Truncated 01/A′–A0 species derived from 5′-ETS accumulate in human cells producing enzymatically deficient exosome-associated nucleases. (A) A scheme of the human 47S pre-rRNA (top) and enlarged portion of 5′-ETS extending from the nucleotide +1 to the processing site A0 (bottom). Positions of the processing sites within the 47S precursor are indicated with vertical thin lines together with the names. Nucleotide numbering is according to GenBank (accession number U13369.1). Gray bars below the primary transcript show positions of northern blot probes and oligonucleotides used for primer extension in this study. (B) Northern blot analysis of pre-rRNA processing across the entire 47S precursor. Total RNA was isolated from model cell lines grown in the presence of tetracycline, separated in a denaturing agarose-formaldehyde gel, and transferred onto nylon membrane, which was then stained with methylene blue and sequentially hybridized with probes targeting various regions of 5′-ETS, ITS1, or ITS2, as indicated at the bottom. Positions of different RNA species are indicated on the right. For the probe h5ETS, the overexposed part of the membrane is additionally shown to better visualize accumulation of the long processing intermediate of unknown origin, containing +1-01/A′ part of the 5′-ETS. (C) Primer extension analysis of pre-rRNA processing at sites 01/A′ and A0 for the same RNA samples as in panel B. (D) Northern blot analysis of the 5′-ETS degradation intermediates with probes hA′ and a–e, complementary to sequences located at the 5′-end or at a varying distance from the 3′-end of the excised 01/A′–A0 processing by-product. The experiment was performed as in panel B. Upper and bottom parts of the panel show results of two independent experimental replicates. The former one represents analysis for the same RNA samples as in panels B and C. In the latter case, smaller RNA species were better separated, revealing length differences between major degradation intermediates detected by probes hA′ and a in comparison to probes b–d, which are marked with dashed lines and referred to on the left as 01/A′–A0x and 01/A′–A0y, respectively. (E) Schematic representation of the 01/A′–A0x and 01/A′–A0y decay intermediates.
