We cotransfected FLAG-tagged human RPL4 with hemagglutinin HA-tagged wild-type, DEVD mutant or SAT mutant form of human Gua into HeLa cells and did immunopreci-pitation with anti-FLAG re
Trang 1ribosomal protein L4
Hushan Yang, Dale Henning and Benigno C Valdez
Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
Ribosome biogenesis is a complicated cellular process
which occurs in the nucleolus [1] The entire scenario
begins at the end of mitosis and includes ribosomal
DNA transcription, pre-ribosomal RNA (pre-rRNA)
modifications and processing as well as assembly of
rRNAs and ribosomal proteins into preribosome
sub-units which are then exported to the cytoplasm to
form the mature ribosomes [2] Errors in this process
are reported to be associated with several diseases
[3–8] The availability of genetic manipulations makes
ribosome biogenesis much better studied in yeast than
in higher eukaryotes such as mammalian and frog
sys-tems, resulting in the identification of more than 80
yeast ribosomal proteins and numerous trans-acting
elements including small nucleolar RNAs (snoRNAs)
as well as nonribosomal proteins However, in
mam-malian systems, ribosome biogenesis is far from being
thoroughly understood due to the increased
complex-ity To date, only a few nucleolus-localized
nonribo-somal proteins have been implicated in pre-rRNA
processing in mammalian cells and include B23⁄ NO38⁄ NPM [9], C23 ⁄ nucleolin [3], fibrillarin [10,11], p120 [12], EBP1 [13], Bop1 [14] and p19Arf [15] No bona fide RNA helicase has been implicated in this process in higher eukaryotes except RNA helicase
II⁄ Gua [16,17]
RNA helicase II⁄ Gua is a multifunctional nucleolar protein with in vitro RNA-dependent ATPase activity, ATP-dependent RNA helicase activity and GTP-stimu-lated RNA foldase activity [17–19] The presence of both RNA unwinding and RNA folding activities in two distinct domains of the same protein highly sug-gests a role of Gua in rRNA biogenesis [19] Using antisense oligodeoxynucleotide and siRNA to down-regulate Gua expression in Xenopus oocytes [16] and mammalian cells [17], respectively, we demonstrated that Gua is important for 18S and 28S rRNA produc-tion in both systems In addiproduc-tion, Gua was also showed
to participate in other major cellular activities such as cell growth and differentiation [19,20], regulation of
Keywords
ribosomal protein; ribosomal RNA
biogenesis; RNA helicase; nucleolus
Correspondence
B C Valdez, Department of Pharmacology,
Baylor College of Medicine, Houston,
TX 77030, USA
Fax: +1 713 798 3145
Tel: +1 713 798 7908
E-mail: bvaldez@bcm.tmc.edu
(Received 11 April 05, revised 19 May 05,
accepted 9 June 05)
doi:10.1111/j.1742-4658.2005.04811.x
RNA helicase II⁄ Gua is a multifunctional nucleolar protein involved in ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells Downregulation of Gua using small interfering RNA (siRNA) in HeLa cells resulted in 80% inhibition of both 18S and 28S rRNA production The mechanisms underlying this effect remain unclear Here we show that
in mammalian cells, Gua physically interacts with ribosomal protein L4 (RPL4), a component of 60S ribosome large subunit The ATPase activity
of Gua is important for this interaction and is also necessary for the func-tion of Gua in the producfunc-tion of both 18S and 28S rRNAs Knocking down RPL4 expression using siRNA in mouse LAP3 cells inhibits the pro-duction of 47⁄ 45S, 32S, 28S, and 18S rRNAs This inhibition is reversed
by exogenous expression of wild-type human RPL4 protein but not the mutant form lacking Gua-interacting motif These observations have sug-gested that the function of Gua in rRNA processing is at least partially dependent on its ability to interact with RPL4
Abbreviations
aa, amino acid; GST, glutathione S-transferase; Gua, RNA helicase II ⁄ Gua; HA, hemagglutinin; IPTG, isopropylthio-b- D -galactoside; NLS, nuclear localization signal; NoLS, nucleolar localization signal; RPL4, Ribosomal Protein L4; rDNA, ribosomal DNA; rRNA, ribosomal RNA; RNP, ribonucleoprotein; siRNA, small interfering RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA.
