Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels.. Lower, Dutp25 ⁄ GAL1::UTP25 strain sh
Trang 1interacts with U3 snoRNP subunits and affects
processing of the 35S pre-rRNA
Mauricio B Goldfeder and Carla C Oliveira
Department of Biochemistry, University of Sa˜o Paulo, SP, Brazil
Introduction
Ribosome biogenesis is a complex and
energy-consum-ing process in eukaryotic cells that demands tight
regulation between rRNA transcription and
process-ing, r-protein translation and rRNA⁄ r-protein
assem-bly Three of the Saccharomyces cerevisiae rRNAs are
transcribed by RNA polymerase I as a polycistronic
35S precursor that undergoes endo- and exonucleolytic
cleavage reactions and nucleotide modifications, before
originating the mature rRNAs 18S, 5.8S and 25S which will be assembled into the small and large ribo-somal subunits, respectively At least 200 factors are predicted to be involved in pre-rRNA processing in yeast, and a large number of them are small nucleolar ribonucleoproteins (snoRNPs) [1,2] Most snoRNPs are classified as members of two major families, box
C⁄ D (that guide 2¢-O-ribose-methylation at specific
Keywords
nucleolus; pre-40S; ribosome synthesis;
rRNA processing; Saccharomyces cerevisiae
Correspondence
C C Oliveira, Department of Biochemistry,
Chemistry Institute, University of Sa˜o Paulo,
Av Prof Lineu Prestes, 748, Sa˜o Paulo, SP,
Brazil CEP 05508-900
Fax: +55 11 3815 5579
Tel: +55 11 3091 3810; Ext 208
E-mail: ccoliv@iq.usp.br
(Received 19 November 2009, revised 31
March 2010, accepted 28 April 2010)
doi:10.1111/j.1742-4658.2010.07701.x
In eukaryotes, pre-rRNA processing depends on a large number of nonribo-somal trans-acting factors that form intriguingly organized complexes Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages
of 35S pre-rRNA processing and give rise to the mature ribosome subunits Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation
Structured digital abstract
l MINT-7889901 : SAS10 (uniprotkb: Q12136 ) physically interacts ( MI:0915 ) with Utp25p (uni-protkb: P40498 ) by pull down ( MI:0096 )
l MINT-7889915 : NIP7 (uniprotkb: Q08962 ) physically interacts ( MI:0915 ) with RRP43 (uni-protkb: P25359 ) by two hybrid ( MI:0018 )
l MINT-7889852 : Utp25p (uniprotkb: P40498 ) physically interacts ( MI:0915 ) with MPP10 (uniprotkb: P47083 ) by two hybrid ( MI:0018 )
l MINT-7890065 : NOP1 (uniprotkb: P15646 ) and Utp25p (uniprotkb: P40498 ) colocalize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7889865 : Utp25p (uniprotkb: P40498 ) physically interacts ( MI:0915 ) with SAS10 (uni-protkb: Q12136 ) by two hybrid ( MI:0018 )
Abbreviations
GFP, green fluorescent protein; GST, glutathione S-transferase; snoRNP, small nucleolar ribonucleoprotein; SSU, small subunit; UTP,
U three-protein complex; YP, yeast extract–peptone medium.
Trang 2positions in nascent rRNAs) and box H⁄ ACA (that
guide pseudouridylation of specific nucleotides in
rRNAs) Some snoRNPs, however, are involved in
endonucleolytic cleavage reactions of the pre-rRNA,
among them the endonuclease MRP (responsible
for the cleavage at site A3 in ITS1) [3], the box C⁄ D
snoRNPs U3 and U14, and the box H⁄ ACA snoRNPs
snR10 and snR30, involved in the cleavage reactions
at sites A0, A1and A2[4–7]
All box C⁄ D snoRNAs are bound by four core
pro-teins, Nop1p, Nop58p, Nop56p and Snu13p [8] In
addition to the core proteins, the U3 snoRNP is
asso-ciated with other proteins specific for this snoRNP
The first proteins to be identified in the U3 snoRNP
complex were Sof1p, Mpp10p, Lcp5p, Imp3p, Imp4p,
Dhr1p and Rrp9p [9–14] Later experiments have
shown that U3 is associated with at least 28 proteins,
forming a large multisubunit complex also known as
the small subunit (SSU) processome [15] The
mecha-nism of U3 snoRNP complex assembly in the 90S
par-ticle is unknown However, recent evidence suggests
that stable subcomplexes bind the nascent 35S
pre-rRNA sequentially [16] Interestingly, electron
microscopy analyses have shown that early
preriboso-mal particles undergo time-dependent changes in size
and shape upon binding to the primary rRNA
pre-cursor, suggesting that their components are
sequen-tially assembled [17] Accordingly, recent studies have
revealed the presence of discrete 90S particle
subplexes that have been named U three-protein
com-plexes (UTP) UTP-A⁄ t-UTP, UTP-B and UTP-C
[18,19] t-UTP complex binds very early during
tran-scription of the pre-rRNA, followed by the UTP-B
complex, U3 snoRNP and the Mpp10p complex, and
later by Rrp5p and the UTP-C complex [16] It is
