TcUBP-1, an mRNA Destabilizing Factor from Trypanosomes, Homodimerizes and Interacts with Novel AU-rich Element- and Poly(A)-binding Proteins Forming a Ribonucleoprotein Complex

Abstract

Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic illness in Latin America. The parasite has a life cycle that includes a vertebrate and an insect vector, with different developmental stages involved in each host. In the insect, two main forms of the parasite are present: replicative epimastigotes and metacyclic trypomastigotes. The latter form is infective to humans after being released on the skin or mucosa with the feces of the bug. Metacyclic trypomastigotes invade host cells and differentiate into a replicative amastigote form that differentiates into bloodstream trypomastigotes. The latter stage is able to invade a wide variety of cells, thus propagating the infection. The cycle closes when the hematophagous vector ingests circulating trypomastigotes with its blood meal. Trypanosomes, protozoan parasites of the order Kinetoplastida, have particular features in terms of mechanisms leading to protein expression. RNA polymerase I promoters were identified in rDNA but also in genes encoding the variable surface antigens from African trypanosomes (1). Conversely, only few RNA polymerase II promoters were described (2). As part of the maturation process, a common 39-nucleotide RNA, named spliced leader, is added to all trypanosome mRNAs by a trans-splicing process. This phenomenon was shown to be couple to 3Ј-poly(A) tail addition during polycistronic RNA processing (3). As a consequence, and at variance with higher eukaryotic cells, the control of protein expression in trypanosomatids is mainly post-transcriptional (4). However, little information is available on the relative importance of these processes and how they operate jointly in the parasite.One of the possible mechanisms to regulate protein expression is through modification of the half-life of mRNAs. Several cis-elements located throughout the mRNA, coding and/or untranslated regions (UTRs), 1 were identified (5-7). However, only few of them were found to be recognized specifically by trans-acting factors (8). We have found previously (9) two distinct cis-elements located in the 3ЈUTR of the mRNAs of a mucin surface antigen family named SMUG of T. cruzi. A 44-nucleotide AU-rich element (ARE) was shown to destabilize SMUG transcripts in the infective non-replicative trypomastigote stage of the parasite. Conversely, a 27-nucleotide G-rich cis-element stabilizes SMUG in the non-infective replicative epimastigote stage of the parasite (8). These results suggest that both elements, ARE and the G-rich cis-element, act coordinately in a developmentally regulated manner and are recognized by specific RNA-binding proteins (8). Recently, we described that this ARE sequence was recognized by a single RRM-type RNA-binding protein named TcUBP-1, for T. cruzi Uridine-binding protein. In vivo, TcUBP-1 was shown to destabilize SMUG mRNAs in the epimastigote stage of the parasite (10). Furthermore, and at variance with what occurs in yeast, the processes of 3Ј-5Ј and 5Ј-3Ј exonucleolityc cleavage were shown to be active in trypanosomes (11). Th...

RNA Recognition Motif-type RNA-binding Proteins in Trypanosoma cruzi Form a Family Involved in the Interaction with Specific Transcripts in Vivo

“…In previous work, we demonstrated the existence of two RNA-binding proteins having RRMs in T. cruzi, named TcUBP1 and TcUBP2 (8,10). In order to elucidate whether they form part of a protein family, we carried out hybridization experiments on a high density filter containing a cosmid library (20), with a probe encompassing the RRM motif of TcUBP1.…”

“…A second gene, named Tcubp2, was also shown to be present in the T. cruzi genome (9). Its product, TcUBP2, is part of an RNA-protein complex together with TcUBP1 and the poly(A)-binding protein (10).…”

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

“…Overexpression of this protein in trypomastigotes resulted in a decreased TcSMUG transcript half-life (D'Orso & Frasch 2001). Together with TcUBP2 and polyadenylation binding protein, TcUBP1 is part of a ribonucleoprotein (mRNP) complex that recognises Urich motifs (D'Orso & Frasch 2002). RNA-protein interactions were analysed by mRNP immunoprecipitation with specific antibodies against each RRM-type protein, which identified 24 mRNAs that encoded known proteins as well as 10 hypothetical genes.…”

“…This element was found to act as a destabilising cis-acting factor in metacyclic trypomastigotes because no difference was observed in CAT activity in epimastigotes transfected with either of these vectors (Di Noia et al 2000). As discussed in the next section, TcUBP1, an RNA-binding protein (RBP), recognises the ARE site in TcSMUG mRNA and forms a large protein complex with TcUBP2 and the poly binding protein to control stage-specific expression by changing mRNA stability in T. cruzi (D'Orso & Frasch 2002).…”

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

“…The RNA-binding domain of TcRBP19 belongs to the RNA-recognition motif family (RRM), which is widely represented in proteins involved in posttranscriptional events, such as RNA processing, transport, translation, degradation and stabilisation (Dreyfuss et al 2002). In the T. cruzi genome, 77 open reading frames that code for putative RRM-containing proteins have been identified (De Gaudenzi et al 2005); however, with few exceptions, the expression and functional roles of these proteins remain to be characterised (D'Orso & Frasch 2002, De Gaudenzi et al 2003, Noe et al 2008, Cassola & Frasch 2009. The low levels of TcRBP19 expression, together with its ability to discriminate between RNA targets, led us to propose the involvement of this protein in specific regulatory steps of gene expression (Perez-Diaz et al 2007).…”