Deadenylation is the first and rate-limiting step during turnover of mRNAs in 
eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' 
exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT 
deadenylase complex, belonging to the DEDD and 
Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has 
been identified as a new member of the EEP family of exonucleases based on 
sequence homology, but its activity and biological roles are presently unknown. 
Here, we show using in vitro deadenylation assays on defined RNA species 
mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' 
exonuclease most active at slightly acidic conditions. We further show that the 
enzyme depends on divalent metal ions for activity and possesses specificity 
towards poly-A RNA similar to what has been observed for cellular deadenylases. 
The results suggest that Ngl3p is naturally involved in processing of 
poly-adenylated RNA and provide insights into the mechanistic variations 
observed among the redundant set of EEP enzymes found in yeast and higher 
eukaryotes.