pre-dicted, however, that the SSU processome interacts with other proteins in order for the cleavage of the pre-rRNA to occur
The S cerevisiae protein coded by the open reading frame YIL091C had not been previously characterized However, analysis of essential yeast proteins had iden-tified in the YIL091C sequence a domain with low homology to RNA helicases These studies have also shown that this protein is localized to the nucleolus [20] Here we show that the protein named Utp25p is involved in pre-rRNA processing Its depletion leads
to accumulation of the pre-rRNA 35S and the aber-rant 23S, and subsequent decrease in the levels of pre-rRNA 20S and mature 18S pre-rRNA Consistent with its subcellular localization and involvement in 18S rRNA formation, Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p Utp25p also co-immu-noprecipitates U3 snoRNA, which strongly indicates that it is a novel SSU processome subunit
Results
Previous global analyses of yeast protein localization have shown that the 83 kDa protein Utp25p, coded by the open reading frame YIL091C, localizes to the nucleolus [20] In order to confirm the subcellular local-ization of Utp25p, the UTP25 gene was cloned into a plasmid, fused to green fluorescent protein (GFP), and cells were analyzed by fluorescence microscopy The GFP–Utp25p signal is restricted to the nucleus and is concentrated in the nucleolus (Fig 1) GFP, by con-trast, is present throughout the cell RFP–Nop1p, used
as a control, is restricted to the nucleolus (Fig 1) The nucleolar localization of Utp25p suggested that this protein is involved in ribosome synthesis
GFP
GFP-Utp25
GFP + RFP + Hoechst
Fig 1 Subcellular localization of GFP–Utp25p Yeast strains expressing RFP–Nop1p and either GFP (upper) or a GFP–Utp25p N-terminal fusion (lower) were analyzed Hoechst, indicates nuclei stained with the DNA dye Hoechst; GFP, indicates the localization of the green fluo-rescent protein; RFP, indicates the localization of the red fluofluo-rescent protein GFP + RFP, merging of green and red signals GFP + RFP + Hoescht, merging of all signals.
Trang 3In order to characterize Utp25p function, we first
obtained a conditional mutant strain A heterozygous
diploid strain (YIL091C⁄ yil091c::KanMX4 –
Euro-scarf) was transformed with a plasmid containing a
copy of the UTP25 gene under control of the inducible
promoter GAL1 After sporulation, a haploid deletion
strain was obtained and its genotype was confirmed by
PCR analysis of UTP25 gene (data not shown) The
conditional Dutp25⁄ GAL1::UTP25 strain was then
analyzed for growth in glucose medium, compared
with the otherwise isogenic parental strain, UTP25
Dutp25⁄ GAL1::UTP25 cells are not able to grow on
glucose plates, showing that Utp25p is essential for
growth (Fig 2A) Dutp25⁄ GAL1::UTP25 cells were
transformed with a second plasmid that harbors an
extra copy of UTP25 under the control of a
constitu-tive promoter, which rescues growth of the conditional
mutant on glucose plates (Fig 2A) As shown here,
the fusion proteins GFP–Utp25p and Gal4AD–Utp25p
(transcription activation domain of Gal4p) are
functional Growth of the conditional strain was also
analyzed in liquid glucose medium and the results
show that after 5 h in glucose, growth of Dutp25⁄
GAL1::UTP25 slows in comparison with the parental
wild-type strain, but the difference in growth rate is
more evident after 14 h in this medium (Fig 2B)
Based on the Utp25p nucleolar localization and its possible involvement in ribosome synthesis, we ana-lyzed the polysome profile of the conditional strain after depletion of Utp25p When growing in galactose medium, Dutp25⁄ GAL1::UTP25 cells show a normal polysome profile on density gradients However, after
20 h of growth in glucose, it is possible to see a severe reduction in the relative amounts of the 40S ribosomal subunit, as well as in 80S ribosomes and polysomes (Fig 3A, lower) Accordingly, free 60S accumulate in the cells, resulting in a large peak that overlaps with 80S ribosomes (Fig 3A, lower) Analysis of free ribo-some subunits in the presence of EDTA confirms a strong decrease in the relative amounts of 40S
ribo-A
B
UTP25
AD-Utp25
GFP-Utp25
–
Glucose
GAL1::UTP25 UTP25
h, Glu
5
4
3
2
1
0
Δutp25/ GAL1::UTP25
Fig 2 UTP25 is an essential S cerevisiae gene (A) Tenfold serial
dilution of UTP25 and Dutp25 ⁄ GAL1::UTP25 strains growing on
glu-cose-containing plates Dutp25 ⁄ GAL1::UTP25 was transformed
with plasmids containing an extra copy of the UTP25 gene under
the control of a constitutive promoter, fused to Gal4AD or GFP.
