Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing

Univ. Sci. 23 (2): 267-290, 2018. doi: 10.11144/Javeriana.SC23-2.cotm Bogotá original article Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing Elizabeth Ruíz1, César Augusto Ramírez1, Julián Camilo Casas1, María Isabel Ospina1, José María Requena2, Concepción J. Puerta1, * Edited by Juan Carlos Salcedo-Reyes () 1. Laboratorio de Parasitología Molecular. Departamento de Microbiología. Facultad de Ciencias. Pontificia Universidad Javeriana. Bogotá, Colombia. 2. Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, España. * Received: 07-02-2018 Accepted: 17-07-2018 Published on line: 25-09-2018 Citation: Ruíz E, Ramírez CA, Casas JC, Ospina MI, Requena JM, Puerta CM. Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing, Universitas Scientiarum, 23 (2): 267-290, 2018. doi: 10.11144/Javeriana.SC23-2.cotm Funding: This work was supported by COLCIENCIAS research project ID PPTA 120356933228, “Caracterización de factores proteicos asociados a la regulación de la proteína mLYT1 de Trypanosoma cruzi” granted to CJP. ERM and JCC were supported by COLCIENCIAS convocatoria doctorados nacionales 647-2014 and convocatoria jóvenes investigadores e innovadores 645-2015, respectively. Electronic supplementary material: Suppl. 1-8. Abstract In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of the mRNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYT1 and kLYT1, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5’ and 3’ UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5’ UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T. cruzi DTU. In addition, for the first time, was unscovered the existence of two types of LYT1 mRNAs transcripts, differing in length by 116 nts, that are generated by alternative polyadenylation. Furthermore, an in-silico analysis of the experimentally obtained UTRs, and ten additional LYT1 sequences retrieved from TritrypDB and GenBank databases, together with a thoroughly search of structural motifs, showed a remarkable conservation of relevant structural motifs previously associated with RNA metabolism in the different UTRs; these elements might be involved in the differential stage-specific expression of each LYT1 isoform. Keywords: Trypanosoma cruzi; Untranslated region [UTR]; RNA binding proteins [RBP]; Regulation of gene expression; LYT1 gene. Introduction Trypanosoma cruzi is the etiological agent of Chagas disease, an illness recognized for the World Health Organization [WHO] as one of the today seventeen-neglected tropical diseases [NTDs]. Indeed, this disease is a worldwide public health problem, with a current prevalence of 6 to 7 million of infected people, of which 0.7 to 1.2 million are in Colombia [1, 2]. Universitas Scientiarum, Journal of the Faculty of Sciences, Pontificia Universidad Javeriana, is licensed under the Creative Commons Attribution 4.0 International Public License 268 Characterization of the mRNA UTRs from LYT1 isoforms of T. cruzi Like other medically important trypanosomatids, T. cruzi has a complex life cycle that must alternate between two types of hosts: insect triatomines and mammals, including humans [3]. To accomplish it, parasites must face and adapt to their hosts’ environments through a fine and delicate regulation of their gene expression. In these organisms, gene expression is largely regulated at the posttranscriptional level because of their special gene arrangement, in which large clusters comprising up to hundreds of genes and having the same transcriptional orientation, are constitutively expressed as polycistronic RNA precursors [4-7]. These polycistronic RNAs are ultimately processed into individual molecules through the addition of a Spliced Leader [SL], a common miniexon sequence present in all the mature mRNAs of trypanosomatids, to the 5’ end and a poly [A] tail to the 3’ end, processes known as trans-splicing and polyadenylation, respectively [8]. In this way, the mechanisms that govern mRNA expression basically operate at the maturation, transport, stability and translation steps through the recognition of RNA motifs, mainly located on the untranslated regions [UTR] of mRNAs, by RNA Binding Proteins [RBP]; both types of molecules, mRNAs and RBP, conform ribonucleoprotein complexes [RNP], which define the fate of the mRNA molecules [4-6, 9, 10]. In trypanosomatids, it has also been demonstrated the existence of alternative trans-splicing in which two or more mature transcripts are generated from the same gene by the use of different trans-splicing acceptor sites. The relevance of alternative trans-splicing is well illustrated in regards with the functional expression of the LYT1 protein, a virulence factor of T. cruzi [11, 12]. This protein was uncovered in a pioneer study by Dr Andrews’ group in which it was described the existence in T. cruzi of a secreted protein, immunologically related to the C9 component of the membrane attack complex of complement, that possesses membrane pore-forming activity at low pH [13]. Given this activity, it was postulated that this protein would be mediating the escape of T. cruzi from the phagosome into the cytosol after cell invasion. Subsequently, Manning-Cela et al. [14], in an outstanding work, undertook the search for the coding gene of this virulence factor by immunoscreening of a T. cruzi cDNA expression library using antibodies against the C9 component. As a result, the LYT1 gene was identified and cloned. These authors, furthermore, demonstrated the cytolytic activity of the LYT1 protein by transfecting Schizosaccharomyces pombe with the LYT1 gene and analyzing its hemolytic effect [14]. Accordingly, deletion of LYT1 resulted in attenuation of T. cruzi infection capacity; however, unexpectedly, the LYT1-deficient epimastigotes transformed into metacyclic trypomastigotes more rapidly than wild-type parasites. Thus, a double function was suggested for LYT1: (i) a pore-forming activity relevant for intracellular survival, and (ii) a regulatory role during stage transition. Interestingly, in a subsequent article [15], these authors suggested the existence of two LYT1 isoforms, each one associated (...truncated)