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 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.
D'Orso
I
I
Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, Consejo Nacional de Investigaciones Cientificas y Técnicas, Universidad Nacional de General San Martin, 1650 San Martin, Provincia de Buenos Aires, Argentina.
Frasch
A C
AC
D000225:Adenine; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006147:Guanine; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D016053:RNA, Protozoan / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development; D014498:Uracil
D012333:RNA, Messenger; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D014498:Uracil; D006147:Guanine; D015500:Chloramphenicol O-Acetyltransferase; D000225:Adenine