Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway
Nucleic Acids Research
Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway
Elodie Choque 1
Claudia Schneider 0
Olivier Gadal 1
Christophe Dez 1
0 Institute for Cell and Molecular Biosciences, Newcastle University , Newcastle upon Tyne NE2 4HH , UK
1 Laboratoire de Biologie Mol e ́culaire Eucaryote, Centre de Biologie Int e ́grative (CBI), Universit e ́ de Toulouse , CNRS, UPS, Toulouse Cedex 9 , France
Ribosome biogenesis requires more than 200 transacting factors to achieve the correct production of the two mature ribosomal subunits. Here, we have identified Efg1 as a novel, nucleolar ribosome biogenesis factor in Saccharomyces cerevisiae that is directly linked to the surveillance of pre-40S particles. Depletion of Efg1 impairs early pre-rRNA processing, leading to a strong decrease in 18S rRNA and 40S subunit levels and an accumulation of the aberrant 23S rRNA. Using Efg1 as bait, we revealed a novel degradation pathway of the 23S rRNA. Coimmunoprecipitation experiments showed that Efg1 is a component of 90S pre-ribosomes, as it is associated with the 35S pre-rRNA and U3 snoRNA, but has stronger affinity for 23S pre-rRNA and its novel degradation intermediate 11S rRNA. 23S is cleaved at a new site, Q1, within the 18S sequence by the endonuclease Utp24, generating 11S and 17S' rRNA. Both of these cleavage products are targeted for degradation by the TRAMP/exosome complexes. Therefore, the Q1 site defines a novel endonucleolytic cleavage site of ribosomal RNA exclusively dedicated to surveillance of pre-ribosomal particles.
Eukaryotic ribosomes are large ribonucleoprotein (RNP)
particles composed of the small (40S) and large (60S)
subunits that assemble on the messenger RNA (mRNA),
allowing its translation into protein. The 40S subunit is
assembled around the 18S rRNA, whereas the 60S particle
contains the 25S/28S, 5.8S and 5S rRNAs. Ribosome
biogenesis is a highly complex process which begins in the nucleolus
with the rDNA transcription by the RNA polymerase I (Pol
I) leading in the yeast Saccharomyces cerevisiae to the
production of the 35S pre-rRNA precursor (Figure 1A). This
precursor contains the mature 18S, 5.8S and 25S rRNA
sequences with external (5 -ETS and 3 -ETS) and internal
(ITS1 and ITS2) transcribed spacers, which are absent from
mature ribosomes. Maturation of this 35S pre-rRNA
involves a well-defined series of endonucleolytic steps (Figure
1A), followed by exonucleolytic maturation (Illustrated in
Figure 1B) that eliminate ETS and ITS sequences in order
to release 18S, 5.8S and 25S mature rRNAs (for reviews,
)). The 5S rRNA is synthesized independently by
RNA polymerase III (Pol III) and is incorporated as part
of a small RNP particle (3).
Many trans-acting factors and some ribosomal proteins
start assembling and stabilize the nascent RNA Pol I
transcript leading to the formation of a large RNP complex
called the small-subunit (SSU) processome or the 90S
); for review, see (
). In S. cerevisiae, more
than 200 trans-acting factors, also called assembly factors,
have been shown to participate directly in co- and
posttranscriptional steps of ribosome biogenesis. Among these
assembly factors, exo- and endonucleases are responsible
for transcribed spacer processing and elimination;
adenosine triphosphate (ATP)-dependent RNA helicases are
involved in RNA folding or remodeling; ATPases, GTPases,
kinases and other energy consuming factors are essential for
the process; and the function(s) of a large class of other
proteins are still unclear (
). The first particle to be assembled,
the SSU processome, is a large RNP complex containing
the U3 small nucleolar RNA (snoRNA). Within this
preribosomal complex, the three early pre-rRNA cleavages at
sites A0, A1 and A2 (
), defining the ‘A2 pathway’, take
place (Figure 1B). Protein-protein and protein–RNA
interaction data lead to the conclusion that pre-ribosomes are
sequentially assembled from multiple independently formed
The SSU processome integrity is of critical importance
for the early, mostly co-transcriptional, endonucleolytic
cleavages, which lead to the separation of the 43S and 66S
A0 A1 18S
1699 008 1510
D A2 A3
particles, precursors of the mature ribosomal subunits. In
consequence, individual loss of most SSU processome
factors inhibits these pre-rRNA cleavages at sites A0, A1 and
A2 and leads to the accumulation of the 23S pre-rRNA (‘A3
pathway’; Figure 1B). Utp24 has been proposed as the
endonuclease enzyme for the A1 and A2 sites (
of residues in the predicted active site of Utp24 leads to
reduced cell growth and defects in cleavage at the A1 and A2
sites. Consistent with a direct involvement in these
cleavages, Utp24 exhibited in vitro endonuclease activity on an
RNA substrate containing the A2 site. Moreover, the
integrity of the Utp24 PINc domain is required for efficient
cleavage at A2 site, both in yeast and human (
Inhibition of cleavages at sites A0, A1 and A2 leads to
the accumulation of the 23S pre-rRNA (see Figure 1). The
23S pre-rRNA results from a direct cleavage of the 35S
prerRNA at site A3, within ITS1, by RNAse MRP (Figure
1B and 8) (
). Depending on the yeast genetic
background and growth conditions, 23S pre-rRNA is
invariably present but in various amounts in wild-type (WT) cells.
23S pre-rRNA markedly accumulates in all ribosome
biogenesis mutants affecting early processing events.
