Co-immunoprecipitation of U3 snoRNA with ProtA-Nop58 in Δnop17
cells. (A) Total cell extracts from strains WT, Δnop17 and Δrsa1 were mixed with IgG-Sepharose beads for co-immunoprecipitation of snoRNAs with ProtA-Nop58. Bound RNA was detected by northern blotting using probes specific to the snoRNA U3. Membrane was washed and re-hybridized against probe specific to the 5S rRNA (internal control). TE, total extract; B, bound fraction. Bands were quantitated using Typhoon equipment and mean values of three biological replicates are shown. U3 bands were quantitated relative to 5S bands in each lane. U3/5S in mutants were then calculated relative to WT strain. (B) Nop58 immunoprecipitates snoRNA U3 chromatin. ChIP assay with A-Nop58 expressed in strains WT, Δnop17 and Δrsa1 was performed, followed by RT-qPCR reactions with primers for amplification of various regions of the U3 snoRNA gene. Mean values are based on three different experiments with two biological replicates.