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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
The Role of the Trypanosoma cruzi TcNRBD1 Protein in Translation
Camila Oliveira1, Paulo Costa Carvalho1, Lysangela Ronalte Alves1
1Instituto Carlos Chagas, Fiocruz-Paraná, Rua Professor Algacyr Munhoz Mader, 3775, Cidade Industrial de Curitiba-CIC, 81350-010, Curitiba, Brasil.
Abstract:
The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. Despite the importance of this type of control in Trypanosoma cruzi, few RNA binding proteins have been characterized. The RRM domain (RNA Recognition Motif) is one of the most abundant domains found in RNA-binding proteins in higher eukaryotes. Proteins containing the RRM domain are involved in the majority of post-transcriptional processes regulating gene expression. In this work, we aimed to characterize the protein TcNRBD1 from T. cruzi. TcNRBD1 is an RNA-binding protein that contains 2 RRM domains and is the ortholog of the P34 and P37 proteins from Trypanosoma brucei. The TcNRBD1 protein is expressed in all developmental stages of T. cruzi, and its localization pattern is concentrated at the perinuclear region. TcNRBD1 is associated with polysomes and with the 80S monosomes. Furthermore, sequencing of the mRNAs bound to TcNRBD1 allowed the identification of several transcripts that encode ribosomal proteins. Immunoprecipitation assays followed by mass spectrometry showed that the protein complexes with several ribosomal proteins from both the 40S and 60S subunits. In summary, the results indicate that TcNRBD1 is associated with different parts of the translation process, either by regulating mRNAs that encode ribosomal proteins or by acting in some step of ribosome assembly in T. cruzi.
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