Adenovirus Type 12-Induced Fragility of the Human RNU2 Locus Requires U2 Small Nuclear RNA Transcriptional Regulatory Elements

Abstract

Infection of human cells with oncogenic adenovirus type 12 (Ad12) induces 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 encoding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, and the RN5S locus encoding 5S rRNA. Recently, an artificial tandem array of the natural 5.8-kb U2 repeat unit has been shown to generate a new Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, and S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating that the U2 repeat unit alone is sufficient for virally induced fragility. To identify elements within the U2 repeat unit that are required for virally induced fragility, we generated cell lines containing artificial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragment spanning the U2 gene alone, or the same 834-bp fragment from which key U2 transcriptional regulatory elements had been deleted. The U2 snRNA coding regions within each artificial array were marked by an innocuous single base change (U to C at position 87) so that the relative expression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both the 5.8-and the 0.8-kb U2 repeat units are fragile but that arrays lacking either the distal sequence element or both the distal and the proximal sequence elements of the promoter are not. Surprisingly, variations in repeat copy number and/or transcriptional activity of the artificial arrays do not appear to correlate with the degree of Ad12-inducible fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRNA transcription per se.

The Microsatellite Sequence (CT)n · (GA)nPromotes Stable Chromosomal Integration of Large Tandem Arrays of Functional Human U2 Small Nuclear RNA Genes

“…In all constructs, the U2 snRNA genes were marked with an innocuous U87C point mutation. This mutation in a phylogenetically variable nucleotide allowed us to use a primer extension assay to monitor steady-state levels of U2 snRNA derived from the transgenes relative to the background of resident U2 snRNA (5).…”

“…Six ligations were performed: (i) 6.0 g of mU2 plus 0.06 g of E1, (ii) 6.0 g of mU2ϩCT plus 0.06 g of E1, (iii) 6.0 g of mU2⌬DSE⌬PSE plus 0.06 g of E1, (iv) 6.0 g of mU2⌬DSE ⌬PSEϩCT plus 0.06 g of E1, (v) 6.0 g of mU2 plus 1.1 g of mU2ϩCT plus 0.06 g of E1, and (vi) 6.0 g of mU2 plus 0.5 g of CT plus 0.06 g of E1. All ligations were carried out in two steps: DNA at 1 mg/ml was incubated for 4 h at room temperature with 100 U of T4 DNA ligase (Boehringer Mannheim) per ml in buffer provided by the manufacturer, to which 50 mM spermidine had been added to promote DNA condensation (5,8); the volume was then doubled, 100 U of additional ligase per ml was added, and the reaction was continued overnight. Dilution was necessary to ensure that the amount of glycerol, introduced with the enzyme, did not exceed 10%; dilution also reduced the viscosity, which increased perceptibly during the first ligation step.…”

“…The E1 selection cassette contains the mouse dihydrofolate reductase gene driven by the simian virus 40 early promoter and the neomycin resistance gene driven by the herpes simplex virus thymidine kinase gene promoter. The E1, mU2, and mU2⌬DSE⌬PSE constructs have been described previously (5). A 700-bp AccI fragment containing the entire CT microsatellite (33) was excised from the iU2 construct, subcloned into the SmaI site of pUC18Bgl, and excised with BglII and BamHI.…”

“…The DNA was phenol extracted, and agarose oligomers were removed by the LiCl precipitation method (16). To ensure one E1 selection cassette per artificial array, BamHI/BglII fragments bearing the U2 gene and the E1 selection cassette were ligated at a mass ratio of 100 to 1 (5). Six ligations were performed: (i) 6.0 g of mU2 plus 0.06 g of E1, (ii) 6.0 g of mU2ϩCT plus 0.06 g of E1, (iii) 6.0 g of mU2⌬DSE⌬PSE plus 0.06 g of E1, (iv) 6.0 g of mU2⌬DSE ⌬PSEϩCT plus 0.06 g of E1, (v) 6.0 g of mU2 plus 1.1 g of mU2ϩCT plus 0.06 g of E1, and (vi) 6.0 g of mU2 plus 0.5 g of CT plus 0.06 g of E1.…”

The p53 Tumor Suppressor Protein Represses Human snRNA Gene Transcription by RNA Polymerases II and III Independently of Sequence-Specific DNA Binding

“…Secondly, infection of human cells by adenovirus serotype 12 causes metaphase fragility at four chromosomal sites, including the U1 snRNA (RNU1) and U2 snRNA (RNU2) loci (1,36), in a process that requires p53 (38,39). It was postulated that fragile site formation occurs during viral infection, because RNA polymerase II stalls at these genes and interferes with chromosome condensation during metaphase (37).…”