Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3′ ETS but not the 5′ ETS

Abstract

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A₀ in the 5′ external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A₀, A₁, and A₂ are kinetically delayed. In contrast, cleavage in the 3′ ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3′ extended form of the 25S rRNA. The 3′ extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3′ ETS but not for cleavage at site A₀. The sites of in vivo cleavage in the 3′ ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3′ end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5′ end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3′ ETS is coupled.