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In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi.

Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.