Surprisingly, despite containing the entire 18S rRNA sequence,
accumulated 23S pre-rRNA does not seem to be further
). When it is produced, 23S pre-rRNA is
efficiently targeted by a surveillance pathway (
This quality-control mechanism involves polyadenylation
of the targeted RNA by the TRAMP (TRf4/5-Air1/2-Mtr4
Polyadenylation) complex and its degradation by the
nuclear exosome (reviewed in (
)). Degradation of aberrant
pre-ribosomes is clearly of paramount importance to ensure
the fidelity of gene expression by avoiding the production
of defective ribosomes. The translation of mRNAs by such
ribosomes would potentially lead to the production of
mutated or truncated proteins with deleterious effects for the
cell. Moreover, degradation of accumulated pre-ribosomes
would also be needed for release and recycling of factors to
make them available to new particles.
In this work, we studied EFG1, a gene encoding a
nonessential and poorly characterized protein localized in the
nucleolus. It was previously described that deletion of EFG1
causes slow growth at 30◦C, thermosensitivity at 37◦C and
alters 18S/25S rRNA ratio (
). We present evidence
that EFG1 encodes a ribosome biogenesis factor involved
in 35S pre-rRNA processing at A0, A1 and A2 sites. Efg1
depletion leads to the accumulation of the 35S pre-rRNA
and the aberrant 23S pre-rRNA with subsequent decrease
of 18S rRNA level. We demonstrate that Efg1 is associated
with the 35S pre-rRNA and U3 snoRNP, but also with 23S
and a novel 11S rRNA. We propose that the 11S and the
previously observed 17S’ rRNAs (
) are the result
of 23S pre-rRNA cleavage at site Q1 by Utp24
endonuclease. This is the first demonstration of the involvement of
Utp24 in the elimination of unprocessed particles and in
consequence, recycling of assembly factors.
MATERIALS AND METHODS
Strains, media, plasmids and cloning
Standard procedures were used for the propagation of yeast
using YPD medium (1% yeast extract, 2% peptone and
2% glucose), YPG medium (1% yeast extract, 2% peptone
and 2% galactose) or YNB medium (0.67% yeast
nitrogen base, 0.5% (NH4)2SO4 and 2% glucose or galactose)
supplemented with the required amino acids. Yeast strains
used in this study were derivatives of S. cerevisiae S288C or
BY4741, originating from the S288C background (MATa,
his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). EFG1::TAP strain was
purchased from Open Biosystems where insertion of the
tandem affinity purification (TAP) cassette was selected
using HIS3MX6 marker. The strain was grown exponentially
and no growth phenotype was observed neither at 30◦C
nor at 37◦C. EFG1Δ strain was purchased from Euroscarf
haplo¨ıd system where EFG1 open reading frame was
replaced by a kanamycin resistance selectable marker (kanR).
The GAL1::3HA::EFG1 strain, expressing 3HA-Efg1
under the control of a GAL1 promoter was constructed as
follows: a polymerase chain reaction (PCR) cassette
containing the kanamycin resistance (kanR) selectable marker,
GAL1 promoter and the 3HA-tag sequence was amplified
by PCR from the plasmid pFA6a-KanMX6-PGAL1–3HA
using primers 580 and 581 (
) (see Table 1). The PCR
fragment was inserted by homologous recombination
upstream of the chromosomal EFG1 open reading frame in
the BY4741 strain. Transformants were selected for
resistance to kanamycin and screened by immunoblotting.
The EFG1::YFP strain containing a plasmid pUN100
NOP1::mCherry was constructed as follows: a PCR
cassette containing the URA3 gene from Kluyveromyces
lactis as selectable marker and YFP-tag sequence was
amplified by PCR from the plasmid pFA6a-YFP-URA using
primers 700 and 701. The PCR fragment was inserted by
homologous recombination in the EFG1::TAP strain using
the Swap-tag method (
). Transformants were selected for
uracil prototrophy and screened by immunoblotting. In a
second step, plasmid pUN100 NOP1::mCherry was
transformed in the resulting strain (
The Tet::UTP24::3HA strain expressing Utp24–3HA
under the control of a Tet promoter was constructed as
follows: a PCR cassette containing the 3HA-tag sequence
and the HIS3 gene as selectable marker was amplified
by PCR from the plasmid pFA6a-3HA-His3-MX6 using
CS-447 and CS-448 primers. The PCR fragment was
inserted by homologous recombination in the Tet::UTP24
strain (pYDR339C::kanR-tet07-TATA, URA3::CMV-tTA,
MATa, his3–1, leu2–0 met15–0; gift from Mike Tyers’s
laboratory). Deletion of RRP6 was performed as previously
Plasmids expressing WT or D68N Utp24 were
constructed as follows. HTP-tagged forms of Utp24 were
initially amplified using CS-327 and CS-332 from
UTP24::HTP strain described in (
). The PCR
fragments were cloned into pRS315 plasmid cut with SalI.
Oligonucleotides used to construct UTP24::D68N
mutant plasmid by site-directed mutagenesis are described in
). HTP tag was next shortened at their C-terminus by
site directed mutagenesis introducing a stop codon
immediately after the TEV cleavage site using CS-599 and
CS600 oligonucleotides. The expression of the protein remains
detectable with the anti-TAP antibody (CAB1001, Thermo
Fluorescence microscopy. For fluorescence microscopy,
cells were grown in YPD medium at 30◦C to an OD600 of
0.6. Aliquots were collected, washed and resuspended in
dextrose-containing YNB supplemented with the needed
amino acids. After washing, the cells were mounted on a
slide and observed using the fluorescence microscope IX-81
(Olympus) equipped with a polychrome V monochromator
and a CoolSNAP HQ camera (Roper Industries). Digital
pictures were processed using Photoshop software (CS3
Western analyses. Proteins from total extracts, obtained
from gradient fractions after trichloroacetic acid (TCA)
precipitation or from immunoprecipitated pellets, were
separated on 4–12% polyacrylamide/SDS gels (Invitrogen) and
transferred to hybond-ECL membranes (GE Healthcare).
