Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Abstract

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

Trypanosoma cruzi; tRNA-derived fragments; subcellular localisation


SHORT COMMUNICATION

Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Larissa ReifurI, III; Maria Rosa Garcia-SilvaII; Saloê Bispo PoubelI; Lysangela Ronalte AlvesI; Paulo AraucoI; Diane Kelly BuiarIII; Samuel GoldenbergI; Alfonso CayotaII; Bruno DallagiovannaI, + + Corresponding author: brunod@tecpar.br

IInstituto Carlos Chagas-Fiocruz, Curitiba, PR, Brasil

IIFunctional Genomics Unit, Institut Pasteur de Montevideo, Mataojo, MV, Uruguay

IIIDepartamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brasil

ABSTRACT

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

Key words:Trypanosoma cruzi - tRNA-derived fragments - subcellular localisation

The non-infective epimastigote form of Trypanosoma cruzi undergoes metabolic and morphological adaptations to differentiate into the pathogenic metacyclic trypomastigote form, which causes Chagas disease in mammals (Figueiredo et al. 2000). Morphological changes are associated with and preceded by a shift in metabolism, which is correlated with the differential expression of several genes (Minning et al. 2009). Although the metabolism of the replicative epimastigote is fully active, with constitutive polycistronic transcription, starvation reduces the transcriptional rates of the RNA polymerases in metacyclics (Ferreira et al. 2008). Despite the reduced level of transcription, translation is detected in these non-replicative forms, indicating that gene expression control in metacyclics occurs primarily at the post-transcriptional level (reviewed in Haile & Papadopoulou 2007).

Considering the almost exclusive post-transcriptional control of gene expression in T. cruzi, it would be surprising if this organism did not have an alternative pathway to compensate for the absence of an RNA interference system. A myriad of small non-coding RNAs have been reported in trypanosomatids (Dumas et al. 2006, Garcia-Silva et al. 2010, Michaeli et al. 2012), canonical microRNAs, and siRNAs have not been detected in T. cruzi (Franzén et al. 2011). Short (20-35 nt) RNAs derived from tRNAs were first observed in cytoplasmic granules in the epimastigote form of T. cruzi after an initial fingerprint sequencing of 348 clones (Garcia-Silva et al. 2010). Although 26% of the sequenced clones represented tRNA-derived fragments, a more thorough sequencing of the small RNAs from epimastigotes showed that 65.3% of more than 282.000 clones represented tRNA-derived fragments (Franzén et al. 2011). The composition of the small RNA population in T. cruzi was strikingly different from that observed in Trypanosoma brucei, reflecting clear differences in the molecular biology of the two parasites (Michaeli et al. 2012). Nonetheless, the relative abundance of tRNA-derived fragments could be related to gene expression control under various types of cellular stress, as has been suggested for prokaryotes, yeast, mammalian cells, the protozoans Giardia lamblia and Tetrahymena thermophila (Lee & Collins 2005, Li et al. 2008, Pederson 2010).

In this study, we conducted further analyses of the relative abundance and subcellular localization of T. cruzi tRNA-derived fragments during the infective stage of the parasite. We used T. cruzi metacyclic trypomastigotes derived from Dm28c epimastigotes cultured in vitro, as described by Contreras et al. (1985). Total RNA was extracted using TRIzol (Invitrogen) and size fractionated on a denaturing 15% polyacrylamide gel electrophoresis (PAGE) gel. Subsequently, 18-40 nt RNAs were excised from the gel, purified and cloned as described by Garcia-Silva et al. (2010). To recover the small RNAs, specific oligonucleotide adaptors containing Ban 1 restriction sites were ligated to the 5' and 3' ends. The RNAs were then reverse transcribed, amplified by polymerase chain reaction for 10 cycles, Ban 1-digested, concatamerized, cloned into the pGEM T-easy vector (Promega Corp) and sequenced. Analyses were performed using the public GenBank (ncbi.nlm.nih.gov/genbank), GeneDB (genedb.org) and TriTrypDB (tritrypdb.org/tritrypdb) databases.

