Trypanosoma cruzi: Molecular characterization of an RNA binding protein differentially expressed in the parasite life cycle

Clemente Estable Biological Research Institute, Ciudad de Montevideo, Montevideo, Uruguay
Experimental Parasitology (Impact Factor: 1.64). 10/2007; 117(1):99-105. DOI: 10.1016/j.exppara.2007.03.010
Source: PubMed


Molecular studies have shown several peculiarities in the regulatory mechanisms of gene expression in trypanosomatids. Protein coding genes are organized in long polycistronic units that seem to be constitutively transcribed. Therefore, post-transcriptional regulation of gene expression is considered to be the main point for control of transcript abundance and functionality. Here we describe the characterization of a 17 kDa RNA-binding protein from Trypanosoma cruzi (TcRBP19) containing an RNA recognition motive (RRM). This protein is coded by a single copy gene located in a high molecular weight chromosome of T. cruzi. Orthologous genes are present in the TriTryp genomes. TcRBP19 shows target selectivity since among the different homoribopolymers it preferentially binds polyC. TcRBP19 is a low expression protein only barely detected at the amastigote stage localizing in a diffuse pattern in the cytoplasm.

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    • "Though some RRM RBPs have been characterized in T. cruzi, their RNA targets as well as their role in gene expression regulation have been reported only for a few cases [8]. TcRBP19 function could not be directly attributed in silico, since orthologues are restricted to kinetoplastids and not characterized so far [7]. As a first approach to understand the differential expression pattern of TcRBP19, we investigated the mechanisms underlying its mRNA abundance. "
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    ABSTRACT: Because of their relevant role in the post-transcriptional regulation of the expression of a multitude of genes, RNA-binding proteins (RBPs) need to be accurately regulated in response to environmental signals in terms of quantity, functionality and localization. Transcriptional, post-transcriptional and post-translational steps have all been involved in this tight control. We have previously identified a Trypanosoma cruzi RBP, named TcRBP19, which can barely be detected at the replicative intracellular amastigote stage of the mammalian host. Even though protein coding genes are typically transcribed constitutively in trypanosomes, TcRBP19 protein is undetectable at the epimastigote stage. Here, we show that this protein expression pattern follows the steady-state of its mRNA. Using a T. cruzi reporter gene approach, we could establish a role for the 3′ UTR of the tcrbp19 mRNA in transcript down-regulation at the epimastigote stage. In addition, the binding of the TcRBP19 protein to its encoding mRNA was revealed by in vitro pull down followed by qRT-PCR and confirmed by CLIP assays. Furthermore, we found that forced over-expression of TcRBP19 in T. cruzi epimastigotes decreased the stability of the endogenous tcrbp19 mRNA. These results support a negative feedback control of TcRBP19 to help maintain its very low concentration of TcRBP19 in the epimastigote stage. To our knowledge, this is the first RBP reported in trypanosomatids capable of negatively regulating its own mRNA. The mechanism revealed here adds to our limited but growing number of examples of negative mRNA autoregulation in the control of gene expression.
    Full-text · Article · Jun 2013 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.
    Full-text · Article · May 2011 · Memórias do Instituto Oswaldo Cruz
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    ABSTRACT: Trypanosoma cruzi is the causative agent of Chagas disease, a neglected disorder that affects millions of people in the Americas. T. cruzi relies mostly upon post-transcriptional regulation to control stage specific gene expression. RNA binding proteins (RBPs) associate with functionally related mRNAs forming ribonucleoprotein complexes that define post-transcriptional operons. The RNA Recognition Motif (RRM) is the most common and ancient family of RBPs. This family of RBPs has been identified in trypanosomatid parasites and only a few of them have been functionally characterized. We describe here the functional characterization of TcRBP40, a T. cruzi specific RBP, and its associated mRNAs. We used a modified version of the recombinant RIP-Chip assay to identify the mRNAs with which it associates and in vivo TAP-tag assays to confirm these results. TcRBP40 binds to an AG-rich sequence in the 3'UTR of the associated mRNAs, which were found to encode mainly putative transmembrane proteins. TcRBP40 is differentially expressed in metacyclogenesis. Surprisingly, in epimastigotes, it is dispersed in the cytoplasm but is concentrated in the reservosomes, a T. cruzi specific organelle, which suggests a putative new function for this parasite organelle.
    Full-text · Article · Mar 2012 · Biochemical and Biophysical Research Communications
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