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Spangenberg L.,Institute Pasteur Montevideo | Shigunov P.,Instituto Carlos Chagas Fiocruz Parana | Abud A.P.R.,Instituto Carlos Chagas Fiocruz Parana | Cofre A.R.,Instituto Carlos Chagas Fiocruz Parana | And 11 more authors.
Stem Cell Research | Year: 2013

Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life. © 2013 Elsevier B.V.

Guerra-Slompo E.P.,Instituto Carlos Chagas Fiocruz Parana | Probst C.M.,Instituto Carlos Chagas Fiocruz Parana | Pavoni D.P.,Instituto Carlos Chagas Fiocruz Parana | Goldenberg S.,Instituto Carlos Chagas Fiocruz Parana | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2012

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. © 2012 Elsevier Inc.

Perez-Diaz L.,Laboratorio Of Interacciones Moleculares | Pastro L.,Laboratorio Of Interacciones Moleculares | Smircich P.,Laboratorio Of Interacciones Moleculares | Dallagiovanna B.,Instituto Carlos Chagas FIOCRUZ Parana | Garat B.,Laboratorio Of Interacciones Moleculares
Biochemical and Biophysical Research Communications | Year: 2013

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. © 2013 Elsevier Inc.

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