Identification of mRNA decapping activities and an ARE-regulated 3' to 5' exonuclease activity in trypanosome extracts.
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 m(7)GDP from mRNAs that contain an m(7)GpppN cap at their 5' end. In yeast, the release of m(7)GDP 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 m(7)GMP from short capped RNA and are inhibited by addition in trans of m(7)GpppG. 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.
Milone
Joseph
J
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.
Wilusz
Jeffrey
J
Bellofatto
Vivian
V
D000818:Animals; D005092:Exonucleases / Q000378:metabolism*; D011061:Poly A / Q000235:genetics; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology; D014351:Trypanosomatina / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D012315:RNA Caps; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011061:Poly A; D005092:Exonucleases