The 27SA3 pre-rRNA copurifies with Noc2-TAP when Rlp7 is depleted, but is degraded during purification. Pre-ribosomal particles were purified from the PGAL1-Rlp7 strain using Noc2-TAP as bait protein. The strain was grown in a galactose-containing medium (wild-type conditions; gal) or incubated in a glucose-containing medium for 16 h (Rlp7 depleted; glu). Additionally, cells were treated with lithium chloride for 3 h (Li+) or RVC was added during purification. (–) Untreated samples. (A) Coomassie-stained gel with TEV eluates of Noc2-TAP particles from the Rlp7 depleted (glu) or control strains (gal). (MWM) PageRuler prestained protein ladder (Thermo Fisher). (B) Northern blot of RNA isolated from the PGAL1-Rlp7 strain by Noc2-TAP pull-downs. Probes indicated on the left; (pre)-rRNA species indicated on the right. (C,D) 1.5% agarose gel separating the products of the Riboprobing workflow. (m) 100-bp ladder (Thermo Fisher). (C) Riboprobing using 5′ linker and C1C2 primer and RNA isolated from the Noc2-TAP pull-downs after Rlp7 depletion (glu). Riboprobing with RNA isolated from the same strain grown in galactose-containing medium (gal) served as a control. (D) RNA isolated from Noc2-TAP pull-downs of the PGAL1-Rlp7 strain grown in galactose-containing medium and treated with LiCl was investigated by Riboprobing using the indicated combinations of linker and primer. (E) Assembly factors strongly coenriched with the Noc2-TAP bait protein in pull-downs from the LiCl-treated strain were identified by qMS. Relative abundance to the Noc2 bait protein was calculated. The mean abundance of assembly factors with a standard deviation of <0.1 and an abundance of >0.2 relative to the bait protein are shown (dots represent individual replicates).
