Spliced segments at the 5 ' terminus of adenovirus 2 late mRNA * ( adenovirus 2 mRNA processing / 5 ' tails on mRNAs /

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5! terminus were not hydrogen bonded to the single-stranded DNA. DNA se uences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17,20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of vira RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA. Most eukaryotic mRNAs bear modifications at both termini; their 3' termini have a tract of poly(A) that ranges in length from 30 to 200 bases (1-4), while their 5' termini are typically capped with a methylated guanine joined through a 5'-5' pyrophosphate linkage to a second nucleotide methylated at its 2' position (5, 6). Both types of modifications of eukaryotic mRNA are known to occur after transcription. All adenovirus mRNAs are thought to contain poly(A) tracts at their 3' termini (7) and be capped with a methylated guanine (8, 9). Specific restriction endonuclease cleavage fragments of adenovirus 2 (Ad2) DNA have permitted the mapping of regions of the genome expressed as mRNA and viral proteins during different stages of the lytic cycle (10-12). Little is known about the molecular mechanisms of viral mRNA synthesis. An important aspect of late mRNA synthesis is thought to be the processing and selection of viral mRNAs from the nucleus (18, 14). We have purified a late Ad2 hexon mRNA and found evidence providing some insight into the mechanism of synthesis of this mRNA. MATERIALS AND METHODS Isolation of Ad2 DNA and RNA. Polyribosomal RNA was prepared from Ad2-infected cells 32 hr after infection as described by Flint and Sharp (14, 15) and selected by chromatography on poly(U)-Sephadex (16). R-Loop Mapping. The R-loop hybridization mixture was essentially that of Thomas et al. (17) and contained 70% (vol/ vol) formamide [Matheson, Coleman, and Bell, 99%, further purified as described by Duesberg and Vogt (18)]; 0.20 M Tris-HCl, pH 7.91; 0.50 M NaCI; 0.01 M EDTA; Ad2 DNA at 10 ,g/ml; and purified hexon mRNA at 1-10 Ag/ml. This mixture was incubated at 52.50 for 2-3 hr and spread on a hyThe costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3171 pophase of water with internal length standards of DNA from bacteriophage qX174, 5375 bases (19). Hybridization to Single-Stranded Ad2 DNA. Hybridizations of either polyribosomal poly(A) or purified hexon mRNA with restriction endonuclease fragments of Ad2 DNA were carried out in reaction mixtures of-80% formamide; 0.40 M NaCl; 0.04 M 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.2; 0.01 M EDTA; DNA at 10 lg/ml; and hexon mRNA at 1.0-10 ,ug/ml (20). The sample was incubated at 57-60° for 2-3 hr.