Nop1 was detected as described in (
proteins were detected using rabbit PAP (Sigma) diluted 10 000
fold. HA-tagged proteins were detected using anti-HA
peroxydase antibodies (Roche) diluted 1000-fold.
Sucrose gradient sedimentation experiments. Sucrose
gradient sedimentation experiments were performed as
previously described (
RNA extractions and northern hybridizations. RNA
extractions and northern hybridizations were performed as
previously described (
). For high molecular weight RNA
analysis, 2 g of total RNA were glyoxal denatured and
resolved on a 1.2% agarose gel. Low molecular weight RNA
products were resolved on 8% Polyacrylamide/8.3M urea
gels. The sequences of oligonucleotides used to detect the
different RNA species are reported in Table 2.
Immunoprecipitations. Total cell extracts were prepared
from strains expressing TAP-tagged proteins or untagged
strains as control. Growing cells were frozen in liquid
nitrogen and broken in a mortar. Immunoprecipitations and
analysis of co-precipitated RNAs were performed as
previously described (
). Tot/IP ratios loaded were 1/20 for
agarose and acrylamide gels.
Tandem affinity purifications. Cell pellets (corresponding
to about 5 × 1010 cells) frozen in liquid nitrogen were
broken in a mortar and resuspended in A200 KCl buffer (20
mM Tris–Cl [pH 8.0], 5 mM MgAc, 200 mM KCl, 0.2%
Triton X-100) supplemented with 1 mM dithiothreitol,
1× Complete ethylenediaminetetraacetic acid (EDTA)-free
protease inhibitor cocktail (Roche), and 0.5 U/ l RNasin
For TAP purifications under native conditions, cell
extracts were clarified by centrifugation at 14 000 rpm (21
000 × g) for 10 min at 4◦C. Clarified extracts were
incubated with 200 l (bed volume) of IgG-Sepharose beads
(GE Healthcare) for 4 h on a rocking table. Beads were
extensively washed with A200. Columns were next
equilibrated with ice-cold TEV cleavage buffer (10 mM Tris–Cl
[pH 8.0], 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 1
mM dithiothreitol). Beads were resuspended with 1 ml of
TEV cleavage buffer and incubated for 2 h at 16◦C on a
rocking table in the presence of 100 units of ActTEV
protease (Invitrogen). Eluted samples (about 1 ml) were mixed
with 3 ml of calmodulin binding buffer (10 mM Tris–Cl [pH
8.0], 150 mM NaCl, 1 mM MgAc, 1 mM imidazole, 2 mM
CaCl2, 0.1% NP-40, 10 mM ß-mercaptoethanol) and 3 l of
1 M CaCl2 and incubated with 200 l of calmodulin beads
(Stratagene) at 4◦C for 1 h on a rocking table. Beads were
washed with 40 ml of calmodulin binding buffer, and
proteins were eluted by the addition of 6 × 200 l of calmodulin
elution buffer (10 mM Tris–Cl [pH 8.0], 150 mM NaCl, 1
mM MgAc, 1 mM imidazole, 2 mM EGTA, 0.1% NP-40, 10
mM -mercaptoethanol). Eluted proteins were precipitated
with 20% TCA, rinsed with acetone, and resuspended with
20 l of 1× sodium dodecyl sulphate (SDS) gel-loading
buffer (40 mM Tris–Cl [pH 6.8], 2% SDS, 10% glycerol, 25
mM dithiothreitol, 0.1% bromophenol blue). Samples were
loaded on 4–12% Bis-Tris gels. Gels were briefly (15 min)
stained with silver staining solution (Fermentas), and pieces
of gels containing the samples were excised. The proteins
contained in these samples were analyzed by mass
spectrometry as described (
Pulse chase analysis. Metabolic labeling of pre-rRNAs
was performed as previously described (
) with the
following modifications. Strains were pre-grown in synthetic
galactose medium lacking adenine and transferred to
glucose medium lacking adenine for 3 h (GAL1::3HA::EFG1).
Cultures at OD600 0.8 were labeled with [2,8–3H]-adenine
(NET06300 Perkin Elmer) for 2 min followed by a chase
with excess cold adenine. The 1 ml samples were collected 1,
2, 5, 10, 20 and 30 min following the addition of cold
adenine and cell pellets were frozen in liquid nitrogen. RNAs
were then extracted and precipitated with ethanol.
Efg1 is a nucleolar protein associated with pre-ribosomes
First described as two distinct open reading frames (ORF),
YGR271C-A and YGR272C, EFG1 (Exit oF G1) encodes a
protein of 233 amino acids and has been identified as a non
essential gene in S. cerevisiae (
). Efg1 deletion resulted
in a slow growth phenotype at all temperatures, and is fully
lethal at 37◦C (22); data not shown). Furthermore, S.
cerevisiae strains containing a large deletion in the 5 domain
of Efg1 exhibited a 18S/25S rRNA ratio imbalance (
suggesting a role in ribosome biogenesis. We first searched
for possible Efg1 orthologs in other organisms or for
conserved protein domains. Efg1 seems to be well conserved in
the ascomycete phylum. Orthologs of Efg1 are also found
in plants. Furthermore, it contains a domain of unknown
function, DUF2361 (pfam10153), which is described as a
putative coiled-coil domain, conserved through eukaryotes
Previous global analyses of yeast protein localization
have shown that the GFP tagged Efg1 protein is enriched
in the nucleus and/or in the nucleolus (
). To better
define the protein localization, a construct expressing a
Cterminal YFP fusion was integrated at the endogenous
locus of EFG1, using a one step PCR strategy. The
resulting strain was transformed with plasmid
pUN100-mCherryNOP1 expressing a mCherry-fused version of Nop1
). Nop1, the yeast fibrillarin ortholog, is a core
component of box C/D snoRNPs required for early,
nucleolar stages of pre-rRNA processing, and is used to
visualize the nucleolus. The strain was grown exponentially
and no growth phenotype was observed neither at 30◦C
nor at 37◦C. Living cells were analyzed by fluorescence
microscopy. The YFP signal observed under these conditions
fully co-localizes with Nop1-mCherry, in a crescent-shaped
region consistent with nucleolar localization (Figure 2B).
