Dataset Information

Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.

ABSTRACT: Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

SUBMITTER: Dallagiovanna B

PROVIDER: S-EPMC2276385 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.

Dallagiovanna Bruno B Correa Alejandro A Probst Christian M CM Holetz Fabiola F Smircich Pablo P de Aguiar Alessandra Melo AM Mansur Fernanda F da Silva Claudio Vieira CV Mortara Renato A RA Garat Beatriz B Buck Gregory A GA Goldenberg Samuel S Krieger Marco A MA

The Journal of biological chemistry 20071204 13

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the pu ...[more]

PMID: 18056709

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Project description:Post-transcriptional gene regulation in Trypanosoma cruzi, the etiological agent of Chagas disease, plays a critical role in ensuring that the parasite successfully completes its life cycle in both of its obligate hosts: insect vector and mammals. This regulation is basically governed by RNA binding proteins (RBPs) through their interactions with cis-elements located in the UTRs of their mRNA targets. LYT1 gene, coding for a virulence factor of T. cruzi, is expressed into two isoforms: kLYT1 and mLYT1, which play different functions according to their cellular location and parasite life-cycle stages. Whereas kLYT1 exhibits a regulatory role during the epimastigote-to-metacyclic trypomastigote stage transition, mLYT1 acts as a pore-forming protein, relevant for host cell invasion and parasite intracellular survival. Considering the LYT1 biological relevance and the fact that this is a protein exclusive of T. cruzi, the protein and its mechanisms regulating the alternative gene expression products are promising targets for therapeutic intervention. In this work, an experimental approach consisting of pull-downs assays followed by proteomic analyzes was carried out to identify the proteins interacting with the different LYT1 mRNAs. The dataset presented here was obtained through three biological replicates using all the different UTRs characterized in the LYT1 mRNAs (i.e., 5´UTR kLYT1, 5´UTR mLYT1, and I and II-type 3´UTRs) as baits, and protein extracts from epimastigotes and trypomastigotes of the 058 PUJ (DTU I) strain. Bound proteins were analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). As a control of non-specificity, the same protein extracts were incubated with Leishmania braziliensis rRNA and the bound proteins also identified by LC/MS. In all, 1,557 proteins were identified, 313 of them were found in at least two replicates and 18 proteins were exclusively associated with the LYT1 baits. Of these, six proteins have motifs related to RNA binding, and seven remain annotated as hypothetical proteins. Remarkably, three of these hypothetical proteins also contain nucleic acid binding motifs. This knowledge, beside expanding the known T. cruzi proteome, gains insight into putative regulatory proteins responsible for alternative LYT1 mRNAs processing. Raw mass spectrometry data are available via MassIVE proteome Xchange with identifier PXD027371.

Dataset Information

Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.

Publications

Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.

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