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. 2014 Feb 1;28(3):220–230. doi: 10.1101/gad.234591.113

Figure 4.

Figure 4.

Neonucleoli recruit DFC and GC nucleolar components and produce mature neo-NOR-derived rRNAs. (A) Combined 3D immuno-RNA-FISH reveals that early pre-rRNA processing factors fibrillarin and U3 snoRNA colocalize with neo-NOR-derived transcripts in clone m1. (B) S1 assays performed on RNA extracted from 3T3 (0.2 μg), HT1080 (20 μg), and neo-NOR (20 μg) cell lines indicate that neo-NOR-derived transcripts are accurately and efficiently cleaved at the A′ site of their 5′ ETS. Protected fragments arising from cleaved (C) and uncleaved (UC) transcripts are indicated. (C) RT–PCR reveals the presence of mouse 18S rRNAs in the cytoplasm of neo-NOR cell lines. Reactions in which reverse transcriptase (RT) was omitted served as negative controls. (D) Combined 3D immuno-RNA-FISH reveals that late pre-rRNA processing factors Nop52 and NPM colocalize with neo-NOR-derived transcripts in clone m1. (E) Northern blots of cellular (Total) and cytoplasmic (Cyto.) RNA extracted from 3T3 (0.5 μg), HT1080 (20 μg), and neo-NOR (20 μg) cell lines probed with a mouse-specific oligonucleotide reveal the presence of cytoplasmic mouse 28S rRNAs in neo-NOR lines.