We concluded that Efg1 accumulates in the yeast nucleolus,
specific locus of ribosome synthesis.
To assess the putative involvement of the Efg1 protein in
ribosome synthesis, we investigated its association with
ribosomal particles. To this end, sedimentation analyses on
a sucrose density gradient were carried out using extracts
from a strain expressing a C-terminal fusion between EFG1,
at its endogenous locus, and the TAP tag. Western blot
analysis of gradient fractions (Figure 3A) demonstrates that
Efg1-TAP sediments all along the gradient but is enriched
within high molecular mass fractions (fractions 11–13), the
density of which is consistent with that of early 90S
preribosomal particles. Efg1-TAP also sediments within
fractions containing polysomes (fractions 13–17) as it is the case
for most of nucleolar proteins involved in early maturation
steps in yeast (
To determine whether Efg1 might associate in vivo
with specific pre-rRNAs and snoRNAs,
immunoprecipitation experiments were performed. Total extracts from
EFG1::TAP and a non-tagged WT strains were incubated
with IgG-conjugated Sepharose beads. Following
immunoprecipitation, total RNAs were extracted and analyzed
by northern hybridization (co-immunoprecipitated proteins
were analyzed independently, Supplementary Figure S1).
Results show that Efg1-TAP efficiently precipitates the 35S
pre-rRNA, and is strongly associated with the aberrant
21S, 22S and 23S pre-rRNAs (Figure 3B, left panel,
compare lane 4 to 2). In contrast, we observed a very poor
coimmunoprecipitation of the 27S and 7S pre-rRNAs as well
as the mature 25S,18S, 5.8S and 5S rRNAs and a mild
association with the 20S pre-rRNA. Association with Efg1
is also observed for U3 and U14 snoRNAs, but not for
SnR31, SnR36 and SnR190 (Figure 3B, middle panel,
compare lane 8–6). We concluded that Efg1 associates with early
pre-ribosomal particles, containing U3 and U14 snoRNAs,
suggesting it could play a role in early steps of ribosome
synthesis. Besides, the tight association of Efg1 with the 23S
pre-rRNA and the poor association with the 20S pre-rRNA
might suggest that the A2 pathway, but not A3 pathway, is
required for the release of the protein from pre-ribosomal
particles (Figure 1B).
One striking feature of our experiment was the fact
that two 1200/1300 nt long fragments were strongly
coprecipitated with Efg1-TAP. We called these RNAs 11S
(Figure 3B; Full lane is shown in Figure 3C). Those
fragments are revealed using a 5 -ETS probe which lies 35 nt
downstream from the RNA Pol I transcription start site
(TSS). In contrast, no signal was detected using a probe
immediately upstream from the Pol I TSS (data not shown).
From these observations, we concluded that Efg1-TAP
strongly co-precipitated RNA fragments starting at the Pol
I TSS and ending 1200/1300 nt downstream, within the 18S
rRNA sequence, ∼500/600 nt downstream of A1 site.
Efg1 is required for biogenesis of the small ribosomal subunit
Efg1 is non-essential for cell viability (
). However, EFG1
deletion results in severely impaired cell growth. We
decided to monitor ribosome biogenesis during its depletion
(and thus during the onset of the phenotype) rather than
in cells lacking the EFG1 gene. For that purpose, we
constructed a yeast strain that conditionally expresses the Efg1
protein comprising a 3HA tag at the N-terminus
(3HAEfg1 protein), allowing its easy detection. The EFG1 open
reading frame was tagged by the 3HA-encoding sequence
and placed under the control of the regulated GAL1–
10 promoter by homologous recombination, creating the
GAL::3HA::EFG1 strain. This strain was propagated in
a medium containing galactose as carbon source and was
then shifted to a glucose-containing medium to allow
depletion of the protein. The kinetics of Efg1 depletion were
assessed by western blot using anti-HA antibodies. The
abundance of 3HA-Efg1 was strongly reduced after transfer to
glucose medium and became almost undetectable after <1h
(see Figure 4A).
On galactose containing medium, the growth rate of
GAL1::3HA::EFG1 strain is almost identical to that of the
WT strain. However, 4 h after transfer to the non-permissive
glucose medium, the growth rate of GAL1::3HA::EFG1
strain was already substantially reduced compared to WT
(Figure 4B). After several hours of depletion, the doubling
time of GAL1::3HA::EFG1 was estimated to be 9 h. In the
same conditions, the WT strain was doubling in 2.4 h. These
results confirmed that even though Efg1 is not essential,
the growth of strains depleted for the protein is largely
To characterize Efg1 function in ribosome biogenesis, we
analyzed the sedimentation profile of ribosomal particles
from a strain depleted of Efg1. Total extracts from depleted
cells and WT strain were loaded on 4.5–45% sucrose
gradients (Figure 4C). Compared to WT, Efg1 depletion led to
a severe reduction in the relative amounts of 40S subunit,
80S ribosomes/90S pre-ribosomes and polysomes.
Accordingly, 60S subunits accumulated in the cell, resulting in a
large peak that partially overlaps with 80S ribosomes. This
phenotype confirms a role of Efg1 in the maturation of the
small ribosomal subunit.
