central  Google Scholar 

  8. Buchan DW, Minneci F, Nugent TC, Bryson K, Jones DT (2013) Scalable web services for the PSIPRED protein analysis workbench. Nucleic Acids Res 41 (web server issue):W349–357 doi:https://doi.org/10.1093/nar/gkt381

  9. 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

    CAS  PubMed  Google Scholar 

  10. DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, Vazquez MP, Levin MJ, Teixeira SM (2004) Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92(2):113–120. https://doi.org/10.1007/s00436-003-1004-5

    Article  PubMed  Google Scholar 

  11. 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(1–2):1–4. https://doi.org/10.1016/j.molbiopara.2015.02.003

    CAS  Article  PubMed  Google Scholar 

  12. Das A, Morales R, Banday M, Garcia S, Hao L, Cross GA, Estevez AM, Bellofatto V (2012) The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism. RNA 18(11):1968–1983. https://doi.org/10.1261/rna.033829.112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Drozdetskiy A, Cole C, Procter J, Barton GJ (2015) JPred4: a protein secondary structure prediction server. Nucleic Acids Res 43(W1):W389–W394. https://doi.org/10.1093/nar/gkv332

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, Aggarwal G, Caler E, Renauld H, Worthey EA, Hertz-Fowler C, Ghedin E, Peacock C, Bartholomeu DC, Haas BJ, Tran AN, Wortman JR, Alsmark UC, Angiuoli S, Anupama A, Badger J, Bringaud F, Cadag E, Carlton JM, Cerqueira GC, Creasy T, Delcher al, Djikeng A, Embley TM, Hauser C, Ivens AC, Kummerfeld SK, Pereira-Leal JB, Nilsson D, Peterson J, Salzberg SL, Shallom J, Silva JC, Sundaram J, Westenberger S, White O, Melville SE, Donelson JE, Andersson B, Stuart KD, Hall N (2005) Comparative genomics of trypanosomatid parasitic protozoa. Science 309(5733):404–409, DOI: https://doi.org/10.1126/science.1112181

  15. Gazestani VH, Yip CW, Nikpour N, Berghuis N, Salavati R (2017) TrypsNetDB: an integrated framework for the functional characterization of trypanosomatid proteins. PLoS Negl Trop Dis 11(2):e0005368. https://doi.org/10.1371/journal.pntd.0005368

    Article  PubMed  PubMed Central  Google Scholar 

  16. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R (2010) A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38(web server issue):W695–W699. https://doi.org/10.1093/nar/gkq313

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Hieronymus H, Silver PA (2003) Genome-wide analysis of RNA–protein interactions illustrates specificity of the mRNA export machinery. Nat Genet 33(2):155–161. https://doi.org/10.1038/ng1080

    CAS  Article  PubMed  Google Scholar 

  18. Jimenez V (2014) Dealing with environmental challenges: mechanisms of adaptation in Trypanosoma cruzi. Res Microbiol 165(3):155–165. https://doi.org/10.1016/j.resmic.2014.01.006

    Article  PubMed  PubMed Central  Google Scholar 

  19. Keene JD (2007) RNA regulons: coordination of post-transcriptional events. Nat Rev Genet 8(7):533–543. https://doi.org/10.1038/nrg2111

    CAS  Article  PubMed  Google Scholar 

  20. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. https://doi.org/10.1038/nprot.2015.053

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Kolev NG, Ullu E, Tschudi C (2014) The emerging role of RNA-binding proteins in the life cycle of Trypanosoma brucei. Cell Microbiol 16(4):482–489. https://doi.org/10.1111/cmi.12268

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Lander N, Li ZH, Niyogi S, Docampo R (2015) CRISPR/Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment. MBio 6(4):e01012–e01015. https://doi.org/10.1128/mBio.01012-15

  23. Marchini FK, de Godoy LM, Rampazzo RC, Pavoni DP, Probst CM, Gnad F, Mann M, Krieger MA (2011) Profiling the Trypanosoma cruzi phosphoproteome. PLoS One 6(9):e25381. https://doi.org/10.1371/journal.pone.0025381

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Nett IR, Martin DM, Miranda-Saavedra D, Lamont D, Barber JD, Mehlert A, Ferguson MA (2009) The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness. Mol Cell Proteomics 8(7):1527–38

  25. Nicholas K, Nicholas H, Deerfield DW (1997) GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4:14

    Google Scholar 

  26. Rassi A, Jr., Rassi A, Marin-Neto JA (2010) Chagas disease. Lancet 375(9723):1388–1402 doi:https://doi.org/10.1016/S0140-6736(10)60061-X

  27. Romaniuk MA, Cervini G, Cassola A (2016) Regulation of RNA binding proteins in trypanosomatid protozoan parasites. World J Biol Chem 7(1):146–157. https://doi.org/10.4331/wjbc.v7.i1.146

    Article  PubMed  PubMed Central  Google Scholar 

  28. Roy A, Yang J, Zhang Y (2012) COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res 40(web server issue):W471–W477. https://doi.org/10.1093/nar/gks372

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7(1):539. https://doi.org/10.1038/msb.2011.75

    Article  PubMed  PubMed Central  Google Scholar 

  30. Tonelli RR, Augusto Lda S, Castilho BA, Schenkman S (2011) Protein synthesis attenuation by phosphorylation of eIF2alpha is required for the differentiation of Trypanosoma cruzi into infective forms. PLoS One 6(11):e27904. https://doi.org/10.1371/journal.pone.0027904

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Trindade S, Rijo-Ferreira F, Carvalho T, Pinto-Neves D, Guegan F, Aresta-Branco F, Bento F, Young SA, Pinto A, Van Den Abbeele J, Ribeiro RM, Dias S, Smith TK, Figueiredo LM (2016) Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice. Cell Host Microbe 19(6):837–848. https://doi.org/10.1016/j.chom.2016.05.002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Urbaniak MD, Martin DM, Ferguson MA (2013) Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei. J Proteome Res 12(5):2233–2244. https://doi.org/10.1021/pr400086y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Vazquez MP, Levin MJ (1999) Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene 239(2):217–225. https://doi.org/10.1016/S0378-1119(99)00386-8

    CAS  Article  PubMed  Google Scholar 

  34. Zhang Y (2009) I-TASSER: fully automated protein structure prediction in CASP8. Proteins 77 Suppl 9(S9):100–113. https://doi.org/10.1002/prot.22588

    Article  PubMed  Google Scholar 

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Correspondence to Vivian Bellofatto.

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Section Editor: Journaliz Badon

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Tyler Weisbarth, R., Das, A., Castellano, P. et al. The Trypanosoma cruzi RNA-binding protein RBP42 is expressed in the cytoplasm throughout the life cycle of the parasite. Parasitol Res 117, 1095–1104 (2018). https://doi.org/10.1007/s00436-018-5787-9

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Keywords

  • Trypanosomes
  • Trypanosoma cruzi
  • mRNA-binding proteins
  • Immunofluorescence
  • Transgenic parasites
  • Chagas disease