–, empty plasmid (B) Growth curve of UTP25 and Dutp25 ⁄
GAL1::UTP25 strains in glucose medium.
A
B
A254 nm
Polysomes
80S
Polysomes
40S 60S
80S
A254 nm
Galactose
Polysomes
40S 60S 80S
Glucose
Polysomes
40S
60S
80S
A254 nm
GAL1::UTP25 UTP25
GAL1::UTP25
Galactose
40S 60S
22.7 16.3
Glucose
40S 60S
22.3
3.4
Fig 3 Analysis of the polysomal profile in strain Dutp25 ⁄ GAL1::UTP25 UTP25 and Dutp25 ⁄ GAL1::UTP25 strains were incu-bated either in galactose or in glucose medium for 20 h for the analysis of polysomal profile through sucrose gradient (A) Upper, UTP25 strain Lower, Dutp25 ⁄ GAL1::UTP25 strain shows very low levels of 40S ribosomal subunit, an accumulation of free 60S sub-unit, and consequent low number of polysomes (B) Analysis of ribosomal subunits through sucrose gradient in the presence of EDTA Levels of 40S subunit are strongly decreased upon depletion
of Utp25p Numbers indicate area quantitation of subunits peaks.
Trang 4somal subunits upon depletion of Utp25p (Fig 3B).
Indeed, estimation of the areas under free subunit
peaks showed a change in the 60S : 40S ratio from
1.4, under permissive conditions, to 6.5, after depletion
of Utp25p (Figs 3B and S1)
To investigate the possible association of Utp25p
with ribosomal particles, the sedimentation profile of
endogenous Utp25p on density gradients was analyzed
Total extracts prepared from wild-type strain UTP25
grown in glucose medium was loaded onto 5–47%
sucrose gradients Proteins isolated from the gradient
fractions were analyzed by western blot using a
poly-clonal antiserum raised against recombinant Utp25p
Total RNA isolated from the same fractions was
ana-lyzed by northern blot to detect U3 snoRNA and the
mature rRNAs 25S and 18S The results show that
endogenous Utp25p is concentrated in the fractions
containing soluble proteins, co-fractionating with free
snoRNA U3, but it is also present in higher molecular
mass fractions (Fig 4A) As a control, antiserum
spe-cific for large ribosomal subunit protein Rpl5p was
used, showing that it is concentrated in the fractions
containing the 60S ribosomal subunits Mature rRNAs
25S and 18S were used as controls for large and small
subunit-containing fractions (Fig 4A)
U3 snoRNA shows a normal sedimentation profile
in these sucrose gradients, being present in the soluble
fractions but concentrated in fractions containing the
90S SSU processome (Fig 4A and Fig 4B, upper)
The strong effect of Utp25p depletion on the 40S subunits levels, and its co-fractionation with free U3 snoRNA suggests that Utp25p is involved in 40S ribo-somal subunit maturation
To analyze whether Utp25p depletion might affect U3 snoRNP association with preribosomes, northern blot hybridization was performed to detect U3 snoRNA from sucrose gradient fractions The results show that, after 20 h of growth in glucose, depletion
of Utp25p leads to a distribution of U3 snoRNA in two different sets of fractions, those corresponding to soluble material and in larger complexes (Fig 4B, lower) Interestingly, the 35S pre-rRNA distribution in these gradients is also shifted to larger complexes in the absence of Utp25p (Fig 4B)
To assess the possible involvement of Utp25p on pre-rRNA processing, the effect of its depletion on this pathway was analyzed by northern hybridization The results show that upon depletion of Utp25p there is an accumulation of the pre-rRNA 35S and the aberrant 23S, and a decrease in pre-rRNA 20S and mature 18S rRNA (Fig 5A) The large ribosomal subunit RNAs 25S, 5.8S and 5S are mostly unaffected by the deple-tion of Utp25p (Figs 5A and S2) The results shown here indicate the involvement of Utp25p in the early nucleolar reactions of pre-40S maturation Pulse-chase RNA labeling experiments with [3H]uracil were also performed with cells grown in glucose for 20 h The results confirm the northern blot data and show that
25S 40S 60S80S Polysomes
25S 18S
18S
U3
U3
35S
35S
40S 60S 80S Polysomes
25S 18S
U3
Utp25p
*
*
Rpl5p
Fig 4 Analysis of Utp25p and U3 snoRNA sedimentation profile on polysomal gradients (A) Sedimentation of endogenous Utp25p was detected by western blot of fractions from the wild-type strain (UTP25) polysomal profile Total RNA was analyzed using northern blotting to detect snoRNA U3 The sedimentation of mature rRNAs 25S and 18S were used as controls Western blot with antiserum against Rpl5p was performed as a control (B) The effect of Utp25p depletion on the sedimentation of U3 snoRNA was analyzed by northern hybridization (Upper) Extract from UTP25 strain (Lower) Dutp25 ⁄ GAL1::UTP25 growing in glucose medium Fractions corresponding to peaks of ribosome subunits are indicated.
