POLY(A) TAIL SHORTENING BY A MAMMALIAN POLY(A)-SPECIFIC 3'-EXORIBONUCLEASE

3'-Exonucleolytic removal of the poly(A) tail is the first and often r ate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplas mic extract from HeLa cells, the poly(A) tail of mRNA was degraded fro m the 3'-end. In agreement with earlier in vivo observations, prominen t decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for th is poly(A) shortening activity was purified from calf thymus. A polype ptide of 74 kDa copurified with the activity. The deadenylating nuclea se (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly d ependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly( A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. A t either salt concentration DAN and PAB I reconstituted poly(A) shorte ning with the same pattern of intermediates seen in cytoplasmic extrac t. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.