Trang 2c-Jun-mediated gene expressions [21] and in vitro
clea-vage by PIAS1 [22] It is unclear whether the effect of
Gua on cell proliferation is due to its involvement
in ribosomal RNA production, c-Jun-mediated gene
expression or, as yet, other undiscovered mechanisms
RNA helicases are believed to function through
regulation of RNA structure rearrangement and
RNA-RNA, RNA-protein or protein–protein interactions
[23,24] To date, at least 18 putative ATP-dependent
RNA helicases have been suggested to contribute to
ribosome production in yeast S cerevisiae [25] It is
hypothesized that RNA helicases interact with other
trans-acting protein factors in preribosomal particles
during pre-rRNA processing and ribosome assembly
to modulate specific intracellular RNA structures
[26–28] To test the hypothesis that Gua may function
by interacting with other nucleolar proteins in rRNA
production, we did immunoprecipitation and identified
ribosomal protein L4 physically interacting with Gua
in an RNA-independent manner It was also
demon-strated that the interaction is necessary for the function
of Gua in 28S rRNA production in mammalian cells
Results
Ribosomal protein L4 physically interacts with
RNA helicase II⁄ Gua
In our search for Gua-interacting proteins, we used
isopropylthio-b-d-galactoside (IPTG) to induce a stable
LAP3 clone expressing FLAG epitope-tagged mouse
Gua protein Two days after induction, the cells were
lysed, treated with RNase A to rule out any protein that
may be in the complex due to binding to the same RNA,
and subjected to immunoprecipitation using anti-FLAG
Ig resin (Sigma, St Louis, MO) The immunoprecipitates
were resolved on a sodium dodecyl
sulfate–polyacryl-amide (10%) gel and analyzed by silver staining Several
stained bands were detected in the FLAG-Gua lane but
not in the control lane (Fig 1A) One of the major
bands with molecular weight of approximately 50 kDa
was recovered and sent for mass spectrometry
sequen-cing It turned out to be ribosomal protein L4 There
were other faster-migrating bands in the FLAG-Gua
lane which were of greater abundance than those in
the control lane (Fig 1A) We did not sequence these
bands, but we suspect they represent other ribosomal
proteins and⁄ or trans-acting factors of a large nucleolar
complex essential for ribosome biogenesis
The yeast two-hybrid system was used to prove the
direct interaction between Gua and RPL4 We
sub-cloned human Gua and RPL4 into pGBKT7 and
pGADT7 yeast expression vectors, respectively The
growth of yeast cells containing both Gua and RPL4
in a triple drop-out medium that lacks tryptophan, leucine, and histidine indicates interaction of the two proteins (Fig 1B, right) The specificity of Gua–RPL4 interaction is shown by the inability of the yeast clones that harbor RPL4 and p68, a DEAD-box helicase implicated in RNA splicing and export [29], or RPL4 and p53, to grow in a triple drop-out medium (Fig 1B, right) The growth of yeast cells containing the above expression constructs in a double drop-out medium that lacks tryptophan and leucine indicates that these constructs were expressed (Fig 1B, left) Because protein–protein interactions shown by yeast two-hybrid system are not always direct, we performed
an in vitro pull down assay using bacterially expressed GST-RPL4 and untagged Gua mixed together and pulled down with GSH-resin Gua was pulled down with GST-RPL4, which further supported the direct interaction between Gua and RPL4 (Fig 1C)
The in vivo association of Gua with RPL4 was shown in both human HeLa cells and mouse LAP3 cells We cotransfected either FLAG-tagged Gua and protein A-tagged RPL4, or FLAG-tagged RPL4 and protein A-tagged Gua into HeLa cells and did immu-noprecipitation using anti-FLAG resin Figure 1D shows that both overexpressed (proA-RPL4) and endogenous RPL4 interact with Gua Figure 1E shows that both overexpressed (proA-Gua) and endogenous Gua interact with RPL4 We tried using either anti-Gua or anti-RPL4 Ig to do similar experiments to show an association of endogenous Gua and endo-genous RPL4 However, neither antibody worked for immunoprecipitation although they performed well in western blot analyses
To determine the expression and cellular localization
of RPL4 protein, anti-FLAG Ig was used for indirect-immunofluorescence of HeLa cells transfected with FLAG-tagged RPL4 GFP-tagged Gua was cotrans-fected as a control to show the positions of nucleoli The localization of RPL4 to the nucleolus further indi-cates the role it may play in rRNA processing and ribosome assembly (Fig 1F) RPL4 is a component of 60S ribosome large subunit with proposed cytoplasmic localization However, we did not observe strong cyto-plasmic fluorescent signal for FLAG-RPL4 This observation has been shown with other reported ribo-somal large subunit proteins such as L23 [30]
A DEVD (Asp-Glu-Val-Asp) mutant of RNA helicase II⁄ Gua does not interact with RPL4 The DEVD motif of RNA helicases is critical for their ATPase activity which is necessary for RNA
Trang 3helicase-mediated reorganization of RNA⁄ protein structure
[23] We previously showed that the DEVD motif of
Gua is important to 18S and 28S rRNA production
[17] Since we surmised that Gua–RPL4 interaction is
necessary for Gua to function in pre-rRNA processing,
we sought to determine if the DEVD motif is also
important to the Gua–RPL4 interaction DEVD was
mutated to ASVD with reported abolishment of both
ATPase and helicase activities [16] An SAT mutant, in
which the SAT motif was mutated to LET, was used
as a control This mutant still retains ATPase activity but not RNA helicase activity [19] We cotransfected FLAG-tagged human RPL4 with hemagglutinin (HA)-tagged wild-type, DEVD mutant or SAT mutant form
of human Gua into HeLa cells and did immunopreci-pitation with anti-FLAG resin Figure 2A shows that FLAG-RPL4 was pulled-down efficiently in all three precipitates However, only wild-type Gua was also present in the precipitate, suggesting the relevance of the DEVD and SAT motifs in its association with
F D
C A
B
E
Supernatant
WB: anti-FLAG FLAG-Guα
Gu α
FLAG-RPL4 ProA-Gu α ProA-RPL4
RPL4 WB: anti-Guα
WB: anti-FLAG
Input Sup’
IP Input Sup’
WB: anti-RPL4
Pellet
Mock GST
GST Mock GST
Gu α
Gu α
RPL4
Other proteins
Double drop-out
(No Trp, No Leu)
Triple drop-out (No Trp, No Leu, No His)
IgG-H
Fig 1 Gua interacts with RPL4 (A) Stable LAP3 clones were induced with 2 m M IPTG for 48 h to express FLAG-tagged mouse Gua RNase A-treated lysates were used in immunoprecipitation using anti-FLAG resin Silver staining shows the precipitation of 50-kDa protein (RPL4)
in cells expressing mouse Gua but not in cells expressing vector alone (B) Yeast two-hybrid analysis showing the interaction of human RPL4 with human Gua and Gub Yeast clones were grown on selection media Growth in the absence of tryptophan and leucine would indi-cate presence of the appropriate vectors used to clone RPL4 and its candidate partner Presence of colonies in the triple drop-out medium (no tryptophan, no leucine, no histidine) would indicate interaction between RPL4 and the other protein (C) In vitro interaction of RPL4 with Gua Purified GST, GST-RPL4 or blank control was mixed with purified untagged Gua in a binding buffer prior to addition of GSH-resin Cen-trifugation separated the supernatant from the resin Both the supernatant and resin were analyzed by western blot analysis using anti-Gua
Ig (D) Overexpressed Gua interacts with both endogenous and overexpressed RPL4 Extracts from HeLa cells cotransfected with FLAG-tagged Gua and protein A-FLAG-tagged RPL4 were immunoprecipitated using anti-FLAG resin and probed with the indicated antibodies (E) Over-expressed RPL4 interacts with both endogenous and overOver-expressed Gua in HeLa cells Extracts from HeLa cells cotransfected with FLAG-tagged RPL4 and protein A-tagged Gua were immunoprecipitated with anti-FLAG resin and probed with indicated antibodies (F) HeLa cells transfected with FLAG-tagged human RPL4 were stained by indirect immunofluorescence using anti-FLAG Ig Anti-mouse IgG coupled
to rhodamine was used as secondary antibody GFP-tagged human Gua was cotransfected and visualized directly under microscope, as a control showing the position of nucleoli.
