Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA.

• DOI: 10.1128/mcb.10.4.1705
• Publication Date: 2015-10-01T21:55:41Z
• Authors: P L Wigley; M D Sheets; D A Zarkower; M E Whitmer; M Wickens
• Keywords: 0301 basic medicine; 0303 health sciences; Base Sequence; Transcription, Genetic; Molecular Sequence Data; Polynucleotide Adenylyltransferase; DNA-Directed RNA Polymerases; Nucleotidyltransferases; Substrate Specificity; Kinetics; 03 medical and health sciences; T-Phages; RNA, Messenger; Oligonucleotide Probes; Poly A

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze reaction. In this study, we identified structural features of RNA substrate critical for polyadenylation. We found a contained only 11 nucleotides, which first six were AAUAAA, underwent AAUAAA-specific is shortest have used supports polyadenylation: removal single nucleotide from either abolished Although appeared strict requirement polyadenylation, number nucleotides between 3' was critical. Substrates with seven fewer beyond received decreased efficiency yet still bound efficiently factor. infer on these shortened substrates, polymerase cannot simultaneously contact RNA. By incorporating 2'-deoxyuridine into U demonstrated 2' hydroxyl binding hence addition. finding may indicate one factors involved interaction protein.