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. 2014 Aug;34(15):2903–2916. doi: 10.1128/MCB.01634-13

FIG 4.

FIG 4

Loss of HPM1 results in early rRNA processing defects and delayed kinetics of pre-rRNA maturation. (A) rRNA precursor processing pathway in S. cerevisiae. The 35S pre-rRNA encodes the 18S rRNA of the small ribosomal subunit and the 5.8S and 25S rRNAs of the large subunit. The predominant pathway for the processing of the 35S pre-rRNA transcript is shown on the left, and the abnormal pathway is shown on the right (dashed arrow). First, the 5′ end of the 35S pre-rRNA is cleaved at sites A0 and A1, generating the mature 5′ end of the 18S rRNA. Cleavage at the A2 site separates the 20S and 27SA2 precursors. The 20S precursor is cleaved at site D in the cytoplasm to yield the mature 18S transcript. The 5′ end of the 27SA2 precursor is cleaved at site A3; this is followed by 5′-to-3′ exonuclease trimming to generate the mature 5′ end of the 5.8S rRNA. C2 cleavage separates the 5.8S and 25S precursors, which are then trimmed by exonucleases to yield the mature 5.8S and 25S rRNAs. (B) Northern blot analysis of rRNA precursor steady-state levels in the WT and the hpm1Δ mutant (lanes 1 and 2). Lanes 3 to 8 represent deletions of various ribosomal protein methyltransferases. The locations of the probes within each rRNA species are indicated on the right. The 25S and 18S blots are from the ethidium bromide fluorescence of the gel prior to transfer. (C) Top, Northern blot analysis of rRNA precursor steady-state levels with the 35S probe in WT and hpm1Δ and rnt1Δ mutant cells; bottom, 25S and 18S rRNAs detected by ethidium bromide fluorescence of the gel prior to transfer. (D) Top, pulse-chase assay with [3H]uracil for analysis of pre-rRNA processing kinetics in WT and hpm1Δ mutant cells. Cultures in the early exponential growth phase were pulsed with tritiated uracil for 2 min and then chased with an excess of nonisotopically labeled uracil. Bottom, analysis of the membrane from the pulse-chase assay by Northern blotting of 25S and 18S rRNAs. The ratios of the 25S to 18S rRNAs are normalized to the 1-min chase lane of the WT.