To assess which mature rRNAs require Efg1 for
accumulation, we decided to analyze all ribosomal RNAs
(rRNAs) from Efg1-depleted cells. Aliquots of WT and
GAL1::3HA::EFG1 cells grown in galactose-containing
medium or grown for 1, 3 and 6 h in glucose-containing
medium were harvested. From these aliquots, total RNAs
were extracted. We then compared pre-rRNA processing in
WT and Efg1-depleted cells by northern analysis (Figure
5A). We observed that after Efg1 depletion, levels of the 35S
pre-rRNA are highly increased. In the absence of Efg1, the
32S, 27SA2 and 20S pre-rRNAs were reduced, suggesting
that A0, A1 and A2 cleavages are impaired. This is supported
by the strong accumulation of the aberrant 23S pre-rRNA,
produced from a direct cleavage of the nascent transcript at
site A3. Note that 22S (A0–A3) and 21S (A1–A3) are also
accumulated, suggesting that A0 or A1 occurs after A3. As
a consequence, after 6 h of Efg1 depletion, 18S rRNA
accumulation is already diminished. We also observed that
depletion of Efg1 leads to a decrease of 25S rRNA level,
together with the accumulation of 27SB pre-rRNA. A2
cleavage inhibition leads to a direct cleavage at A3 site. In
consequence, a 27SA2 accumulation defect is observed
concomitantly with an accumulation of 27SA3 pre-rRNA which is
rapidly processed to produce the short form of 27SB
prerRNA. This defect may explain a 27SBS accumulation.
Altogether, these observations emphasize a role of Efg1 in
early steps of ribosome biogenesis, in particular in the
efficiency of A0, A1 and A2 cleavages.
Effects of reduced 3HA-Efg1 levels on rRNA
processing were also assessed by in vivo [2,8- 3H]-adenine
pulsechase labeling (Figure 5B). As 3H-adenine can be
incorporated into pre-rRNA only during ongoing transcription,
any radioactive labeling indicates de novo synthesis.
Following this initial pulse labeling, outcome of transcripts can be
followed after chase with an excess of cold adenine.
Pulsechase labeling was performed 30 min after transfer of the
GAL1::3HA::EFG1 strain to glucose medium. After
depletion of Efg1, we still observe 35S pre-rRNA production.
This suggests that rDNA transcription by Pol I is not
affected in absence of Efg1. We also note that, compared to
the WT, pre-rRNA processing is delayed during Efg1
depletion. Indeed, we see that the production of 27SA2
prerRNA seems greatly reduced (A2 pathway). Accordingly,
we have a greater production of 27SB pre-rRNA. However,
maturation of this 27SB pre-rRNA appears to be delayed as
de novo synthesis of mature 25S rRNA is slightly reduced.
In parallel, the absence of Efg1 strongly inhibits 18S rRNA
production, which is probably the consequence of reduced
20S pre-rRNA production detected by this approach. These
observations are consistent with the accumulation of newly
synthesized 23S pre-rRNA, with a slow decay rate (detected
from 1 to 20 min following chase). Note that 5.8S and 5S
production appears mostly unaffected by Efg1 depletion.
These results confirmed that cleavages at sites A0, A1 and
A2 are strongly impaired in cells lacking Efg1.
23S pre-rRNA is targeted by endo- and exonucleolytic
pathways for its degradation
We identified two 1200/1300 nt long fragments which are
strongly co-precipitated with Efg1-TAP (Figure 3). The 5
extremity of the fragment corresponds to the Pol I TSS
and the 3 extremity lies within the 18S rRNA sequence
approximately 500/600 nt downstream of A1 site, hereafter
called 11S rRNA. Because Efg1 is a nucleolar protein and
the 3 extremity of the 11S rRNA ends within the sequence
of the mature 18S rRNA, we hypothesized that such
fragment would be a degradation product of the 35S pre-rRNA
or the 23S pre-rRNA. Degradation pathways of aberrant
or excised (pre-)rRNA primarily involve TRAMP and
exosome complexes. The 11S rRNA 3 extremity could be the
result of incomplete exosome-dependent 3 exonucleolytic
degradation of the 23S rRNA. On the other hand, the
production of this RNA could also result from an
endonucleolytic cleavage within the first third of the 18S rRNA. This
would lead to the production of another rRNA
byproduct extending from within the 18S rRNA sequence to A3
site or Bo, when processed from 23S or 35S, respectively.
To discriminate between these two possibilities, we checked
the existence of this complementary fragment by
northern blot in WT strains or in five strains in which
exonucleolytic degradation pathways are deficient: single rrp6Δ,
trf4Δ, trf5Δ and double rrp6Δ/trf4Δ, rrp6Δ/trf5Δ
deletion mutants (Figure 6A). Polyadenylated forms of 23S and
11S rRNAs were observed in strains lacking Rrp6 or both
Rrp6 and Trf4, suggesting that these two RNAs are targeted
by the TRAMP/exosome pathway for degradation (Figure
6A, lanes 8 and 11). In the same RNA samples, using a
D-A2 probe, we identified a ∼1500 nt long rRNA. Similar
signal was obtained using A2–A3 probe but was not
visible using a probe hybridizing downstream of A3 (data not
shown) establishing that the 3 extremity of this rRNA is A3.
This particular rRNA fragment was already observed by
the Tollervey and Lafontaine laboratories (
they called 17S’. According to the hybridization specificity,
17S’ and 11S have no common sequence, and could
result from the cleavage of the 23S transcript. Moreover, the
size of both fragments (∼1200/1300 nt and ∼1500 nt)
peared compatible with the full size of the 23S pre-rRNA
(∼2800 nt). From these results we concluded that part of
the 23S pre-rRNA is endonucleolytically cleaved within the
sequence of the 18S rRNA resulting in 11S and 17S’
As previously shown (Figure 3B), the A2 pathway leads
to the dissociation of Efg1 from pre-RNAs. In contrast,
when direct A3 cleavage occurs, Efg1 stays associated with
23S and 11S (but not 17S’) pre-RNAs. From this
observation, we tested whether Efg1 would be required for 11S
and 17S’ production or alternatively, would tag these
prerRNAs for degradation. To explore these possibilities, we
decided to monitor the accumulation of the different
deadend products in absence of Efg1 (Figure 6B) and we
compared with cells expressing endogenous Efg1 (Figure 6A).