Trang 5the depletion of Utp25p slows the processing of 35S
pre-rRNA, strongly inhibiting formation of mature
18S rRNA, although little affecting 25S rRNA
forma-tion (Fig 6)
To gain insight into the effect of Utp25p depletion
on early pre-rRNA cleavage reactions, primer
exten-sion reactions were performed with total RNA
extracted from either wild-type cells or Dutp25⁄
GAL1::UTP25 grown in glucose for 16 h Reaction with
a primer complementary to the 5¢ region of the mature
18S rRNA shows that the early cleavage reactions are
strongly inhibited after the depletion of Utp25p, leading
to an increased concentration of bands corresponding to
the pre-35S rRNA 5¢-end (Fig 7A) Although the
accumulation of 35S and 23S rRNAs was detected by
northern hybridization, little effect on cleavage at A1
was observed by primer extension (Fig 7A) This may
be because of the high stability of mature 18S rRNAs
formed prior to the depletion of Utp25p
Reaction with primer P3, which hybridizes
down-stream of site D in ITS1, also shows the accumulation
of pre-rRNAs, with increased extension bands corre-sponding to regions in the mature 18S rRNA upon depletion of Utp25p (Fig 7B; asterisk) A primer extension reaction with an oligo complementary to a region downstream of A2 shows that depletion of Utp25p causes a strong inhibition in the cleavage at this site (Fig 7), and consequently the accumulation of extended products that correspond to regions within the mature 18S rRNA (Fig 7C, asterisk) These results further indicate the involvement of Utp25p in the early steps of processing of pre-40S
To determine whether Utp25p might associate in vivo with pre-rRNAs, co-immunoprecipitation experiments were performed using a ProtA–Utp25p fusion Total extract from cells expressing ProtA–Utp25p was sub-jected to affinity chromatography with IgG–Sepharose beads Following co-immunoprecipitation, RNA was extracted from the different fractions and analyzed by northern hybridization, compared with RNAs recov-ered in parallel from the strain expressing only ProtA The results show that ProtA–Utp25p co-precipitates
B A
0 12 16 0 12 16 h, Glu
P1
35S
23S
5S
GAL1::UTP25 UTP25
D
33S 32S
18S 5.8SS 25S 5.8SL 25S
27SA3
27SBS 27SBL
D
35S
or
Fig 5 Northern blot analysis of pre-rRNA processing (A) Total RNA (20 lg) extracted from cells incubated in glucose medium for different periods and hybridized against specific oligonucleotide probes The relative positions of the probes on the 35S pre-rRNA are indicated in (B) Bands corresponding to the major intermediates and to the mature rRNAs are indicated on the right-hand side The lower panel shows hybridization with a probe against the 5S rRNA, used as an internal control (B) Structure of the 35S pre-rRNA and major intermediates of the rRNA processing pathway in S cerevisiae The positions of the probes used for northern hybridizations are indicated below the 35S pre-rRNA Processing of 35S pre-rRNA starts with endonucleolytic cleavages at sites A 0 and A 1 in the 5¢-ETS, generating 32S pre-rRNA Subse-quent cleavage at site A2, in ITS1, generates the 20S and 27SA2pre-rRNAs The 20S pre-rRNA is then processed at site D to the mature 18S rRNA The major processing pathway of the 27SA2pre-rRNA involves cleavage at site A3, producing 27SA3, which is digested quickly
by exonucleases to generate the 27SB short (27SB s ) pre-rRNA The subsequent processing step occurs at site B 2 , at the 3¢-end of the mature 25S rRNA Processing at sites C1and C2separates the mature 25S rRNA from the 7SSpre-rRNA This pre-rRNA is subsequently pro-cessed exonucleolytically to generate the mature 5.8SSrRNAs A fraction of the 27SA2pre-rRNA is processed at the 5¢-end by a different mechanism and, following processing at the remaining sites, gives rise to the 5.8S long (5.8S L ) rRNA, which is 6-8 nucleotides longer than the 5.8S S rRNA at the 5¢-end.
