Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs.
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.
D000469:Alkaline Phosphatase; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D009694:Nucleic Acid Precursors / Q000235:genetics* / Q000378:metabolism; D011061:Poly A / Q000032:analysis*; D012322:RNA Precursors; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases; D013539:Simian virus 40 / Q000235:genetics*
D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012333:RNA, Messenger; D011061:Poly A; D012260:Ribonucleases; D000469:Alkaline Phosphatase