In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs.

35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.