Trang 4RPL4 This experiment further proved the specificity
of Gua–RPL4 interaction since B23, an abundant
nucleolar phosphoprotein which is also implicated in
ribosomal RNA processing [31], was not pulled-down
by RPL4 (Fig 2A, bottom)
In a similar experiment using mouse cell line,
Xpress-tagged mouse RPL4 was transfected into a
sta-ble LAP3 clone which expressed either IPTG-induced
FLAG-tagged wild-type, DEVD mutant or SAT
mutant form of mouse Gua Immunoprecipitation was
again carried out using anti-FLAG resin and the
pre-cipitate was analyzed by western blot analysis
Figure 2B shows that all three forms of Gua
pulled-down Xpress-tagged mouse RPL4 but with different
efficiencies Wild-type Gua interacted with RPL4 with
the highest efficiency, while the DEVD mutant showed
the least This is demonstrated by comparing the signal
intensities of the precipitates with those of the original
inputs (Fig 2B) The ratio (input : IP) is
approxi-mately 1 : 5 for wild-type, 1 : 1 for SAT mutant and
5 : 1 for DEVD mutant The observed difference in the sensitivity of the two experiments (Fig 2A,B) might be attributed to difference in the levels of expression of FLAG-Gua and FLAG-RPL4 The sig-nal intensity of the FLAG-Gua (Fig 2B) is greater than FLAG-RPL4 (Fig 2A), which is possibly due to higher expression level of FLAG-Gua in the LAP3 stable cell line that was induced with IPTG compared
to FLAG-RPL4 that was expressed by transient trans-fection
The DEVD motif is important for the function of Gua in both 18S and 28S rRNA production
We have demonstrated that in Xenopus oocytes, wild-type Gua can reverse the aberrant rRNA processing pattern while the DEVD mutant cannot [16], highlight-ing the importance of the DEVD motif to the function
of Gua in both 18S and 28S rRNA production in Xenopus In the mammalian system, we were able to demonstrate that an SAT mutant, which lacks helicase activity, can restore 28S but not 18S rRNA production
in mouse LAP3 cells [17], which suggests that the SAT motif is important in 18S but not 28S rRNA produc-tion Because the helicase activity is dependent on the presence of the ATPase activity of Gua [18,19], it is reasonable to expect that mutation of the DEVD motif would consequently result in defects of 18S matur-ation However, whether or not the DEVD motif is necessary for 28S production in mammalian cells is unknown Here, a rescue experiment was performed exactly as described [17] to address this issue Briefly, a stable LAP3 clone was induced with IPTG to over-express a DEVD mutant form of the human Gua, after which the cells were treated with si935, an effect-ive siRNA that specifically targets mouse Gua mRNA but not human Gua mRNA Figure 3 shows that treat-ment of the cells with si935 effectively inhibited the production of both 18S and 28S rRNAs (lane 3, com-pared with lanes 1 and 2), which conforms to our pre-vious results [17] However, in this experiment the expression of a DEVD mutant form of human Gua protein did not restore 18S nor 28S rRNA (Fig 3 lane 4) as the wild-type did [17] Thus, we conclude that the DEVD motif is indispensable for the function of human Gua in both 18S and 28S rRNA production, consistent with our results in the Xenopus oocyte [16]
Amino acids 264–333 of human RPL4 is important
to its interaction with Gua Human RPL4 has not been extensively studied after its cloning [32] Human and mouse RPL4 are 90%
A
B
Input Sup’
Input IP Input IP Input IP
Input Sup’
IP Input Sup’
WB: anti-FLAG
FLAG-RPL4
HA-Gu (WT)
FLAG-RPL4 HA-Gu (SAT-M)
FLAG-RPL4 HA-Gu (DEVD-M) IP: anti-FLAG
IP: anti-FLAG
WB: anti-HA
WB: anti-B23
WB: anti-FLAG
WB: anti-Xpress
WB: anti-B23
FLAG-Guα
Xpress-RPL4
Xpress-RPL4
FLAG-Guα (WT) FLAG-Guα (SAT-M)Xpress-RPL4 FLAG-Gu (DEVD-M)Xpress-RPL4
B23
FLAG-RPL4
HA-Guα B23
Fig 2 The DEVD motif of Gua is important to Gua–RPL4
interac-tion (A) HeLa cells were cotransfected with FLAG-tagged
human-RPL4 and plasmids encoding HA-tagged wild-type (WT), SAT
mutant (SAT-M) or DEVD mutant (DEVD-M) form of human Gua.