We introduced the GAL1::3HA::EFG1 allele in the five
strains in which degradation pathways are deficient. Efg1
was depleted by adding glucose in the media for 3 h, RNAs
were extracted and polyadenylated RNAs were purified.
17S’ rRNA is still detected and polyadenylated in absence
of Efg1. We conclude that Efg1 is not required for the
endonucleolytic cleavage of 23S. However, the 17S’
polyadenylation profile is different. Indeed, in the presence of Efg1,
17S’ RNA is mainly targeted by the TRAMP5 complex
as it was poorly polyadenylated in the absence of Trf5. In
the absence of Efg1, the 17S’ rRNA was polyadenylated in
absence of either Trf4 or Trf5, suggesting that TRAMP4
can substitute for TRAMP5 under these conditions. We
also noticed the accumulation of shorter polyadenylated
rRNAs corresponding to 5 -truncated forms of the 17S’
rRNA (annotated *). This modification of polyadenylated
species was even more striking for 11S. Compared to cells
expressing Efg1, GAL1::3HA::EFG1 cells failed to
accumulate polyadenylated 11S in absence of Rrp6 and both Rrp6
and Trf4 (Figure 6B, lanes 8 and 11).
We conclude that 23S is endonucleolytically cleaved, and
subsequently degraded by the TRAMP exosome pathway
under the regulation of Efg1 protein.
23S pre-rRNA is cleaved at Q1 site and the PIN domain of
Utp24 is required for efficient cleavage
We next decided to map precisely the major 23S
endonucleolytic cleavage site using the 5 extremity of the 17S’. For
this purpose, we extracted total RNAs from WT and Rrp6
deficient cells and performed serial reverse transcription
assays all along the 18S rRNA sequence (data not shown).
We identified one major site in the first third of the 18S
rRNA, and two minor sites (data not shown). The precise
5 extremity of the 17S’ was mapped at position +618
relative to A1 site (Figure 7A). We named this cleavage site
Q1 (for Quality control cleavage site 1). Q1 site is located
within the central domain, between helix 19 and 20
(Figure 7B), within a very flexible A-rich sequence (
sequence is not known to be the target of any
endoribonuclease activity. However, recent RNA–protein crosslinking
data (CRAC analysis (39)) showed that the Utp24
endoribonuclease was predominately associated with sequences
within the 18S rRNA sequence (
). The main peak of
association was located around position +1103 relative to A1,
in the 3 part of the central domain. Even though the Q1
cleavage site (+618) and +1103 are separated by ∼500 nt,
secondary and tertiary structure predictions position them
in close vicinity, on both sides of helix 19. These
observations render Utp24 a good candidate for being involved
in the cleavage at Q1. To assess the putative function of
Utp24 in Q1 cleavage in vivo, we decided to monitor the
accumulation of 11S and 17S’ rRNA in cells deficient for
Utp24 enzymatic activity. For this purpose, we constructed
a strain lacking the RRP6 gene, strongly accumulating 23S,
in which HA-tagged Utp24 is expressed under the
control of the tetracycline repressible promoter (pTetO7). This
strain was next transformed with a plasmid expressing
either WT, Utp24 PIN mutant allele (Utp24-D68N) (
or no protein. Ten hours following addition of doxycycline,
fully functional genomic Utp24 is largely depleted
(Supplementary Figure S2) and the only available source of Utp24
in the cell is the version expressed from the plasmid (WT,
PIN mutant or mock). In these conditions, strains
expressing only WT Utp24, PIN mutant Utp24 or no protein, were
grown in liquid media, harvested and total RNAs were
extracted and polyadenylated RNAs were enriched. Figure 7C
shows northern blot analyses using D-A2 (lanes 1–6) and
5 -ETS (lanes 7–12) probes. In cells expressing WT Utp24,
11S (lanes 7 and 10) and 17S’ (lanes 1and 4) were
accumulated. Cells containing empty vector showed a typical
early pre-rRNA processing defect due to depletion of
endogenous Utp24 with an inhibition of all early cleavage (A0,
A1 and A2), leading to 35S and 23S accumulation and
inhibition of 20S production (lanes 3 and 9). Note that
contrary to the situation where WT Utp24 is expressed, 11S and
17S’ are almost undetectable. Results are slightly different
when the Utp24-D68N PINc mutant is expressed.
According to previous observations (13), Utp24 D68N is
enzymatically deficient but is probably still associated with an
assembled SSU processome (
). In these conditions, A0 is
accurately cleaved whereas A1 and A2 are blocked leading
to the accumulation of the 22S pre-rRNA, an
intermediate extending from A0 to A3. Cells depleted of Utp24 or
expressing Utp24-D68N failed to accumulate the 11S and
17S’ fragments. Note that some residual 11S and 17S’ are
still present in the polyadenylated fractions. This is
consistent with the fact that fully functional Utp24 is not
completely depleted in our system (Supplementary Figure S2:
see residual amounts of Utp24 after 10 h of depletion). We
concluded that the integrity of the PINc domain of Utp24
is required for the normal accumulation of the 11S and 17S’
RNAs strongly suggesting that Utp24 is responsible for the
cleavage at Q1.
