Functionally different AU- and G-rich cis-elements confer developmentally regulated mRNA stability in Trypanosoma cruzi by interaction with specific RNA-binding proteins

Post-transcriptional regulatory mechanisms have been suggested to be the ma in point of control of gene expression in kinetoplastid parasites. We have previously shown that Trypanosoma cruzi SMUG mucin mRNA steady-state level is developmentally regulated by post-transcriptional mechanisms, being stab le in the epimastigote insect vector stage, but unstable in the trypomastig ote infective stage of the parasite, Its turnover is controlled by an AU-ri ch element (ARE) localized in the 3'-untranslated region, since a reporter gene lacking this sequence was stable in the trypomastigote stage (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000) J. Biol. Chem. 275, 10218-10227). Here, we show by gel mobility shift assay that the 44-n t ARE sequence interacts with a set of stage-specific AU-rich element RNA-b inding proteins (ARE-BPs). The epimastigote stage AU-rich element RNA-bindi ng protein, named E-ARE-BP, and the trypomastigote stage ARE-BPs, named T-A RE-BPs, are efficiently competed by poly(U). UV cross-linking analysis show ed that E-ARE-BP has an apparent molecular mass of 100 kDa and is different from the 45-50-kDa ARE-BPs present in other stages of the parasite. Transf ection experiments allowed the identification of a novel cia-element that m ight be responsible for a positive effect on mRNA stability. It is a G-rich element, named GRE, composed by two contiguous CGGGG pentamers. The factor s that recognize GRE were different from the ones that bind to ARE, in both molecular masses and subcellular localization. Thus, ARE and GRE are funct ionally different cis-elements, which might regulate mucin expression throu ghout the parasite life cycle.