Whole cell extracts were immunoprecipitated using anti-FLAG resin
and probed with anti-FLAG, anti-HA or anti-B23 Ig (B) LAP3 cells
were transfected with Xpress-tagged mouse RPL4 and induced
with 2 m M IPTG for 48 h to express FLAG-tagged wild-type, SAT
mutant or DEVD mutant of mouse Gua Whole cell extracts were
immunoprecipitated using FLAG resin and probed with
anti-FLAG, anti-Xpress or anti-B23 Ig.
Trang 5homologous in their cDNA-derived amino acid
sequences The 98 amino acid C-terminal of human
RPL4 protein has little homology with its mouse
homologue However, their N-termini (amino acids 1–
333) are 99% identical with differences in only three
amino acid residues In higher eukaryotes, other than
the proposed involvement in ribosome assembly, RPL4
has been implicated in cell proliferation and
differenti-ation during rat neurogenesis [33] with unknown
mech-anism In yeast, ribosomal protein RPL2 is most
homologous to human RPL4 The yeast RPL2 has
two copies, RPL2A and RPL2B [34] A decrease in the
expression of RPL2A leads to reduced production of
60S large subunits and mature ribosomes, which
conse-quently results in slower growth rates [34] These data
suggest the relevance of RPL4 in lower and higher
eukaryotes We hypothesize that the function of RPL4
is partly regulated by protein–protein interactions
To determine the RPL4 domains involved in Gua
interaction, we generated FLAG-tagged human RPL4
deletion mutants (Fig 4A,D), expressed them in HeLa cells and tested their ability to bind with Gua via immunoprecipitation Analysis of the overexpressed proteins by indirect immunofluorescence showed that wild-type RPL4 (amino acids 1–428), N1 mutant (amino acids 1–264) and C1 mutant (amino acids 131– 428) predominantly localize to the nucleolus but the C2 mutant (amino acids 264–428) is dispersed within the nucleus but not in the nucleolus (Fig 4B), indica-ting the region of amino acids 131–264 probably con-tains both the nuclear (NLS) and nucleolar (NoLS) localization signals while amino acids 264–428 may harbor another NLS but no NoLS
Figure 4C reveals that RPL4 C1 and C2 mutants, but not its N1 mutant form, coimmunoprecipitate with Gua, suggesting the Gua-interacting domain resides in amino acids 264–428 of RPL4 We speculated that if Gua–RPL4 interaction is important to cellular func-tions, then the chance should be high that the Gua-interacting domain in RPL4 would be in a conserved region As the region of amino acids 333–428 is not highly conserved among different species, we focused
on amino acids 264–333 as a possible Gua-interacting motif in RPL4 This hypothesis was proved to be cor-rect by coimmunoprecipitation of three mutants har-boring amino acids 264–333 (Fig 4F, M3, M5, M6) The other three RPL4 mutants that lack amino acids 264–333 did not coimmunoprecipitate with Gua (Fig 4F, M1, M2, M4) We observed that two bands are recognized by the anti-FLAG Ig in mutant M1 The lower band should be the correct deletion mutant expression product according to its expected molecular size The identity of the upper band remains to be determined
Localizations of M2 (amino acids 131–264) and M3 (amino acids 131–333) mutants to the nucleolus are in accordance with the finding that both NLS and NoLS are within amino acids 131–264 Mutant M1 (amino acids 131–196) is dispersed within the whole cell but with stronger signal intensity in the cytoplasm than in the nucleus, suggesting that both the NLS and NoLS should be in the region of amino acids 196–264 Because both M4 (amino acids 204–264) and M5 (amino acids 204–333) mutants localize to the nucleo-plasm but not to the nucleolus, it would follow that the major NoLS for human RPL4 is within amino acids 196–204 For mutant M4, the fluorescent signal
is mainly in the nucleoplasm, however, a significant proportion was also found in the cytoplasm The locali-zations of M5 and M6 mutants, consistent with that of C2 mutant, are predominantly in the nucleus excluding the nucleolar region (Fig 4E) Thus, we suspect that
a strong NLS is within amino acids 264–333 while a
IPTG
si935
47S/45S
32S
28S
28S
18S
18S
–
–
+ –
– +
+ +
Fig 3 The DEVD motif of Gua is important to both 18S and 28S
rRNA production LAP3 cells were induced with 2 m M IPTG to
express DEVD mutant of human Gua Cells were then treated with
si935 for 48 h followed by pulse-labeling with [ 32 P]orthophosphate
for 1.5 h and a chase for 3 h with normal growth medium Total
RNAs were extracted, resolved on a 1.2% agarose-formaldehyde
gel and blotted onto a membrane for phosphorimager analysis.
