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Trypanosoma cruzi, the agent of Chagas disease, does not seem to control gene expression through regulation of transcription initiation and makes use of post-transcriptional mechanisms. We report here a 43-nt U-rich RNA element located in the 3'-untranslated region (3'-UTR) of a large number of T. cruzi mRNAs that is important for mRNA abundance in the intracellular amastigote stage of the parasite. Whole genome scan analysis, differential display RT-PCR, Northern blot, and RT-PCR analyses were used to determine the transcript levels of more than 900 U-rich-containing mRNAs of large gene families as well as single and low copy number genes. Our results indicate that the 43-nt U-rich mRNA element is preferentially present in amastigotes. The cis-element of a protein kinase 3'-UTR but not its mutated version promoted the expression of the green fluorescent protein reporter gene in amastigotes. The regulatory cis-element, but not its mutated version, was also shown to interact with the trypanosome-specific RNA-binding protein (RBP) TcUBP1 but not with other related RBPs. Co-immunoprecipitation experiments of TcUBP1-containing ribonucleoprotein complexes formed in vivo validated the interaction with representative endogenous RNAs having the element. These results suggest that this 43-nt U-rich element together with other yet unidentified sequences might be involved in the modulation of abundance and/or translation of subsets of transcripts in the amastigote stage.