9] Isolation and characterization of yeast ribosomal RNA precursors and preribosomes

Abstract

This chapter describes the identification and characterization of the precursor RNAs and ribonucleoproteins (RNPs) from the 35 S pre-rRNA operon. In addition to 35 S prerRNA, 20 and 27 S pre-rRNAs are the predominant intermediates. The 32 S pre-rRNA is found in variable yields depending on the genetic background of the particular strain and the conditions of growth and harvesting. The major part of the ribosome biogenesis pathway is located in the nucleolus. Shortly after, or possibly already during transcription, 35 S pre-rRNA is subjected to a large number of modifications, including methylation and pseudouridylation. High-molecular-weight pre-rRNAs (20-35 S) are fractionated on agarose gels after glyoxylation or on standard formamide–formaldehyde gels and then are blotted onto a solid support. Filters are hybridized with probes containing ETS, ITS1, or ITS2 sequences, but lacking sequences corresponding to the mature and S rRNAs. Spheroplasts have to be poured into a 10-fold excess of ice-cold 1 M sorbitol containing unlabeled uracil or methionine.