Trang 6weak NLS may be within amino acids 196–264
Com-bined with the NoLS, this weak NLS is capable to
cause most RPL4 molecules to enter the nucleolus It
is not uncommon to have more than one nuclear
local-ization signal within a protein [35,36]
Downregulation of RPL4 inhibits rRNA production
in mouse LAP3 cell line
RPL4 is a component of the 60S ribosome large
sub-unit To date, there is no report showing a direct
involvement of RPL4 in pre-rRNA processing
Ribo-somal proteins are always produced in the cytoplasm,
and then imported into the nucleoli to participate in
preribosome assembly The ribosomes are then
expor-ted back into the cytoplasm where they direct protein
production [37] As we hypothesize the interaction between RPL4 and Gua is important to the function of Gua in rRNA production, it will aid to our hypothesis
if we could determine whether downregulation of RPL4 has any effect on this process A sequence near the 3¢ end of mouse RPL4 was used to design a small interfering RNA (si-L4-M1), which targets mouse but not human RPL4 mRNA (Fig 5A) Downregulation effects were examined at both mRNA and protein lev-els, using RT-PCR and western blot analysis, respect-ively Treatment of LAP3 cells with 100 nm si-L4-M1 for 48 h resulted in a decrease of the mouse RPL4 mRNA level by 70% (Fig 5B, lanes 7 and 8) This decrease was dose-dependent When 5 nm or 10 nm si-L4-M1 was used, the mRNA level decreased by about 42% or 55%, respectively (Fig 5B, lanes 3–6)
C
F
E
D A
B
WT 1
1
131
Constructs Guα-binding Localization
264
WT
anti-FLAG
IP: anti-FLAG
WB: anti-FLAG
FLAG-C1 FLAG-N1
FLAG-C2
HA-Guα
FLAG-M3
FLAG-M5 FLAG-M2
FLAG-M4
HA-Guα
WB: anti-HA
WB: anti-FLAG
WB: anti-HA
IP: anti-FLAG
Hoechst
Phase
anti-FLAG
Hoechst
Phase
M1 M2 M3 M4 M5 M6
264
333
428
131 131 131
196 264 333 264 204 204 264
– – + – + +
Nucleoplasm and cytoplasm Nucleoli Nucleoli
Nucleoplasm and cytoplasm Nucleoplasm
Nucleoplasm 333
333
428 428
+ – + +
Nucleoli Nucleoli Nucleoli Nucleoplasm
N1
C1
C2
M1 M2 M3
M6 M5 M4
Constructs Guα-binding Localization
Fig 4 Mapping of Gua-binding domain in human RPL4 (A) Schematic representation of wild-type and mutant forms of human RPL4 The open bar represents regions conserved between human and mouse RPL4 The shaded bar represents nonconserved regions (B) Cellular localization of human RPL4 mutants HeLa cells transfected with FLAG-tagged human RPL4 and various mutants were stained by indirect immunofluorescence using anti-FLAG Ig Anti-mouse IgG coupled to FITC was used as secondary antibody Nuclei were visualized by Hoe-chst stain The phase images show dark phase nucleoli (C) Whole cell extracts from HeLa cells cotransfected with HA-tagged human Gua and FLAG-tagged human RPL4 deletion mutants were immunoprecipitated using anti-FLAG resin and blotted as indicated (D) Schematic rep-resentations of human RPL4 mutants M1 to M6 and their (E) cellular localization (F) Whole cell extracts from HeLa cells cotransfected with HA-tagged human Gua and FLAG-tagged human RPL4 deletion mutant shown in (D) were immunoprecipitated using anti-FLAG resin and blotted as indicated.
Trang 7Lacking an antibody against mouse RPL4 protein,
we instead used an indirect way to determine the effect
of si-L4-M1 on the protein level of mouse RPL4 We
cotransfected HeLa cells with si-L4-M1 and an
Xpress-tagged mouse RPL4 construct In this
experi-ment, the remaining exogenously expressed mouse
RPL4 protein level after si-L4-M1 treatment could be
measured with anti-Xpress Ig Figure 5C shows that
mouse RPL4 mRNA level was decreased by 83% after
treatment of the cell with 100 nm si-L4-M1 for 48 h
while the exogenously expressed mouse RPL4 protein
level was downregulated by 72% The siRNA did not
have significant influence on mRNA levels of human
RPL4 and U1C, and the protein levels of human
RPL4 and B23
We hypothesized that downregulation of RPL4
would lead to an aberrant rRNA processing pattern if
Gua–RPL4 interaction is necessary for the function of
Gua in rRNA production After 48 h of si-L4-M1
treatment, LAP3 cells were pulse-labeled with [32
P]-orthophosphate and chased with growth medium for
3 h As a negative control, a nonrelated siRNA
(si934Scr) was included [17] Total RNA was extracted,
resolved on a 1.2% agarose-formaldehyde gel and
transferred to a hybond-N nitrocellulose filter for
phos-phorimager analysis We found that all four main
visible species of rRNA (47⁄ 45S, 32S, 28S and 18S) were dramatically decreased in samples treated with si-L4-M1 (Fig 5D) However, the decreases in 47⁄ 45S rRNAs were not as great as those in mature 28S rRNA (Fig 5D), indicating that only part of the decrease in 28S was due to less precursors while the remaining changes resulted from the influence by downregulation
of RPL4 on other pathways involved in rRNA produc-tion Ethidium bromide-stained gel (Fig 5D, bottom)
is shown to indicate equal loading of the RNA
Wild-type RPL4 but not its mutant form which lacks the Gua-interacting domain reverses inhibition of rRNA production
We constructed a deletion mutant of human RPL4, D264-333, which lacks the Gua-interacting domain amino acids 264–333 and another mutant D204-264 as
