The final stage in the formation of the two large
subunit rRNA species in Saccharomyces cerevisiae
is the removal of internal transcribed spacer 2 (ITS2)
from the 27SB precursors. This removal is initiated by
endonucleolytic cleavage approximately midway in ITS2.
The resulting 7S pre-rRNA, which is easily detectable,
is then converted into 5.8S rRNA by the concerted action
of a number of 3′ → 5′ exonucleases, many
of which are part of the exosome. So far the complementary
precursor to 25S rRNA resulting from the initial cleavage
in ITS2 has not been detected and the manner of its conversion
into the mature species is unknown. Using various yeast
strains that carry different combinations of wild-type
and mutant alleles of the major 5′ → 3′
exonucleases Rat1p and Xrn1p, we now demonstrate the existence
of a short-lived 25.5S pre-rRNA whose 5′ end is located
closely downstream of the previously mapped 3′ end
of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature
25S rRNA by rapid exonucleolytic trimming, predominantly
carried out by Rat1p. In the absence of Rat1p, however,
the removal of the ITS2 sequences from 25.5S pre-rRNA can
also be performed by Xrn1p, albeit somewhat less efficiently.