From a total of 844 clones analysed, 509 sequences aligned with the T. cruzi genome, whereas 2.16% showed no matches, indicating that these sequences may correspond to regions of the genome that have not been sequenced, exogenous contaminating DNA or differences between the reference strain and the strain used in this study. No sequences matched small nuclear RNA sequences, but 0.98% mapped to small nucleolar RNAs, 2.95% mapped to intergenic regions, 6.09% mapped to mRNAs and 24.56% mapped to rRNAs. Most of the cloned sequences (63.26%) were fragments derived from tRNAs that appeared to be the result of a specific cleavage at or around the anticodon loop (). The tRNA fragments averaged 33 nt in length and were mostly derived from the 3' end (86.96%) of a restricted group of isoacceptors. In contrast with the RNAs obtained from an epimastigote population sequenced by Garcia-Silva et al. (2010), the tRNA-derived fragments obtained in this study from metacyclics exhibited differences with respect to abundance (tRNA fragments composed 63.26% of the small RNAs in metacyclics versus 26% in epimastigotes), orientation (most of the tRNA fragments were derived from the 3' side of the tRNAs in metacyclics vs. the 5' side in epimastigotes) and origin (in metacyclics, fragments were derived mostly from tRNAGlu, tRNAThr and tRNAVal; in epimastigotes, they were generally derived from tRNAAsp and tRNAGlu). The relative abundances of most fragments did not correspond to either tRNA gene copy number or codon usage (Horn 2008, Padilla-Mejia et al. 2009), consistent with previous reports (Franzén et al. 2011). As reported by Franzén et al. (2011), we observed that the majority of the fragments were derived from the 3' side of the tRNAs and a significant proportion of these fragments (24.53%) carried the 3' CCA sequence, indicating that both mature and pre-tRNAs undergo the cleavage process. The biological importance of these tRNA fragments is unknown, but we expected that a higher percentage of these fragments would be observed in metacyclics because stressed epimastigotes showed only a slight increase in tRNA-derived fragments (Garcia-Silva et al. 2010). This phenomenon is likely observed because tRNA cleavage is a conserved process in cells under various types of stress and metacyclic trypomastigotes are the product of epimastigote differentiation triggered by nutritional stress (Contreras et al. 1985). A comparison of our results with those of Garcia-Silva et al. (2010) demonstrates that the percentage of tRNA fragments is clearly higher in metacyclics. In contrast, this difference is not observed by Franzén et al. (2011). Moreover, the epimastigote tRNA-derived fragments sequenced by Franzén et al. (2011) are mostly derived from the 3' arm of tRNAHis. These discrepancies could be due to differences in the strains analysed in the two studies and to the cloning and sequencing methods used. Franzén et al. (2011) used a different strain (CL Brener) and analysed a much larger number of clones using RNAseq, which provided a higher coverage of the parasite genome.

The cloned RNA sequences were aligned using the LocARNA server (Will et al. 2007) and the secondary structures identified using the RNAalifold server (Bernhart et al. 2008) from the Vienna RNA package and were adjusted manually according to the secondary structural domains of canonical tRNAs. The predicted secondary structures adopted by the most abundant 3' tRNA fragments revealed that, upon cleavage around the anticodon nucleotides, the tRNA fragment maintained the TψC loop conformation and was extended a few extra base pairs into the double-stranded stem, resulting in an energetically favourable structure (). Although this result is based only on computational calculations and does not confirm the true molecular structure in vivo, the fact that the most abundant tRNA-derived fragments can assume a similar, relatively stable configuration suggests a structural (or functional) significance.