a control (Fig 6A) We had already localized the NoLS of RPL4 to amino acids 196–204, which was supported by the nucleolar localization of both mutants (Fig 6B) The immunoprecipitation experi-ment shows their Gua-binding activity (Fig 6C), and supports our earlier findings (Fig 4F) Because the region including amino acids 264–333 seems to be the RPL4-Gua-interacting domain, the D204-264 mutant,
A
D
Human 1120
Mouse 1129
mRPL4
mU1C
si-L4-M1
47S/45S
32S 28S
18S
18S 28S
RT-PCR Western
hU1C hRPL4
hRPL4
hB23
Fig 5 Downregulation of mouse RPL4 using si-L4-M1 resulted in aberrant rRNA processing (A) Comparison of human and mouse RPL4 partial cDNA sequences containing the si-L4-M1 region Underscored nucleotides differ from human to mouse (B) siRNA-mediated down-regulation of mouse RPL4 mRNA LAP3 cells were transfected with increasing concentrations of si-L4-M1 Total RNA was isolated after
48 h and analyzed by RT-PCR to determine the mRNA levels of mouse RPL4 and mouse U1C (C) siRNA-mediated downregulation of mouse RPL4 protein HeLa cells were cotransfected with Xpress-tagged mouse RPL4 and si-L4-M1 Total RNA was isolated after 72 h using TRIzol Reagent (Invitrogen) and analyzed by RT-PCR to determine the mRNA levels of mouse RPL4, human RPL4 and human U1C (left panel) A parallel experiment was done to analyze changes in the protein levels of mouse RPL4, human RPL4 and human B23 after si-L4-M1 treat-ment (D) LAP3 cells were treated with 100 n M si-L4-M1 for 48 h Total 32 P-labeled RNA was analyzed as described in the legend to Fig 3 Ethidium bromide staining of both 18S and 28S rRNA is shown at the bottom.
Trang 8but not the D264-333 mutant form,
coimmunoprecipi-tated with Gua (Fig 6C)
To determine the significance of Gua–RPL4
inter-action in rRNA biogenesis, we first used si-L4-M1 to
downregulate endogenous mouse RPL4 expression and
then exogenously expressed the human orthologue and
looked for reversal of inhibition of rRNA production
Figure 6(D) (lanes 1 and 2) shows that si-L4-M1
effectively inhibited production of all four species of
rRNAs, which is consistent with the results in
Fig 5(D) This effect was reversed by the exogenous
expression of human wild-type RPL4, suggesting that
the human orthologue can functionally replace mouse
RPL4 (Fig 6D, lane 3) However, the expression of
mutant human RPL4 lacking the Gua-interacting
domain could not reverse the aberrant rRNA
process-ing pattern as effectively as the wild-type while the
mutant lacking amino acids 204–264 had a similar
effect to that of the wild-type, indicating that the
Gua–RPL4 interaction is important to the function of
Gua in rRNA processing (Fig 6D, lanes 4 and 5)
Human RPL4 associates with 28S but not 18S
rRNA
To determine if RPL4 associates with 18S or 28S
rRNA, we performed RNA immunoprecipitation using
HeLa cells transiently transfected with either FLAG vector or FLAG-tagged human RPL4 RNA–RPL4 complexes in the nucleolar extracts were immunopre-cipitated with anti-FLAG resin, and the RNA compo-nents were resolved on a 1.2% agarose-formaldehyde gel, blotted onto a nitrocellulose membrane and sub-jected to northern blot analysis Figure 7 shows that
A
D
∆204-264
∆204-264
anti-FLAG
Hoechst
Phase
IP: anti-FLAG
WB: anti-FLAG
WB: anti-FLAG
1 st transfection si934Scr
FLAG FLAG FLAG-RPL4
(WT) FLAG-RPL4 (∆204-264) FLAG-RPL4(∆264-333) si-L4-M1 si-L4-M1 si-L4-M1 si-L4-M1
2 nd transfection
18S 28S 18S 28S 32S 47S/45S
54 ±5 84±1084 ±10 73±683 ±5 42±747 ±6
100 Total±SE
WB: anti-HA
204 264
428
428
333
264 333
∆264-333
∆264-333
∆204-264 ∆264-333
FLAG-∆204-264
FLAG-RPL4 (WT) FLAG-RPL4 (∆204-264) FLAG-RPL4 (∆264-333)
FLAG-∆264-333 HA-Guα
1
1
Fig 6 Reversal of inhibition of rRNA production (A) Schematic representation of human RPL4 deletion mutants D204-264 and D264-333 The open bar represents regions conserved between human and mouse RPL4 The shaded bar represents nonconserved regions (B) Indi-rect immunofluorescence showing both D204-264 and D264-333 are localized to nucleoli (C) Whole cell extracts from HeLa cells
cotransfect-ed with HA-taggcotransfect-ed human Gua and FLAG-taggcotransfect-ed human RPL4 deletion mutant were immunoprecipitatcotransfect-ed using anti-FLAG resin and blottcotransfect-ed
as indicated (D) LAP3 cells were transfected with either 100 n M si934Scr or 100 n M si-L4-M1 as indicated After 48 h, cells were next trans-fected with FLAG-vector, FLAG-tagged wild-type human RPL4 or FLAG-tagged human RPL4 mutants as indicated After an additional 48 h, cells were pulse-labeled with [ 32 P]orthophosphate for 1.5 h and chased with cold medium for 3 h Total RNA was extracted and analyzed as described in Fig 3 Ethidium bromide staining for 18S and 28S rRNAs is shown in the middle panel The lowest panel shows expression of the FLAG-tagged human wild-type RPL4 (WT) and its mutant forms (D204-264 and D264-333) by western blot analysis using anti-FLAG Ig The numbers below the upper panel correspond to the amount of 28S rRNA or total rRNA ± standard error relative to samples in lane 1 (set
at 100) calculated with IMAGE - QUANT software Results were average of three independent experiments ± SE.
