Accessibility of the region flanking cleavage site D to DMS modification during rRNA processing. (A) DMS modification in vivo. The 4TU labeling was performed for 2, 3, 5, and 7 min, with DMS added to the culture for the final minute. 4TU labeling and DMS modification were quenched together by addition of ice-cold β-mercaptoethanol and isoamyl alcohol. RNA was extracted and purified as described in Materials and Methods. Primer extension products were resolved on 12% PAA/7M UREA gel. The results of primer extension of total RNA and newly synthesized RNAs (4TU RNA) are presented on the autoradiogram. The lane described as “no 4TU” represents unlabeled RNA, which was mock-purified. The result from one experiment is presented on two autoradiograms. The first nucleotide at the 5′ side of the D cleavage site is numbered as position 1. The D cleavage site is indicated by the arrow. (Dotted line along the autoradiogram) The region that was quantified. Two adenosines in H45 dimethylated by Dim1 in vivo are marked by “M.” (B) DMS modification of RNA presented in purified pre-40S complex. The purification was performed using Tsr1-HTP (His6-TEV-ProtA) or PTH-Nob1 (ProtA-TEV-His6) as bait. The experiment was performed as described by Granneman et al. (2010). Primer 103 (complementary to ITS1) was used for primer extension through the site D region. (C) Semi-quantitative analysis of the DMS modification of total RNA relative to newly synthesized RNA. The columns represent averages, with error bars showing standard deviations from three independent in vivo experiments. In vitro structure probing was repeated twice. Due to degradation in position 11 of RNA probed in vitro, the intensity of modification was not measured. (D) DMS-modified nucleotides are marked on the secondary structure model of the 5′ end of ITS1 and 3′ end of the 18S rRNA region. The surrounding site D is accessible to modification in total RNA as well as in newly synthesized pre-rRNA. There are no statistically significant changes between the labeling of total RNA relative to newly synthesized RNA, so all positions are marked by gray dots.