Trang 6the 35S pre-rRNA, the aberrant rRNAs 23S and 22S, and much less efficiently, the pre-rRNA 20S (Fig 8) Mature 18S rRNA was not efficiently co-immunopre-cipitated with ProtA–Utp25p, further indicating that this protein is associated only with the early pre-rRNAs This is in accordance with the hypothesis of the involvement of Utp25p in the early cleavages of the 35S pre-rRNA, ProtA–Utp25p co-immunoprecipi-tated U3 snoRNA (Fig 8)
Based on the above results, it seemed likely that Utp25p might interact with protein subunits of the SSU processome To determine whether that interac-tion could occur, the two-hybrid assay was performed using Utp25p fused to the lexA DNA-binding domain and its interaction with Sas10p⁄ Utp3p, Mpp10p, Imp3p and Imp4p was investigated [10,12,15] Expres-sion of the reporter genes HIS3 and lacZ indicates a strong interaction of Utp25p with Sas10p⁄ Utp3p, a weaker interaction with Mpp10p and no interaction with Imp3p or Imp4p (Fig 9A, upper) The direct interaction between Utp25p and Sas10p was confirmed after expressing recombinant proteins in
Escherichi-a coli and performing pull-down assays The results show that glutathione S-transferase (GST)–Sas10p, immobilized in glutathione–Sepharose beads pulls
25S 35S
18S
20S
27S 23S
UTP25 GAL1::UTP25
Fig 6 Metabolic labeling of rRNA Pulse-chase labeling with
[3H]uracil was performed after incubating Dutp25 ⁄ GAL1::UTP25
and control strain in glucose medium for 20 h Total RNA (20 lg)
was loaded onto agarose gel after [ 3 H]uracil labeling The figure
shows autoradiograph of RNA transferred to nylon membrane.
Bands corresponding to major intermediates and mature rRNAs are
indicated on the right-hand side.
A 1
A 0
P2
5’
G A T C
0 16 0 16 h, Glu
A
*
P3
GAT C
B
G A T C
0 16 0 16 h, Glu
C
*
A 2
P8
Fig 7 Early cleavage reactions in 35S pre-rRNA were analyzed through primer extension reactions of total RNA extracted from cells grow-ing in media containgrow-ing either galactose (0 h) or glucose (16 h) Relative positions of the primers used in the primer extension reactions are shown in Fig 5B (A) Primer extension with the primer P2allows the detection of the sites A0and A1 Processing inhibition in Dutp25 ⁄ GAL1::UTP25 strain allows the detection of the 5¢-end of 35S pre-rRNA (B) Reaction with primer P 3 that hybridizes between sites D and A2 shows the accumulation of pre-rRNA after depletion of Utp25p (C) Primer extension reaction with primer P 8 shows that processing at site
A2is inhibited upon depletion of Utp25p Asterisks indicate longer extensions of the reactions due to inefficient processing.
Trang 7down His–Utp25p, whereas the negative control GST
does not (Fig 9B) These results strongly suggest that
Utp25p is part of the SSU processome, participating in
the early stages of pre-rRNA maturation In order to
determine the portion of Sas10p that is responsible for
the interaction with Utp25p, two Sas10p truncated
mutants were fused to Gal4-AD and the interaction
with Utp25p was investigated through the two-hybrid
assay The results show that the N-terminal portion of
Sas10p is sufficient for interaction with Utp25p
(Fig 9A, lower)
Many of the SSU processome protein subunits are conserved throughout evolution In order to identify possible Utp25p orthologs in other organisms, a BLAST search was performed Utp25p homologs are present in many organisms, including humans
0
10
20
30
40
50
60
70
80
90
100
ProtA
ProtA ProtA-Utp25
TE
U3
5S 25S 18S
TE
35S
23S 22S/21S 20S
ProtA A-Utp25
21S
23S
A 0 A 1 D A 2 A 3
22S
A 0 A 1 D A 2 A 3
21S
D
20S
2
D
A
B
C
Fig 8 RNA co-immunoprecipitation with ProtA–Utp25p Total
extracts from cells expressing either ProtA or ProtA–Utp25p were
incubated with IgG–Sepharose beads (A) After
immunoprecipita-tion, RNA was extracted from fractions of total extract (TE), flow
through (FT), wash (W) and bound material (B), separated by
elec-trophoresis and subjected to northern hybridization with probes
specific for the RNAs indicated on the right The structures of the
detected pre- and aberrant rRNAs are shown on the left (B)
Pro-teins isolated from the same fractions were subjected to western
blot for detection of ProtA and ProtA–Utp25p (C) Quantitation of
the bands obtained from RNA co-ip by phosphorimaging Ratio of
bound ⁄ input is shown for all RNAs tested.
