Title

Antisense regulation in polyoma virus

Date of Completion

January 1999

Keywords

Biology, Molecular|Biology, Microbiology

Degree

Ph.D.

Abstract

Antisense RNA may regulate the expression of a number of eukaryotic genes, but little is known about its prevalence or mechanism of action. We have used a model system in which antisense control can be studied both genetically and biochemically. Late in polyoma virus infection, early-strand mRNA levels are downregulated by nuclear antisense RNA from the late strand. Analysis of early-strand transcripts isolated late in infection revealed extensive base modifications. In many transcripts almost half of the adenosines were altered to inosines or guanosines. These results suggest modification of RNA duplexes by dsRNA adenosine deaminase or a related enzyme. Probes that only detect modified RNAs revealed that these molecules are not highly unstable, but accumulate within the nucleus and are thus inert for gene expression. Further characterization of the modified transcripts revealed that both the early (sense) as well as the late (antisense) strands are modified. Moreover, majority of the early-strand modified transcripts are polyadenylated while majority of the late-strand modified transcripts are nonpolyadenylated. Quantitation experiments revealed that almost all of the newly synthesized early-strand transcripts at late times in infection are modified and there is essentially no more accumulation of unmodified early-strand RNAs. Antisense-induced modifications can account for most or all of the observed regulation, with the lowered levels of early-strand RNAs commonly observed late in infection resulting from the fact that many transcripts are invisible to standard hybridization probes. This work suggests that similar antisense-mediated control mechanisms may also operate under physiological conditions in uninfected eukaryotic cells, and leads to the proposal that there is a novel pool of nuclear RNAs which cannot be seen with many molecular probes heretofore used. ^