Ribosome biogenesis is a very complex and error-prone
pathway that is actively surveyed by quality control
mechanisms. In fast growing cells, the nucleolar RNA surveillance
pathway is active and the non-productive 23S pre-rRNA
is rapidly targeted for degradation (
). This involves its
recognition and polyadenylation by the TRAMP5 complex
followed by its 3 -5 degradation by the exosome and
particularly Rrp6 (
). Indeed, absence of either Trf5 or
Rrp6 led to a 23S pre-rRNA accumulation.
In this study, we report the characterization of EFG1,
encoding a nucleolar protein involved in early cleavages of the
35S pre-rRNA leading to the production of the small
ribosomal subunit. We also present the identification of a new
pathway leading to the degradation of the non-productive
23S pre-rRNA. We show that 23S pre-rRNA is initially
targeted by the PINc endonuclease Utp24 resulting in its
cleavage at position +618 relative to A1, a new cleavage site we
called Q1. Both resulting fragments (11S and 17S’) are
subsequently targeted by TRAMP5 and the exosome for their
degradation. We also show the requirement of Efg1 for the
TRAMP5 targeting of 17S’ and the polyadenylation of 11S
RNA by the TRAMP complex.
In S. cerevisiae, Efg1 accumulates in the nucleolus which
is the specific place of early steps of ribosome biogenesis.
Efg1 is a non essential protein, however upon Efg1
depletion, we observed a rapid decrease in the accumulation of
the mature 18S rRNA. This decrease is the consequence
of a defect in A0, A1 and A2 cleavages as Efg1 depletion
resulted in the accumulation of the 35S and the 23S
prerRNAs concomitantly with a strong decrease of the 20S
pre-rRNA, the direct precursor of the mature 18S rRNA.
It is noticeable that 21S and 22S pre-rRNAs are
accumulated in absence of Efg1. This observation as well as the
fact that residual amounts of 20S pre-rRNA persist in
absence of Efg1, strongly suggest that even if the cleavages are
strongly delayed, they are not fully inhibited in these
conditions. Consistent with these observations, pulse chase
experiments clearly revealed that small amounts of 20S
prerRNA (leading to 18S rRNA) are still produced in absence
of Efg1. We hypothesize that this residual production is
sufficient to ensure cell viability. Furthermore, global genetic
screens to identify Efg1 mutants alleviating or aggravating
the growth phenotype did not identify other RNA
processing factors (data not shown).
Co-immunoprecipitation experiments revealed that Efg1
interacts physically with numerous RNA components
(including 35S pre-rRNA and U3 snoRNA) and proteins of
the 90S pre-ribosomal particles (see Supplementary Figure
S1). However, our results showed that Efg1 is not
10 11 12
sary for the assembly of the U3 processome. Indeed,
gradient analyzes showed that Efg1 is not required for the
association of UTP-A (Utp17p), UTP-B (Pwp2p),
UTPC (Utp22p) complexes as well as Rrp5 with the 90S
preribosomes suggesting that these modules assemble
independently of Efg1 (Supplementary Figure S3). Conversely,
components of UTP-A, UTP-B and UTP-C complexes as
well as Rrp5 are not required for the association of Efg1
with the 90S pre-ribosome (Supplementary Figure S3).
Altogether, these results strongly suggest that Efg1 is not
a bona fide structural component of the U3 processome as
its assembly with the pre-ribosomes appears independent
of the assembly of UTP modules. Therefore, Efg1 associates
very early with pre-ribosomes, probably during rDNA
transcription. This is in accordance with recent results showing
that Efg1 associates with nascent transcripts, and more
precisely with a region corresponding to the 5 domain of 18S
). In contrast to 35S pre-rRNA, very low levels of
20S pre-rRNA are co-immunoprecipitated with Efg1
suggesting that Efg1 is released from the particle during or
immediately after A2 cleavage.
One interesting observation of the co-precipitation data
was the strong recovery of the aberrant 23S pre-rRNA
with Efg1. This pre-rRNA is detectable in WT cells, but
at very low levels, and is strongly enriched in the
Efg1TAP immunoprecipitates. We concluded that A0, A1 and
A2 cleavages are required for the release of Efg1 from
preribosomes. In absence of these cleavages, Efg1 stays
associated with the particle. Another striking feature was the
strong co-immunoprecipitation of a RNA we called 11S,
extending from the 5 -TSS to the first third of the 18S rRNA
sequence. The previously observed 17S’ rRNA (
starting from position +618nt relative to A1 (Q1 site) and
extending to A3 site is not associated with Efg1. The 23S
pre-rRNA is processed to the 17S’ and 11S rRNAs through
an endoribonucleolytic cleavage at Q1 site. This cleavage is
achieved by the endonuclease Utp24 which binds within the
central domain of the 18S rRNA (
), in close vicinity to the
Q1 cleavage site, on the opposite side of helix 19.
As well as the nonproductive 23S pre-rRNA, 11S and
17S’ are enriched in polyadenylated fractions and
accumulated in absence of Rrp6. This observation strongly suggests
that 11S and 17S’ are also targeted for degradation by the
TRAMP/Exosome pathway. In absence of Rrp6, the
levels of these two degradation fragments are strongly
diminished in poly(A+) fractions when Trf5 is absent
suggesting they are mainly targeted by the TRAMP5 complex for
polyadenylation. We propose that cleavage at Q1 site is likely
part of a surveillance pathway that removes pre-ribosomes
that have failed to undergo correct assembly and/or
processing during the early steps of their biogenesis. Cleaving
23S pre-rRNA within the sequence of the mature 18S rRNA
would definitely avoid the maturation of 23S containing
particles. Our results suggest that 23S pre-rRNA could be
either directly targeted by the TRAMP/exosome pathway
to promote its 3 -5 degradation or primarily cleaved at Q1,
leading to the production of 11S and 17S’ that will next
undergo TRAMP/exosome elimination. This redundancy
probably enhances the overall efficiency of the surveillance
pathway. Indeed, taking advantage of Q1 cleavage, 5
sequences of 23S would presumably be even more rapidly
degraded, given that the degradation machinery is slowed by
strong secondary structures in its 3 sequences.