28S probe
28S rRNA
18S rRNA 18S probe
Fig 7 Human RPL4 associates with 28S but not 18S rRNA HeLa cells were transfected with either vector only or FLAG-tagged human RPL4 After 48 h, cells were collected and RNA-RPL4 complexes were immunoprecipitated from nucleolar extracts using anti-FLAG resin as described under Experimental procedures RNA components were isolated and resolved in a 1.2% agarose-formaldehyde gel and blotted onto a nitrocellulose membrane, which was subjected to northern blot analysis as described under Experimental procedures.
Trang 9overexpressed RPL4 pulled-down 28S but not 18S
rRNA in a dose-dependent manner (lanes 5 and 6)
We did not observe any signal from 47⁄ 45S, 36S and
32S pre-rRNAs, the precursors of 28S rRNA Based
on our previous experience [16], a single
oligodeoxy-nucleotide probe we used to detect 28S could not
detect higher molecular weight pre-rRNAs under our
hybridization conditions for unknown reasons As we
used nucleolar extract as the starting material for
the immunoprecipitation experiment, the 28S rRNA
pulled down by RPL4 should be newly produced
Discussion
RNA helicase II⁄ Gua is the first nucleolar RNA
heli-case shown to be directly involved in rRNA processing
in both the metazoan and mammalian systems [16,17]
There are several other nonhelicase nucleolar proteins
which have been demonstrated to also function in
rRNA processing in higher eukaryotes including
B23⁄ nucleophosmin, C23 ⁄ nucleolin, Bop1, p120 and
p19Arf Each of these proteins was found to function
at least partially through RNA–protein or protein–
protein interactions [12,14,38–42] Among the 18 RNA
helicases which have been directly implicated in yeast
ribosome biogenesis, at least nine were demonstrated
to functionally interact with other protein factors
[25,37,43] Based on the bona fide helicase activity of
Gua and its demonstrated role in rRNA processing, it
is conceivable that Gua will be shown to have partners
that facilitate its function in the ribosome biogenesis
pathway
In this paper, we report the identification of
ribo-somal protein L4 as a Gua-interacting partner through
immunoprecipitation in mouse LAP3 cells (Fig 1A)
We noticed that several other fast migrating bands
were also pulled down by anti-FLAG resin (Fig 1A,
other proteins), which we suspect to be proteins
associ-ated with either Gua or RPL4 The high concentration
of RNase A (200 lgÆmL)1), which was used in
previ-ous reports to isolate specific target-associated proteins
[31,44], suggests that these additional interactions
might not be RNA-mediated The Gua–RPL4
inter-action was further confirmed by immunoprecipitation
from HeLa cells (Fig 1D,E), yeast two-hybrid analysis
(Fig 1B) and in vitro binding assay (Fig 1C) It is
noteworthy that Gub also interacts with RPL4 as
shown by the two-hybrid analysis (Fig 1B, lower
right) As a paralogue of Gua, Gub also possesses
in vitro ATPase and helicase activities, but no RNA
foldase activity [45] The current data suggest that
both paralogues arose through gene duplication but
the resulting genes are differentially regulated and
might possess different functions [46] Overexpression
of Gub in mouse LAP3 cells leads to inhibition of total rRNA production, suggesting contrasting roles for Gub and Gua [17] It would be valuable to deter-mine whether the inhibitory effect of Gub on rRNA biogenesis is through its competitive interaction with RPL4 Indirect immunofluorescence showed a predom-inant localization of newly produced FLAG-tagged RPL4 protein to the nucleolus (Fig 1F) which is con-sistent with the published report that most newly formed ribosomal proteins are highly concentrated in the nucleolus [47] Burial of FLAG epitope within the highly structured mature ribosome subunit might account for the absence of strong fluorescent signal in the cytoplasm The distribution of RPL4 in the nucleo-lus seems more localized compared with the more dis-persed localization of Gua throughout the entire nucleolus (Fig 1F) This subtle discrepancy between the localizations of the two proteins may indicate that Gua interacts with other partners in different sub-nucleolar regions, which is consistent with the presence
of other additional bands shown in Fig 1A Moreover, our previous immunoelectron microscopy experiments showed that rat Gua is localized to the dense fibrillar component (DFC) and granular component (GC) within the nucleolus [16]
An interesting finding was the importance of the DEVD motif in Gua–RPL4 interaction (Fig 2) We previously showed that in Xenopus, the DEVD motif
of Gua was important for both 18S and 28S rRNA production [16] However, in mammalian cells, we were only able to prove that SAT motif is necessary for 18S maturation [17] As the unwinding activity is dependent on the ATPase activity, we speculated that the DEVD motif of Gua is also necessary for 18S pro-duction in mammalian cells In this report, we showed the conserved importance of the DEVD motif of Gua
to 28S maturation in mouse LAP3 cells (Fig 3) Because DEVD is important for 28S production as well as Gua–RPL4 interaction, and because RPL4 is a component of the ribosome large subunit which con-tains 28S but not 18S rRNA, it is reasonable to sus-pect that RPL4 might be involved in the function of Gua in 28S rRNA production
Through a series of deletion mutants of RPL4 used
in the immunoprecipitation and indirect immunofluo-rescence experiments, we identified the NLS and NoLS
