Functionally Different AU- and G-rich cis-Elements Confer Developmentally Regulated mRNA Stability inTrypanosoma cruzi by Interaction with Specific RNA-binding Proteins

Iván D'Orso,Alberto C.C Frasch

doi:10.1074/jbc.m010959200

Abstract

Post-transcriptional regulatory mechanisms have been suggested to be the main 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 stable in the epimastigote insect vector stage, but unstable in the trypomastigote infective stage of the parasite. Its turnover is controlled by an AU-rich 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-nt ARE sequence interacts with a set of stage-specific AU-rich element RNA-binding proteins (ARE-BPs). The epimastigote stage AU-rich element RNA-binding protein, named E-ARE-BP, and the trypomastigote stage ARE-BPs, named T-ARE-BPs, are efficiently competed by poly(U). UV cross-linking analysis showed 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. Transfection experiments allowed the identification of a novel cis-element that might be responsible for a positive effect on mRNA stability. It is a G-rich element, named GRE, composed by two contiguous CGGGG pentamers. The factors that recognize GRE were different from the ones that bind to ARE, in both molecular masses and subcellular localization. Thus, ARE and GRE are functionally different cis-elements, which might regulate mucin expression throughout the parasite life cycle.

Highlights

Viewed in Refs. 2 and 3). ␣-Amanitin-sensitive RNA polymerase II from trypanosomes transcribes large polycistronic units containing a number of coding sequences [4]
To further determine if the lack of a strong shifted band in the polysome fraction was due to the presence of some endogenous U-rich RNA competitor that might be sequestering part of E-AU-rich element (ARE)-BP, the extract was pre-treated with ribonuclease A (RNase A) as described previously [37], and the nuclease was inactivated prior to perform the in vitro binding reaction with the SMUG-L-AU RNA probe
In this work we have obtained evidence for the existence of novel cis-elements localized in the 3Ј-untranslated regions (UTRs) of SMUG mucins from T. cruzi that control both mRNA stability and translation efficiency

Summary

Introduction

Viewed in Refs. 2 and 3). ␣-Amanitin-sensitive RNA polymerase II from trypanosomes transcribes large polycistronic units containing a number of coding sequences [4]. We have identified a novel G-rich element, named GRE, which might be responsible for a stage-specific stabilization of SMUG mRNA family in the epimastigote form of the parasite.

Results

Conclusion