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Regulatory elements in the 3 untranslated region of the GP82 glycoprotein are responsible for its stage-speciﬁc expression in Trypanosoma cruzi metacyclic trypomastigotes
Ethel Bayer-Santos 1 , Luciana Girotto Gentil 1,2 , EstebanMaurício Cordero 2 , Paulo Roberto Ceridório Corrêa, José Franco da Silveira ∗
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, UNIFESP, Rua Botucatu 862, CEP 04023-062 São Paulo, Brazil
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Gene expression in Trypanosoma cruzi is regulated at the post-transcriptional level and cis-acting elements present in the 3untranslated region (3 UTR) play an important role by interacting with regulatory proteins. Previous studies demonstrated that the GP82 surface glycoprotein, which is involved in host cell invasion, is up-regulated in the infective metacyclic trypomastigote form, and that GP82 mRNA half-life is longer in this form compared to the non-infective epimastigote form. Here, we demonstrate that the 3 UTRof the GP82 transcript is involved in this developmental regulation, promoting higher expression of the green ﬂuorescent protein (GFP) reporter in metacyclic trypomastigotes than in epimastigotes. A series of stepwise deletions in the 3 UTR was created and results suggest that the mechanism regulating GP82 expression involves multiple elements in the 3 UTR. © 2012 Elsevier B.V. All rights reserved.Article history: Received 25 January 2012 Received in revised form 13 March 2012 Accepted 14 March 2012 Available online 8 May 2012 Keywords: Untranslated region Post-transcriptional control Trans-sialidase Surface protein Trypanosoma cruzi
1. Introduction Transcription in trypanosomatids occurs in a constitutive fashion and initiates bi-directionally in regions between two divergent geneclusters (Martinez-Calvillo et al., 2003). Different sets of genes are transcribed as large polycistronic pre-mRNA units that are processed into mature mRNAs by a coupled mechanism involving trans-splicing and polyadenylation (Matthews et al., 1994). As a consequence of polycistronic transcription, regulation of gene expression in trypanosomatids takes place at the posttranscriptional level, wheremRNAs interact with different sets of regulatory proteins that adjust mRNA levels according to cellular demands. Transcriptomic microarray analysis revealed that 50% of Trypanosoma cruzi genes are regulated during the parasite’s life cycle (Minning et al., 2009). Regulatory elements in mRNA 5 and 3 untranslated regions (UTRs) have been shown to be involved in controlling transcript abundancethrough interaction with RNAbinding proteins (RBPs) (reviewed in Araujo and Teixeira, 2011).
Metacyclic trypomastigotes express a surface glycoprotein with an apparent molecular weight of 82 kDa called GP82 (Teixeira and Yoshida, 1986), which is a member of the trans-sialidase family and is involved in the parasite attachment to and internalization into mammalian cells (Ramirez et al., 1993, 1999).It was demonstrated that GP82 transcripts accumulate in metacyclic forms and that treatments with translation inhibitors increased GP82 mRNA half-life in epimastigotes, suggesting that protein factors act by destabilizing transcripts in the epimastigote stage (Gentil et al., 2009). To better understand the mechanisms regulating GP82 stage-speciﬁc gene expression, we transfected epimastigotes withconstructs containing the native and truncated versions of GP82 3 UTR fused down-stream of the green ﬂuorescent protein (GFP) reporter, and submitted them to in vitro metacyclogenesis. Their GFP mRNA and protein levels were then analyzed. 2. Materials and methods All GFP reporter constructs were generated by PCR ampliﬁcation using a set of primers described in Supplemental Table I. Amplicons...