ADENOVIRUS TYPE 12-INDUCED FRAGILITY OF THE HUMAN RNU2 LOCUS REQUIRESU2 SMALL NUCLEAR-RNA TRANSCRIPTIONAL REGULATORY ELEMENTS

Infection of human cells with oncogenic adenovirus type 12 (Ad12) indu ces four specific chromosome fragile sites. Remarkably, three of these sites appear to colocalize with tandem arrays of genes encoding small , abundant, ubiquitously expressed structural RNAs-the RNU1 locus enco ding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, a nd the RN5S locus encoding 5S rRNA. Recently, an artificial tandem arr ay of the natural 5.8-kb U2 repeat unit has been shown to generate a n ew Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, an d S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating th at the U2 repeat unit alone is sufficient for virally induced fragilit y. To identify elements within the U2 repeat unit that are required fo r virally induced fragility, we generated cell lines containing artifi cial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragmen t spanning the U2 gene alone, or the same 834-bp fragment from which k ey U2 transcriptional regulatory elements had been deleted. The U2 snR NA coding regions within each artificial array were marked by an innoc uous single base change (U to C at position 87) so that the relative e xpression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both th e 5.8- and the 0.8-kb U2 repeat units are fragile but that arrays lack ing either the distal sequence element or both the distal and the prox imal sequence elements of the promoter are not. Surprisingly, variatio ns in repeat copy number and/or transcriptional activity of the artifi cial arrays do not appear to correlate with the degree of Adl2-inducib le fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRN A transcription per se.