The subcellular localization of the tRNA-derived fragments in the metacyclic forms was evaluated through fluorescence in situ hybridisation (FISH) using probes complementary to the 5' and 3' ends of tRNAGlu-UUC (the most abundantly cloned tRNA fragment). FISH assays showed that these fragments are dispersed throughout the cytoplasm in metacyclics, whereas stressed epimastigotes exhibited the same posterior granular distribution as non-stressed epimastigotes (Fig. 1). To further analyze the cellular localization of the tRNA-derived fragments, metacyclic cells were gently lysed in lysis buffer (300 mM KCl, 10 mM MgCl2, 10 mM Tris-HCl 7.4 pH and 0.5% NP40) for 5 min. Two fractions were collected: the supernatant, containing only the soluble cytosol contents (S), and the insoluble fraction, or pellet (P), containing insoluble organelles, vesicles and cellular membranes. Both fractions were loaded on a sodium dodecyl sulphate-PAGE (10%) and transferred onto Hybond-C membranes (Amersham). Western blots were performed using antibodies to TcPUF6 (1/250), a cytosolic RNA binding protein (Dallagiovanna et al. 2005), and dynamin (1/200), a membrane protein present in endocytic vesicles (Pucadyil & Schmid 2009) (Fig. 2F, G). Northern blots were conducted using total RNA extracted from the two cell fractions (S and P) and a radiolabeled probe specific to the tRNAGlu-UUC fragment. The tRNA-derived fragments were detected in both fractions, supporting the dispersed pattern observed in the immunofluorescence assays (Fig. 2E). The signal observed for the insoluble cellular fraction could indicate the presence of these RNAs in smaller intracellular vesicles. Nonetheless, their dispersed pattern in the cytoplasm in metacyclics contrasts with the granular and posterior localization in epimastigotes, in which a partial co-localization with reservosomes has been inferred. The dismantling of the tRNA fragments in metacyclics could be correlated with the absence of reservosomes in this life stage (Figueiredo et al. 2000) and is consistent with the described cytoplasmic localization of the type II tRNA-derived fragments. Type II tRNA-derived fragments are most likely generated in the cytosol by RNaseZ cleavage and by RNA polymerase III termination and these fragments have been found to coimmunoprecipitate with Argonaute proteins (Elbarbary et al. 2009, Haussecker et al. 2010). This dramatic change in subcellular localization is puzzling and suggests distinct roles for these molecules during the different stages of the parasite life cycle.

To determine whether the dispersed tRNA-derived fragments were associated with translating polysomes, metacyclic and epimastigote forms of T. cruzi were treated with cycloheximide and the polysomes were purified and separated on sucrose gradients, as previously described (Nardelli et al. 2007) (Fig. 2A, D). Parasite extracts were also treated with puromycin as a negative control (not shown). The polysomes, monosomes and ribosome-free fractions were pooled and the total RNA was extracted. After acrylamide gel separation and staining, a high concentration of small RNAs was observed in the ribosome-free pool for the metacyclic and epimastigote forms and a faint small RNA signal was observed in the ribosome-containing fractions (Fig. 2B, E). Northern blot analysis confirmed that tRNA-derived fragments from metacyclics and epimastigotes were concentrated in the ribosome-free fractions (Fig. 2C, F). Therefore, tRNA-derived fragments may not be related to the repression of the translational machinery. The knockdown of the tRNA-derived fragments and structural analysis should be used to help uncover the biological importance of these RNAs.

ACKNOWLEDGEMENTS

To Dr Donna J Koslowsky, Dr Laura E Yu and the reviewers, for critical reading of the manuscript, to Stenio Fragoso and Rosana Gonçalves, for the anti-Dynamin serum, and Nilson Fidencio, for technical assistance.

Received 20 December 2011

Accepted 10 May 2012

Financial support: FIOCRUZ, Araucaria Foundation (18.456) (to LR)

SG, LRA and BD received fellowships from CNPq, SBP received a fellowship from CAPES and DKB received a fellowship from UFPR/TN.

