Identification of mRNA decapping activities and an ARE-regulated 3′ to 5′ exonuclease activity in trypanosome extracts

Abstract

mRNA turnover is a regulated process that contributes to the steady state level of cytoplasmic mRNA. The amount of each mRNA determines, to a large extent, the amount of protein produced by that particular transcript. In trypanosomes, there is little transcriptional regulation; therefore, differential mRNA stability significantly contributes to mRNA levels in each stage of the parasite life cycle. To investigate the enzymatic activities that contribute to mRNA turnover, we developed a cell-free system for mRNA turnover using the trypanosome Leptomonas seymouri. We identified a decapping activity that removed m7GDP from mRNAs that contain an m7GppN Cap at their 5′ end. In yeast, the release of m7GDP by the pyrophosphatase Dcp1p/Dcp2p is a rate-limiting step in mRNA turnover. A secondary enzymatic activity, similar to the human cap scavenger activity, was identified in the trypanosome extracts. Both the human and trypanosome scavenger activities generates by addition in trans of m7GpppG. A third enzymatic activity uncovered in the parasite extracts functioned as a 3′ to 5′ exonuclease. Importantly, this exonuclease activity was stimulated by an AU-rich element present in the RNA. In summary, the cell-free system has defined several RNA turnover steps that likely contribute to regulate mRNA decay in trypanosomes.