AD-Sas10 AD-Mpp10
AD L40-61
AD-Imp3 AD-Imp4
A
B
FT1 FT2 B
GST + His-Utp25
GST-Sas10 + His-Utp25
His-Utp25
GST-Sas10 GST
AD-Sas10 (1–227) AD-Sas10
AD–Sas10 (226–610) AD
Fig 9 Utp25p interacts with SSU processome subunits (A) Utp25p was fused to lexA DNA-binding domain (BD) and tested for interaction with Mpp10p, Sas10p, Imp3p and Imp4p, which were fused to Gal4p transcription activation domain (AD) Sas10p trun-cated mutants fused to Gal4p-AD are inditrun-cated by the amino acid positions relative to the full-length protein [Sas10(1–227) and Sas10(226–610)] Protein interactions were analyzed using the two-hybrid system, testing for expression of the reporter genes HIS3 (left) and lacZ (right) BD–UTP25 + AD, negative control; strain L40-61, which harbors plasmids encoding BD–Nip7p and AD–Rrp43p, was used as a positive control (B) Western blot for detection of proteins after pull-down assay Total extract from cells expressing either GST or GST–Sas10p (TE 1 ) was incubated with a glutathione–Sepharose resin, the flow-through fraction was collected (FT 1 ) and after washing, total extract of cells expressing His–Utp25p (TE 2 , not shown) was loaded The flow-through fraction was collected again (FT2), resin was washed, and bound fraction obtained (B) His–Utp25p is only pulled-down by GST–Sas10p His– Utp25p was detected with monoclonal anti-His IgG2a GST–Sas10p and GST were detected with anti-GST serum.
Trang 8(Fig S3) Utp25p and hUtp25p (human Utp25p) show
37% sequence similarity and 28% sequence identity
Both proteins contain the domain of unknown
func-tion DUF1253, which shows low similarity to DEAD
box helicases [20] To investigate whether hUtp25p
and Utp25p might perform similar functions in the
cell, the human gene (C1ORF7⁄ DEF) was cloned and
expressed in strain Dutp25⁄ GAL1::UTP25 under the
control of a constitutive promoter
(MET25::GFP-hUTP25) Expression of the human protein could not
rescue Dutp25⁄ GAL1::UTP25 growth under the
restric-tive condition (Fig 10A) To obtain higher levels of
expression of hUtp25p in yeast cells, the gene was
cloned under control of a stronger constitutive
pro-moter, PGK1, without the GFP tag, but still could not
rescue growth of the conditional strain in glucose
med-ium (Figs 10A and S4), indicating that, although these
proteins show sequence similarity in the C-terminal
portion, divergences in the remaining sequence of the
protein would render hUtp25p nonfunctional and⁄ or unstable in yeast
To analyze the possibility that the C-terminal DUF1253 domain of Utp25p might be sufficient for the protein function, truncation mutants were fused to GFP, cloned in a plasmid under the control of a con-stitutive promoter and transformed into Dutp25⁄ GA-L1::UTP25 strain The results show that the DUF1253 domain does not complement growth of the condi-tional strain (Figs 10A and S4) The GFP-fused dele-tion mutants were also analyzed by western blot and the results show that all were expressed in the cell (Fig 10B) Sequence analysis also predicted a possible phosphorylation site in Utp25p Indeed, high-through-put analysis showed that Ser196 was phosphorylated [21] To investigate whether this modification was important for function, a point mutation was intro-duced in Utp25(S196V) that would prevent phosphory-lation at this specific residue The more conserved
B
25
40 50 60 80 115 kDa
A
GFP GFP-Utp25 GFP-Utp25Δ243 GFP-Utp25Δ287 GFP-Utp25Δ411
DUF1253
Glu
GFP
UTP25
GFP-Utp25 GFP-Utp25 (S196V) *
GFP-hUtp25 GFP-Utp25 (S198A) *
hUtp25
Fig 10 Schematic representation of the
different clones of Utp25p, full-length,
truncated and the human ortholog, that
were tested for complementation of growth
of the conditional strain Dutp25 ⁄
GA-L1::UTP25 in glucose (A) Tenfold serial
dilution of UTP25 and Dutp25 ⁄ GAL1::UTP25
strains growing on glucose-containing
plates Dutp25⁄ GAL1::UTP25 was
trans-formed with a plasmid containing an extra
copy of the UTP25 gene, truncated mutants
or hUtp25p under control of a constitutive
promoter, fused to GFP hUtp25 indicates
PGK1::hUTP25 (without a GFP tag) (B)
Analysis of GFP–Utp25p mutants and
GFP–hUtp25p by western blot with anti-GFP
serum, compared with wild-type
GFP–Utp25p Arrowheads indicate
full-length proteins Right, Coomassie
Brilliant Blue-stained poly(vinylidene
difluoride) membrane used in the
immunoblot assay.