as well as the Gua-interacting domains in RPL4 (Figs 4 and 5) However, it is worth mentioning that the use of deletion mutants may not accurately reflect the exact functional states of protein inter-actions since the possibility exists that the shortened proteins may be unfolded and thus nonfunctional
Trang 10Subtle point mutations in the identified Gua-interacting
domain might lend more support to our conclusions
Downregulation of RPL4 via si-L4-M1 resulted in the
inhibition of production of all four rRNA species
(Figs 5D and 6D lane 2), strongly suggesting a general
mechanism whereby RPL4 modulates rRNA
biogen-esis through rDNA transcription, rRNA turn over,
ribosome production rate, ribosome stability or rRNA
degradation It is possible that the amount of RPL4 in
the cell correlates with the assembly or stabilization of
pre-rRNA processing machineries or preribosomal
par-ticles Perhaps RPL4 is actually a component of the
pre-rRNA processing machinery If so, a dramatic
change in the amount of RPL4 protein level might
lead to disassembly of the specific machineries or
parti-cles, which would send feedback signals to RNA
polymerase I to advance more slowly or even to
rRNA-degrading complexes to degrade the unincorporated
mature rRNA [17] This hypothesis might help to
interpret the involvement of other nucleolar proteins in
pre-rRNA production such as p19Arf[15] It might also
explain why there is a decrease of 18S rRNA
More-over, inhibition of the 28S pathway might
concomit-antly result in the reduction of 18S rRNA through an
unidentified mechanism For example, many yeast
mutants with 25S rRNA production defects also show
an inhibition in 35S pre-rRNA cleavages which lead to
decrease in 18S biogenesis [48]
Other than the proposed general function of RPL4
in overall rRNA production, we also hypothesize that
RPL4 plays a direct role in 28S production through its
interaction with Gua Several arguments and lines of
evidence support this: (a) our rescue experiment
showed that wild-type human RPL4 reversed the
aber-rant rRNA processing pattern (Fig 6D, compare lanes
2 and 3) but the mutant lacking the Gua-interacting
domain had no effect (Fig 6, compare lanes 2 and 5);
(b) inhibition of 28S rRNA production is more
signifi-cant than that of 47⁄ 45S when RPL4 is downregulated
(Fig 5D, lanes 1 and 2); (c) RPL4 is an important
component of the 60S ribosome subunit which
con-tains the 28S but not the 18S rRNA; (d) RNA
immu-noprecipitation revealed coprecipitation of RPL4 with
28S but not with 18S rRNA (Fig 7); (e) ATPase
activ-ity of Gua is important for both 28S production and
Gua–RPL4 interaction These five lines of evidence
support a more direct role for the Gua–RPL4
inter-action in 28S production than a possible general
mechan-ism, although it is likely that both mechanisms coexist
It is not uncommon for a nucleolar protein to function
in different pathways For example, the function of
C23 in ribosome biogenesis is reflected in almost all
steps of the process including rDNA transcription,
pre-rRNA processing, preribosome assembly and nucleocytoplasmic transport [39]
What then could be a mechanism whereby the Gua– RPL4 interaction facilitates 28S rRNA biogenesis? The fact that Gua and RPL4 have been identified in ribo-nucleoprotein (RNP) particles [31,49] indicates that their interaction might cause them to be localized into pre-rRNA processing machineries essential for pre60S ribosome particles It is known that interruption of early assembly steps results in disassembly of the parti-cles and destabilization of pre-rRNAs [43] Moreover,
we did observe several other bands in the immunopre-cipitation assay (Fig 1A) coimmunoprecipitating with Gua, which may represent other proteins in the same processing machinery as Gua Once Gua has been incorporated into the RNP particle, it might function
in early rRNA processing steps such as regulating interactions between guide snoRNAs and pre-rRNAs, helping the endo- and exo-nucleases in removing inter-nal or exterinter-nal transcribed spacer sequences as well as modulating the numerous trans-acting factors and ribosomal proteins in the pre60S particles through regulation of RNA-RNA, RNA–protein and protein– protein interactions [43] In yeast S cerevisiae, involve-ment of ATP-dependent RNA helicase has been implicated in each of these possible roles [50–54] The functional diversity of an RNA molecule is based on its extreme flexibility With the help of proteins, RNA retains or loses its active configuration in response to various different cellular signals [55] RNA helicase is
a candidate to function in an energy-dependent manner in this process In addition, Gua has another activity, GTP-stimulated RNA folding activity which resides within a domain separate from the ATPase⁄ helicase activity Proteins utilizing GTP as an energy source have recently been found to participate in ribo-some biogenesis [56,57] In addition, it was recently reported that GTP-binding state might influence the nucleolar targeting of nucleostamin, a nucleolar pro-tein which shuttles between nucleoplasm and nucleolus with suspected roles in cell cycle and cell proliferation regulation [58–60] Interestingly, the C-terminal FRGQR-containing region of Gua has also been reported to be critical for both GTP-stimulated RNA foldase activity and nucleolar localization [20,61] It remains to be identified if the FRGQR region of Gua
is relevant to GTP–binding or RPL4 interaction It would not be surprising if the Gua–RPL4 interaction
is found to be important in all three roles mentioned considering the complexity of ribosome assembly, which highly demands versatile energy producers and consumers The multifunctional property of Gua makes it a good candidate