Unusual Properties of Adenovirus E2E Transcription by RNA Polymerase III

The double-stranded DNA genomes of subgroup C human adenoviruses such as adenovirus type 5 (Ad5) contain the coding sequences for over 40 proteins, organized into eight RNA polymerase II transcription units (47). Multiple mRNAs specifying different proteins are produced from the primary transcripts of all but two of these transcription units by alternative splicing and polyadenylation (18, 47). The densely packed adenoviral genome also contains genes transcribed by cellular RNA polymerase III, the enzyme responsible for the synthesis of small cellular RNAs that function in such processes as translation (tRNAs, 5S RNA), pre-mRNA splicing (U6 snRNA), and protein trafficking (7SL RNA) (23, 42, 57). Two such RNA polymerase III transcription units, encoding virus-associated (VA) RNA I and VA RNA III, are located within an intron of the Ad5 major late RNA polymerase II transcription unit (37, 44). Although the number, size, and genomic location of VA RNA genes vary among the Adenoviridae, the majority of adenoviral genomes examined to date contain at least one such gene. Both VA RNAs synthesized in Ad2- or Ad5-infected cells are very abundant and accumulate in the cytoplasm (37). The major species, VA RNA I, is necessary for efficient translation of viral late mRNAs, as it blocks activation of RNA-dependent protein kinase, and hence inhibitory phosphorylation of elF2-α, in infected cells (35, 37). In initial studies of low-molecular-mass adenoviral RNAs, species other than the VA RNAs were not detected by methods with a lower sensitivity limit estimated to be on the order of 103 RNA molecules/cell (36). However, an additional class III gene is present in the viral genome, superimposed on the 5′ end of the E2E RNA polymerase II transcription unit. In both in vitro transcription systems and Ad5-infected cells, RNA polymerase III synthesizes small E2E RNAs of some 45 and 90 nucleotides (28, 43). This enzyme initiates E2E transcription in the immediate vicinity of the major initiation site for RNA polymerase II (by definition, position +1), most frequently at position +2, but terminates transcription at two sites designated t1 (+45) and t2 (+90) (Fig. ​(Fig.1)1) (16, 28, 43). In contrast to the VA RNAs and many cellular RNAs made by RNA polymerase III, the small E2E RNAs, which have been called E2E RNA I and E2E RNA II (Fig. ​(Fig.1),1), accumulate to only very low concentrations (28). This property suggests that the E2E RNA polymerase III transcripts function in a manner previously unknown among viral RNAs synthesized by this enzyme. FIG. 1. Organization of the E2E RNA polymerase II and III transcription units. The Ad5 genome is represented to scale by the solid horizontal line at the top of the figure, upon which are shown the major site of initiation of RNA polymerase II transcription (by ... The E2E RNA polymerase II promoter includes a TATA-like sequence and binding sites for the cellular transcriptional activators ATF and E2F located within the 90-bp sequence upstream of the initiation site (47, 48) (Fig. ​(Fig.1).1). Each of these sequences is required for stimulation of RNA polymerase II transcription by viral E1A proteins in infected cells (32, 53). Sequences located between positions +1 and +30 are also necessary for maximally efficient E2E transcription by RNA polymerase II in vitro (16). The results of mutational analysis of the organization of the E2E RNA polymerase III promoter indicate that it too comprises both internal and upstream sequences (16, 43). The internal sequences conform to those present in the majority of promoters recognized by RNA polymerase (23): the E2E promoter contains an A box with typical sequence and functional properties and a B box that binds the human RNA polymerase III initiation protein TFIIIC. The A box sequence partially overlaps an internal sequence of the RNA polymerase II promoter (16). Maximally efficient E2E transcription by RNA polymerase III also requires an upstream sequence located in the vicinity of the TATA box (16, 43). Sequences of the RNA polymerase II and RNA polymerase III promoters are therefore superimposed between positions −30 and +30 of the adenoviral E2E transcription units. Although unusual, this arrangement of transcription units and control sequences for RNA polymerases II and III is not unprecedented. An RNA polymerase III transcription unit has been reported to be superimposed on the 5′ end of the c-myc gene transcribed by RNA polymerase II (3, 11, 50). While the c-myc RNA polymerase III promoter is active in in vitro systems and Xenopus oocytes, its transcripts have not been detected in mammalian cells in which c-myc mRNA is made (3, 4, 50). However, the 5′ transcriptional control region of the Xenopus class II gene encoding TFIIIA contains an RNA polymerase III transcription active in vivo (34). The superimposition of RNA polymerase II and III promoters and transcription units in the adenoviral genome raises the issue of whether recognition of its E2E promoter by components of the RNA polymerase III system interferes with E2E transcription by RNA polymerase II. Such a phenomenon could influence significantly the course of the infectious cycle: E2E mRNAs encode all three of the viral replication proteins (47), and their production is therefore rate limiting for the onset of viral DNA and the transition into the late phase of infection. To assess the potential for such transcriptional interference and possible functions of E2E transcription by RNA polymerase III, we have examined the properties of both E2E transcription by RNA polymerase III and its products during the Ad5 infectious cycle.