Trang 9Ser198 was also mutated, originating Utp25(S198A).
Interestingly, cells expressing Utp25(S196V) showed a
growth rate similar to that of the wild-type strain,
indi-cating that phosphorylation at Ser196 of Utp25p is not
essential for function (Figs 10A and S4) Polysomal
profile analysis of Dutp25⁄ GAL1::UTP25 ⁄
GFP-utp25(S196V) strain confirms that this mutant is fully
functional (Fig S4) Cells expressing Utp25(S198A),
on the other hand, are not able to grow in glucose
medium (Figs 10A and S4)
Discussion
Although various proteins have already been identified
as components of the SSU processome [15,16,22], it is
possible that many subunits remain to be isolated
Here, we report the characterization of Utp25p as a
novel nucleolar protein required for efficient cleavage
of 35S pre-rRNA at sites A0, A1and A2 Depletion of
Utp25p causes the accumulation of the pre-rRNA 35S
and the aberrant 23S, a consequent decrease in the
lev-els of mature 18S rRNA and strong depletion of 40S
ribosomal subunit In accordance with its nucleolar
localization and effects on pre-rRNA processing,
Utp25p interacts with the protein subunits of the SSU
processome Sas10p and Mpp10p and
co-immunopre-cipitates U3 snoRNA
High-throughput assays identified Utp25p in
com-plexes with Mpp10p and Sas10p [23] Mpp10p has
been characterized as a nucleolar protein that interacts
with the U3 snoRNP, depletion of which causes
inhibi-tion of cleavages at sites A0, A1 and A2, leading to
decreased levels of 18S rRNA [10] Mpp10p is part of
a ternary complex with Imp3p and Imp4p, and these
proteins show interdependence for binding to U3
snoRNA [24] Because Utp25p showed no interaction
with Imp3p and Imp4p and was not isolated in the
Mpp10p ternary complex, it is possible that its
interac-tion with Mpp10p is transient or might occur in the
context of the assembled SSU processome
Sas10-p⁄ Utp3p is also part of the SSU processome,
co-immu-noprecipitates U3 snoRNA and interacts with Mpp10p
[15] Depletion of Sas10p also causes a severe
reduc-tion in 18S rRNA levels, without affecting 25S rRNA
[15] Interestingly, individual depletions of either U3
snoRNA or the U3 snoRNP protein subunits Nop1p,
Nop58p, Mpp10p, Imp3p, Imp4p, Sof1p, Lcp5p,
Utp23p, Utp24p and Enp1p all result in accumulation
of the pre-rRNA 35S and the aberrant 23S, and
decreased levels of the 20S pre-rRNA and the mature
18S rRNA, although to different degrees of severity
[10–12,14,25–28] These results indicate that the SSU
processome must be fully assembled for the cleavage
reactions at sites A0, A1and A2to occur The observa-tion that depleobserva-tion of Utp25p leads to similar pheno-types and its interaction with U3 snoRNA, Mpp10p and Sas10p strongly indicate that this is a novel com-ponent of the SSU processome The direct interaction between Utp25p and Sas10p was confirmed through protein pull-down assays, further indicating that Utp25p is a subunit of that complex
As shown here, in addition to interacting with SSU processome subunits, Utp25p co-immunoprecipitates the pre-rRNAs 35S, the aberrant rRNAs 23S and 22S, and much less efficiently the pre-rRNA 20S Co-immunopre-cipitation of aberrant rRNAs with SSU processome com-ponents has been reported previously [29,30] Utp25p does not co-immunoprecipitate the mature 18S rRNA, however, which is in agreement with its involvement in the early cleavage of the 35S pre-rRNA
Analysis of endogenous Utp25p sedimentation on polysomal gradients shows that it is concentrated in the fractions corresponding to soluble material, frac-tions that also contain U3 snoRNA, which is consis-tent with Utp25p being part of U3 snoRNP complex SSU processome subunits from different U3 snoRNP subcomplexes have also been reported to concentrate
in the soluble fractions of polysomal gradients [16,31] Combined, these results indicate that although Utp25p interacts with the SSU processome and is involved in pre-rRNA maturation, its interaction with the complex may be labile or transient