Compared to cells expressing Efg1, GAL::3HA::EFG1
cells grown in presence of glucose failed to accumulate
polyadenylated 11S in absence of Rrp6 or both Rrp6 and
Trf4 (Figure 5B, lanes 8, 11 and 12). These observations
suggested that TRAMP was unable in these conditions to
target the 11S rRNA. In contrast, the 17S’ rRNA is still
polyadenylated in absence of Efg1. This is correlated by the
fact that Efg1 is associated with 11S and not with 17S’ (this
study and (
)). The presence of Efg1 within the particle
appeared required to target 11S RNA to the TRAMP
complex. It is possible that the absence of Efg1 leads to
conformational changes at the 3 extremity of the 11S
making it poorly accessible to TRAMP. On the other hand,
Efg1 could actively recruit the TRAMP complex on the
11S RNA containing particle in order to direct it to
degradation. The existence of such adaptor protein has already
been reported. Nop53 and Utp18 contain the same Arch
Interaction Motif (AIM) consensus motif that interacts with
the arch domain of Mtr4 (
). This interaction leads to the
docking of TRAMP on specific substrates. Efg1 is
presumably not a similar adaptor factor as such AIM motif was
not found within its sequence. However, Trf5 peptides were
found in Efg1 immunoprecipitates (see Supplementary
Figure S2) suggesting a potential interaction.
Moreover, in Efg1 depleted cells, the global
polyadenylation profile is slightly different. Indeed, in the presence of
Efg1, 17S’ is mainly targeted by the TRAMP5 complex as
it was poorly polyadenylated in absence of Trf5. In absence
of Efg1, the 17S’ rRNA was polyadenylated in absence of
either Trf4 or Trf5, suggesting that TRAMP4 can
substitute for TRAMP5 in these conditions. Globally, TRAMP5
polyadenylation seems less efficient in absence of Efg1.
Ribosome biogenesis is an extremely complex pathway
with at least 200 assembly factors, 75 snoRNPs, 79
ribosomal proteins and a multistep processing pathway leading to
the production of mature ribosomes ensuring the fidelity of
translation. The possibilities for errors are obviously
plentiful and it seems imperative that surveillance occurs at
different checkpoints during the process. A2 cleavage is
presumably one of these as it splits the 90S pre-ribosome in
pre-40S and pre-60S particles that will undergo independent
maturation. Using Utp24 as a main checkpoint actor would
make plenty of sense. Utp24 could be the key element
allowing the shift between productive and non-productive
pathways. Utp24 would commit 90S particles (i) to maturation
by using A2 cleavage site, (ii) to degradation by cleaving at
Q1 site or (iii) to accumulation of 23S containing particles
by utilizing none of them (
) (see Figure 8). How Utp24
would choose between the different pathways remains
unclear. The correct loading of Utp24 onto the particle may
be an important feature. The environment of Q1 cleavage
site appears very ‘flexible’ (
) and may differ depending
on the maturation state of the pre-ribosome. Particles that
would have failed to undergo correct assembly and/or
processing would be in one conformation that exposes Q1
cleavage site to Utp24 instead of A1 and A2. However, we cannot
exclude an active and precise modulation of Utp24
cleavage activity by specific regulation factors. During rapid
exponential growth, TORC1 is active and yeast uses normal
A0, A1 and A2 cleavages to produce mature ribosomes.
Under unfavorable growth conditions, such as environmental
stress or lack of fermentable carbon source (diauxic shift),
TORC1 becomes inactive and yeast uses direct A3 cleavage
instead of A0, A1 and A2 cleavages. This leads to the
accumulation of 23S RNA containing particles with no further
detectable production of new ribosomes (
parallel, rDNA transcription and production of ribosomal
proteins or assembly factors are strongly reduced as the cells
prepare their entry into quiescence (reviewed in (45)). This
contributes to the general downregulation of global protein
synthesis. When conditions improve, cells have to rapidly
switch to exponential growth. Being very reactive in making
new ribosomes would be a prerequisite to start again
making proteins and resume growth. This capability should
provide great evolutionary advantages to compete for resources
with other organisms. Usage of the Q1 pathway would lead
to disassembly of stocked 23S RNA containing particles.
This would immediately release and provide most of the
ribosomal proteins and assembly factors required to rapidly
prime the ribosome pump and greatly increase yeast
adaptability to environment.
We propose that Utp24 is a key element allowing the shift
between productive and non-productive ribosome
biogenesis. Fine tuning of Utp24 activity and cleavage specificity
would allow the control of ribosome biogenesis at the
posttranscriptional level in response to assembly defects or
Supplementary Data are available at NAR Online.
We are very grateful to Yves Henry and Marie-Kerguelen
Sarthou for critical reading of the manuscript. We
acknowledge members of the Gadal’s lab for help, advice and
discussion. This work also benefited from the assistance of the
microscopy facility of the imaging platform of Toulouse TRI.
Agence Nationale de la Recherche (ANR)
[ANR-13BSV5–0010––ANDY]; IDEX [ATS, NudGene]; E. C. was
supported by a Ph.D. Fellowship from the Ministe`re
de l’Education Nationale Ph.D. Fellowship (E.C.); de
l’Enseignement Supe´rieur et de la Recherche Fellowship
(E.C.); ‘l’Association pour la Recherche contre le Cancer’
Fellowship (to E.C.). Royal Society [UF100666, RG110357,
UF150691 to C.S.]. Funding for open access charge: ANR.
Conflict of interest statement. None declared.
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