Processing of the yeast pre-rRNA at sites A₂ and A₃ is linked

Cleavage of the yeast pre-rRNA at site A₂ in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A₃, located 72 nt 3' in ITS1, requires RNase MRP. Analyses of mutations in the pre-rRNA have revealed an unexpected link between processing at A₂ and A₃. Small substitution mutations in the 3' flanking sequence at A₂ inhibit processing at site A₃, whereas a small deletion at A₃ has been shown to delay processing at site A₂. Moreover, the combination of mutations in cis at both A₂ and A₃ leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between +482 and +496. The simultaneous interference with an snoRNP processing complex at site A₂ and an RNase MRP complex at site A₃ may activate a pre-rRNA breakdown pathway. The same aberrant pre-rRNA species are observed in strains with mutations in the RNA component of RNase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by RNase MRP and subsequent exonuclease digestion. We conclude that an snoRNP-dependent processing complex that is required for A₂ cleavage and that recognizes the 3' flanking sequence at A₂, interacts with the RNase MRP complex bound to the pre-rRNA around site A₃.