The question of whether Utp25p binds directly to the snoRNA U3 or associates via interaction with the proteins Sas10p and Mpp10p remains to be addressed The fact that no known RNA-binding motifs can be distinguished in the Utp25p sequence, however, indicates that the latter is more likely Analysis of the Utp25p sequence also shows that this protein contains the domain DUF1253, which occurs
in several hypothetical eukaryotic proteins of unknown function and shows remote homology to DEAD box RNA helicases [20] Attempts to gain insight into the role of the DUF1253 domain on Utp25p function, made by testing the complemen-tation of growth of the conditional strain Dutp25⁄ GAL::UTP25 with deletion mutants expressing only the DUF1253 domain, gave negative results Interest-ingly, Utp25p shows some amino acid residues that are possible targets for phosphorylation Indeed, Utp25p Ser196 has been previously shown to be phosphorylated [21] Our data show that a point mutation in which Ser196 was replaced by a valine had no effect on Utp25p function Interestingly, how-ever, substitution of Ser198 by alanine resulted in a nonfunctional protein
Trang 10During the final preparation of this article, a study
was published on Utp25p [32] In that work, a
network-guided genetics approach was used to identify proteins
involved in ribosome biogenesis, and Utp25p was
char-acterized as a nucleolar protein associated with the 40S
ribosomal subunit Analysis of pre-rRNA processing
also showed that Utp25p depletion causes an
accumula-tion of 35S pre-rRNA Those results are consistent with
those shown here Our data complement that study by
showing the direct interaction of Utp25p with SSU
pro-cessome subunits, and the analysis of the 35S pre-rRNA
cleavage reactions that are affected by the depletion of
Utp25p Furthermore, we show that although a
puta-tive human ortholog of Utp25p was identified, it does
not complement the yeast protein function
Materials and methods
DNA manipulation and plasmid construction
The plasmids used in this study, described in Table 1, were
constructed according to the cloning techniques described
by Sambrook et al [33] and sequenced by the Big Dye
method (PerkinElmer, Waltham, MA, USA) Cloning
strat-egies were as follows UTP25 gene, encoded by the
YIL091C open reading frame, was PCR amplified from
S cerevisiae genomic DNA using primers specific for
UTP25: 5¢-CCCGGGTGGATCCATGAGTGACAGTTCT
GTGAG-3¢ and 5¢-CTCGAGTTATTTAAATTCATAAAT
TTCCTTTTGTGC-3¢ For the two-hybrid assays, the PCR product was digested with SmaI and XhoI and cloned into pBTM116 [34] and pGAD-C2 [35] digested with the same enzymes, generating pBTM–UTP25 and pGAD–UTP25 (which code for the fusions BD–Utp25p and AD–Utp25p respectively, where BD refers to the lexA DNA-binding domain and AD refers to the Gal4p transcription activation domain) MPP10 and SAS10 genes were PCR amplified, the products were digested with the enzymes PvuII and SmaI and cloned into pBTM116 and pGAD-C2 digested with SmaI To obtain Sas10p truncation mutants, plasmid pGAD–SAS10 was cleaved with EcoRI, resulting in a frag-ment coding for Sas10p amino acid residues 1–226, which was cloned into pGADC2 generating pGAD–SAS10(1–226) The plasmid previously digested with EcoRI was religated, generating pGAD–SAS10(227–610) For the pull-down assays, BamHI–XhoI fragments of UTP25 and MPP10 genes were cloned into pET28a (Merck KGaA, Darmstadt, Ger-many) and pGEX-4T1 (GE Healthcare, Little Chalfont, UK), respectively YCp111GAL–UTP25, which carries UTP25 under the control of GAL1 promoter, was obtained
by inserting an EcoRV–SalI fragment into YCp111-GAL digested with NdeI (following T4 DNA polymerase treat-ment) and SalI To determine the subcellular localization of yEGFP3–Utp25p by fluorescence microscopy, plasmid pUG34–UTP25 was constructed by inserting a BamHI–XhoI fragment into pUG34 (U Gueldener & J H Hegemann, unpublished) digested with BamHI and SalI Plasmid pUG36 (U Gueldener & J H Hegemann, unpublished) was used to
Table 1 List of plasmid vectors used.
unpublished
unpublished