Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 13536–13541, December 1997
Functional separation of pre-rRNA processing steps revealed by
truncation of the U3 small nucleolar ribonucleoprotein
SARAH J. LEE* AND SUSAN J. BASERGA†‡
Departments of *Molecular Biophysics and Biochemistry and†Therapeutic Radiology and Genetics, Yale School of Medicine, New Haven, CT 06520-8040
Communicated by Sidney Altman, Yale University, New Haven, CT, October 8, 1997 (received for review August 8, 1997)
(snoRNP) is required for three cleavage events that generate
the mature 18S rRNA from the pre-rRNA. In Saccharomyces
cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein,
halts 18S rRNA production and impairs cleavage at the three
U3 snoRNP-dependent sites: A0, A1, and A2. We have iden-
tified truncation mutations of Mpp10 that affect 18S rRNA
synthesis and confer cold-sensitivity and slow growth. How-
ever, distinct from yeast cells depleted of Mpp10, the mutants
carrying these truncated Mpp10 proteins accumulate a novel
precursor, resulting from cleavage at only A0. The Mpp10
truncations do not alter association of Mpp10 with the U3
snoRNA, nor do they affect snoRNA or protein stability. Thus,
the role in processing of the U3 snoRNP can be separated into
cleavage at the A0 site, which occurs in the presence of
truncated Mpp10, and cleavage at the A1?A2 sites, which
occurs only with intact Mpp10. These results strongly argue
for a role for Mpp10 in processing at the A1?A2 sites.
The U3 small nucleolar ribonucleoprotein
Four rRNAs and many proteins comprise the eukaryotic
ribosome: the 60S subunit contains 25S (Saccharomyces cer-
evisiae), 5.8S, and 5S rRNAs, and the 40S subunit houses 18S
rRNA. In the nucleolus RNA polymerase I transcribes long,
25S, 5.8S, and 18S rRNAs. A schematic of pre-rRNA process-
ing (Fig. 1) shows that the rRNA sequences are flanked by two
external transcribed spacers (5? and 3?) and separated by two
internal transcribed spacers (ITS1 and ITS2). Complexes of a
small nucleolar RNA and proteins, called snoRNPs, and other
nucleolar proteins process this original transcript into the
mature rRNAs. The depletion or mutation of many of these
processing molecules disrupts pre-rRNA processing (1, 2).
Many small nucleolar RNAs (snoRNAs) have been identi-
fied but U3 is the most abundant in vertebrate cells and exists
in a wide evolutionary range of species (from trypanosomes to
humans), making it an attractive molecule for studies aimed at
elucidation of pre-rRNA processing. In vivo and?or in vitro
studies in three different systems, mouse, Xenopus laevis
oocytes, and S. cerevisiae, have demonstrated that it is as an
essential component for production of 18S rRNA (3–9).
In yeast, processing of the pre-18S rRNA occurs before the
cleavage steps that release the mature 5.8S and 25S rRNAs
(Fig. 1). The U3 snoRNA has been shown to be necessary for
the early processing events that begin at the A0 site and then
proceed to the A1 and A2 sites (4, 6, 7, 9). However, other
small nucleolar ribonucleoprotein (snoRNPs), including U14
and snR30, are required for formation of 18S rRNA (10, 11).
Thus, pre-rRNA processing may be mediated by a multi-
snoRNP ‘‘processome,’’ similar to the spliceosome that carries
out splicing of pre-mRNAs (12).
Two U3 snoRNA-pre-rRNA base pairing interactions have
been identified in yeast (6, 7, 9, 13). One of these interactions,
between the U3 snoRNA and a site within the 5? external
transcribed spacers (?150 nucleotides upstream of the A0
cleavage site), is essential for pre-18S rRNA processing. Base
complementarity in this interaction is essential as mutations in
the 5? external transcribed spacer that disrupt the U3-pre-
rRNA association impair 18S rRNA production. Processing
can be restored with these by making the compensatory
mutations in the U3 snoRNA. The sizable distance of this
necessary, base paired region from any of the cleavage sites led
to a model characterizing the U3 snoRNA as an RNA chap-
erone. Instead of catalyzing the cleavage, U3 snoRNA may
help fold the pre-rRNA and expose cleavage sites to the
enzymatically active molecule. Indeed, Rnt1, the yeast ho-
molog of Escherichia coli RNase III (14), is sufficient for
cleavage at this site in vitro and cleaves in vivo at this site. The
components responsible for cleavage at A1 and A2 have not
yet been identified.
