Synthesis and Processing of Adenoviral RNA in Isolated Nuclei

Abstract

Adenoviral RNA sequences synthesized in nuclei isolated during the late phase of productive infection comprise, besides virus-associated, VA, RNA, five major species, ranging in size from approximately 13S to 55S. The latter RNA species is of the length predicted if the major transcriptional unit expressed during the late phase were completely copied in vitro. Some 30% of the RNA sequences labeled in vitro are polyadenylated, and about one-third of the polyadenylated RNA is virus specific. Hybridization analysis of the sequences immediately adjacent to polyadenylic acid in late RNA labeled in isolated nuclei suggests that polyadenylation in vitro occurs at the same sites recognized within the cell. The polyadenylic acid-containing viral RNA sequences made in isolated nuclei are found in three major species of RNA, sedimenting at approximately 28S, 18S, and 13S. These sizes are remarkably similar to those reported for late mRNA species, suggesting that additional processing steps can occur in isolated nuclei. Hybridization of RNA to Xho I fragments of adenovirus type 2 DNA transferred to nitrocellulose filters reveals that sequences complementary to the region from 22.0 to 26.5 units present in 55S RNA are absent from all smaller species, suggesting that the smaller RNA species labeled in isolated nuclei are generated by splicing. The splicing events necessary to generate the 5′ leader segment common to the majority of late adenoviral mRNA species are shown to be performed correctly in isolated nuclei.