Supplementary data

Click to enlarge

Click to enlarge

  • Bernhart SH, Hofacker IL, Will S, Gruber AR, Stadler PF 2008. RNAalifold: improved consensus structure prediction for RNA alignments. BMC Bioinformatics 9: 474.
  • Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S 1985. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol Biochem Parasitol 16: 315-327.
  • Dallagiovanna B, Perez L, Sotelo-Silveira J, Smircich P, Duhagon MA, Garat B 2005. Trypanosoma cruzi: molecular characterization of TcPUF6, a Pumilio protein. Exp Parasitol 109: 260-264.
  • Dumas C, Chow C, Muller M, Papadopoulou B 2006. A novel class of developmentally regulated noncoding RNAs in Leishmania Eukaryot Cell 5: 2033-2046.
  • Elbarbary RA, Takaku H, Uchiumi N, Tamiya H, Abe M, Takahashi M, Nishida H, Nashimoto M 2009. Modulation of gene expression by human cytosolic tRNase Z(L) through 5'-half-tRNA. PLoS ONE 4: e5908.
  • Ferreira LR, Dossin F de M, Ramos TC, Freymuller E, Schenkman S 2008. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis. An Acad Bras Cienc 80: 157-166.
  • Figueiredo RC, Rosa DS, Soares MJ 2000. Differentiation of Trypanosoma cruzi epimastigotes: metacyclogenesis and adhesion to substrate are triggered by nutritional stress. J Parasitol 86: 1213-1218.
  • Franzén O, Arner E, Ferella M, Nilsson D, Respuela P, Carninci P, Hayashizaki Y, Aslund L, Andersson B, Daub CO 2011. The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi PLoS Negl Trop Dis 5: e1283.
  • Garcia-Silva MR, Frugier M, Tosar JP, Correa-Dominguez A, Ronal-te-Alves L, Parodi-Talice A, Rovira C, Robello C, Goldenberg S, Cayota A 2010. A population of tRNA-derived small RNAs is actively produced in Trypanosoma cruzi and recruited to specific cytoplasmic granules. Mol Biochem Parasitol 171: 64-73.
  • Haile S, Papadopoulou B 2007. Developmental regulation of gene expression in trypanosomatid parasitic protozoa. Curr Opin Microbiol 10: 569-577.
  • Haussecker D, Huang Y, Lau A, Parameswaran P, Fire AZ, Kay MA, 2010. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16: 673-695.
  • Horn D 2008. Codon usage suggests that translational selection has a major impact on protein expression in trypanosomatids. BMC Genomics 9: 2.
  • Lee SR, Collins K 2005. Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila J Biol Chem 280: 42744-42749.
  • Li Y, Luo J, Zhou H, Liao JY, Ma LM, Chen YQ, Qu LH 2008. Stress-induced tRNA-derived RNAs: a novel class of small RNAs in the primitive eukaryote Giardia lamblia Nucleic Acids Res 36: 6048-6055.
  • Michaeli S, Doniger T, Gupta SK, Wurtzel O, Romano M, Visnovezky D, Sorek R, Unger R, Ullu E 2012. RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs. Nucleic Acids Res 40: 1282-1298.
  • Minning TA, Weatherly DB, Atwood J 3rd, Orlando R, Tarleton RL 2009. The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi BMC Genomics 10: 370.
  • Nardelli SC, Avila AR, Freund A, Motta MC, Manhaes L, de Jesus TC, Schenkman S, Fragoso SP, Krieger MA, Goldenberg S, Dallagiovanna B 2007. Small-subunit rRNA processome proteins are translationally regulated during differentiation of Trypanosoma cruzi Eukaryot Cell 6: 337-345.
  • Padilla-Mejia NE, Florencio-Martinez LE, Figueroa-Angulo EE, Manning-Cela RG, Hernandez-Rivas R, Myler PJ, Martinez-Calvillo S 2009. Gene organization and sequence analyses of transfer RNA genes in trypanosomatid parasites. BMC Genomics 10: 232.
  • Pederson T 2010. Regulatory RNAs derived from transfer RNA? RNA 16: 1865-1869.
  • Pucadyil TJ, Schmid SL 2009. Conserved functions of membrane active GTPases in coated vesicle formation. Science 325: 1217-1220.
  • Will S, Reiche K, Hofacker IL, Stadler PF, Backofen R 2007. Inferring noncoding RNA families and classes by means of genome-scale structure-based clustering. PLoS Comput Biol 3: 680-691.

Publication Dates

  • Publication in this collection
    14 Sept 2012
  • Date of issue
    Sept 2012

History

  • Received
    20 Dec 2011
  • Accepted
    10 May 2012
Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
E-mail: memorias@fiocruz.br