Although several U3-specific proteins have been identified
in vertebrate cells through immunoprecipitation experiments,
only one has been purified and none of their genes have been
cloned (15, 16). The genes for two proteins associated with the
yeast U3 snoRNP, however, have been cloned: NOP1 (17–19),
the yeast homolog of the vertebrate fibrillarin protein, and
SOF1 (20). Unlike Nop1, a vertebrate homolog for Sof1
remains unknown. Both proteins are required for yeast via-
bility and production of 18S rRNA (21).
Nop1 is a protein component common to a number of
snoRNAs (22), so the phenotypes resulting from mutations in
NOP1 may not reflect a function associated with U3. Sof1, on
the other hand, is a U3-specific protein that was identified in
yeast as an extragenic suppressor of a temperature-sensitive
fibrillarin mutant (20). Cells lacking Sof1 mimic the phenotype
observed in cells depleted of U3, with impaired cleavage of
pre-18S rRNA at the three U3-dependent sites but normal
production of 5.8S and 25S rRNAs. Sof1, it appears, plays a
necessary role in U3 snoRNP function. Without more detailed
studies, however, one cannot acquire further insight into
whether Sof1 affects U3 snoRNP assembly and?or the role of
the snoRNP in cleavage of the three sites.
Recent studies from our laboratory have characterized a
novel protein in yeast and vertebrates, Mpp10, that associates
specifically with the U3 snoRNA (J. M. Westendorf, K. N.
Konstantinov, S. Wormsley, M. D. Shu, N. Matsumoto-Taniura,
F. Pirollet, F. G. Klier, L. Gerace and S.J.B., unpublished
results) (23). MPP10 is essential for cell survival in yeast and
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© 1997 by The National Academy of Sciences 0027-8424?97?9413536-6$2.00?0
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Abbreviations: snoRNA, small nucleolar RNA; snoRNP, small nucle-
‡To whom reprint requests should be addressed at: Therapeutic
Radiology and Genetics, Yale School of Medicine, HRT 317, P.O.
Box 208040, New Haven, CT 06520-8040. e-mail: susan.baserga@
rRNA. Although both mpp10–1 and mpp10–2 strains show
inefficient processing at these sites at 30°C, only the double
mutant, the mpp10–2 strain, incurs a growth defect. Northern
analysis on steady-state levels of rRNA reveals that both the
mpp10–1 and mpp10–2 strains have decreased levels of 18S
rRNA, mpp10-2 more severely affected than mpp10–1. From
wild type to mpp10–1 to mpp10–2, a successive under-
accumulation of both 32S and 20S rRNA is observed, indic-
ative that processing efficiency also declines in this graded
fashion among the three strains. This suggests that there may
be a level of processing deficiency that is tolerable for the cells
and allows them to divide normally, as in the mpp10–1 strain.
Rates of rRNA production in mpp10–1 seem to fall above this
level and the strain grows as quickly as wild-type yeast.
However, if the processing efficiency falls below this permis-
sible level, the rate of rRNA production is no longer able to
match the demands of a cell dividing at wild-type rates.
Therefore, even though processing in the mpp10–2 strain is not
drastically different from the mpp10–1 strain, the decrease in
efficiency compared with wild-type must exceed the threshold,
causing the double mutant to show a sharp two-fold decrease
in cell division rate.
Of note is the accumulation of the primary transcript, the
35S rRNA, in the double truncation mutant strain (mpp10–2),
but not in the single truncation strain (mpp10–1). It is possible
that the appearance of an increase in the 35S precursor occurs
when pre-rRNA processing is only severely affected, as an
increase in the 35S precursor is also observed for yeast
individually depleted of three U3 snoRNP components (9, 20,
23). This is in contrast to results obtained after disrupting
pre-rRNA processing in Xenopus oocytes, where levels of the
primary transcript remain constant following depletion of the
U22 snoRNA, which is also required for pre-18S rRNA
processing (34, 35). This may reflect a difference between
Although our results indicate that part of the Mpp10 protein
is required for U3 snoRNA association, it is not known with
which part of the U3 snoRNA it is associated. Experiments in
vertebrates have indicated that only the 3? half of U3 is
required for fibrillarin binding, and this association requires an
intact box C (36). A 55 kDa U3 snoRNP protein identified in
hamster cells is also associated with the 3? half of the U3
snoRNA (16). Determination of the U3 nucleotides required
for binding of Mpp10 may strengthen our view that it is
involved in the formation of the pseudoknot at the 5? end of
the 18S rRNA, if similar sequences are required for U3
snoRNP association and pre-rRNA processing.
We wish to thank these members of the Baserga lab for their help
and advice: Steven Wormsley, David Dunbar, and Tina Agentis. We
thank Peter Glazer and Chris Yoo for critical reading of the manu-
script. S.J.B. is a member of the Yale Cancer Center.
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Biochemistry: Lee and BasergaProc. Natl. Acad. Sci. USA 94 (1997)13541