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.