Novel deletion mutants that enhance a distant upstream 5′ splice in the E3 transcription unit of adenovirus 2

Abstract

Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site.

Non‐Classical Export of an Adenovirus Structural Protein

“…When analyzing various Ad2 mutants (dL762, dL712, ts1) that all carry intact Pb genes, we observed that trafficking of the Pb protein was never affected (Figure 2C). dL712 is an Ad2/Ad5 hybrid virus with a deletion of the E3 Ad death protein (ADP), (25,26), and the dL762 mutant has a deletion in the E3 14.7 K protein (27). Likewise, Ad2 ts1 bearing a point mutation in the L3/p23 protease gene which renders the protein incapable of packaging into newly synthesized virions at the restrictive temperature (28) clearly showed cell‐to‐cell spreading of its Pb protein.…”

A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus

“…5). mRNAfis overproduced by dn712 (3,13) and dM763 (Brady et al, in preparation). It is probably also overproduced by d1764 because d1764 has nearly the same deletion as 14- Cys-labeled 10.4K from mRNA purified by hybridization from pm760-infected KB cells.…”

Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

“…A study by Deutscher et al (12) suggests a testable mechanism for alternative selection of Drosophila MHC exons that depends on selective repression of the 5' splice junction of the thorax-specific exon. They found that a 400-base region of the adenovirus E3 transcription unit represses use of a particular 5' splice site, perhaps by formation of an RNA stem-loop structure which prevents recognition of this splice site by the splicing machinery (55).…”