Figure 3.

Neo-NORs are undercondensed during mitosis and transcribed by Pol I into 45S pre-rRNA in interphase. (A) Schematic representation of the neo-NOR cassette. (B) Metaphase chromosome spreads from neo-NOR clone m1 were hybridized with an XEn probe. In clone m1, an array of neo-NOR cassettes is inserted into the p-arm of chromosome 4 (Supplemental Fig. S4). Arrowheads indicate novel secondary constrictions at the site of neo-NOR insertion. (C) RNA-FISH using mouse (green) and human (red) 5′ ETS probes identifies neo-NOR-derived transcripts (arrowheads) in clone m1. (D) Combined 3D immuno-RNA-FISH reveals that UBF and PAF49 colocalize with neo-NOR-derived transcripts in clone m1. (E) S1 nuclease protection assays on RNA extracted from HT1080 and neo-NOR cell lines (20 μg each) were performed at nucleotide resolution. These assays indicate that neo-NOR-derived transcripts initiate from the expected nucleotide +1 of neo-NOR 5′ ETS, resulting in the protection of 41 base pairs (bp) from the 60-bp probe. (F) Northern blotting of RNA extracted from 3T3 cells (1 μg), HT1080 cells (10 μg), and neo-NOR clones a1 and m1 (10 μg each) probed with mouse 5′ ETS sequences identifies neo-NOR-derived 45S pre-rRNAs and 34S pre-rRNA processing intermediates.