Formation of mRNA 3' termini involves cleavage of an mRNA precursor and 
polyadenylation of the newly formed end. Cleavage of simian virus 40 late 
pre-mRNA in a crude nuclear extract generated two RNAs, 5' and 3' 
half-molecules. These RNAs were unmodified and linear. The 5' half-molecule 
contained sequences upstream but not downstream of the poly(A) site and ended in 
a 3'-terminal hydroxyl. The 3' half-molecules comprised a family of RNAs, each 
of which contains only sequences downstream of the poly(A) site, and ends in a 
5'-terminal phosphate. These RNAs differed only in the locations of their 5' 
terminus. The 3' terminus of the 5' half-molecule was the adenosine 10 
nucleotides downstream of AAUAAA, at the +1 position. The 5' terminus of the 
longest 3' half-molecule was at +2. Thus, these two RNAs contain every 
nucleoside and phosphate of the precursor. The existence of these half-molecules 
demonstrates that endonucleolytic cleavage occurs near the poly(A) site. 5' 
half-molecules generated in the presence of EDTA (which blocks polyadenylation, 
but not cleavage) ended at the adenosine at position +1 of the precursor. When 
incubated in the extract under suitable conditions, they became polyadenylated. 
5' half-molecules formed in 3'-dATP-containing reactions contained a single 
3'-deoxyadenosine (cordycepin) residue added onto the +1 adenosine and were poor 
polyadenylation substrates. We infer that the +1 adenosine of the precursor 
becomes the first A of the poly(A) tract and provides a 3' hydroxyl group to 
which poly(A) is added posttranscriptionally.