Depletion of Rlp7 leads to the accumulation of 27SA3 pre-rRNA, which is shifted to 27SA2 pre-rRNA after nuclease inhibition by lithium chloride treatment. The PGAL1-Rlp7 strain was grown in galactose-containing medium (wild-type condition; gal) or incubated in glucose-containing medium for 16 h (Rlp7 depleted; glu). Additionally, both conditions were combined with lithium chloride treatment for 3 h (Li+). (–) Untreated samples. (A,B) 1.5% agarose gel separating the products of the Riboprobing workflow. (m) 100-bp ladder (Thermo Fisher). (A) Depletion of Rlp7 for 16 h resulted in a band with 460 nt length that was confirmed to arise from 27SA3 pre-rRNA by Sanger sequencing. 27SB pre-rRNA has two forms that can be detected in the overlapping sequencing results. (B) Although depletion of Rlp7 led to 27SA3 pre-rRNA accumulation, additional treatment with lithium chloride led to a strong signal arising from the earlier 27SA2 pre-rRNA. (C) Treatment scheme for the PGAL1-Rlp7 strain. (D) Northern blot of the PGAL1-Rlp7 strain grown in galactose or glucose-containing medium and treated with LiCl. An untreated culture served as control. Probes indicated on the left; (pre)-rRNA species on the right. (E) The different 27S pre-rRNA species were quantified using either signals from the northern blot (see Fig. 4C) or signals from the Riboprobing experiment (see Fig. 4B). For all conditions in each experimental approach, we normalized the signal intensities to the total 27S pre-rRNA signal in the untreated wild-type strain grown in galactose-containing media. The mean values of two biological replicates are shown.
