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Nucleic Acids Research, 2002, Vol. 30, No. 18 4040-4050
© 2002 Oxford University Press

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

Joseph Milone, Jeffrey Wilusz and Vivian Bellofatto*

Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, International Center for Public Health, 225 Warren Street, Newark, NJ 07103, USA

*To whom correspondence should be addressed. Tel: +1 973 972 4406; Fax: +1 973 972 3644; Email: bellofat{at}umdnj.edu

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 m7GpppN 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 generate m7GMP from short capped RNA and are inhibited 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 regulated mRNA decay in trypanosomes.


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