|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 50, 48490-48500, December 13, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, July 3, 2002, and in revised form, October 13, 2002
The 15.5K protein directly binds to the 5'
stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA
box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C
motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box
B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of
15.5K to the box B/C motif. The association of hU3-55K with the U3BC
RNA was found to be also dependent on a conserved RNA structure that
flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our
data support a new structural model of the box B/C region of the U3
snoRNA in which the box B/C motif is base-paired to form a structure
highly similar to that of both the U4 5' stem-loop and the box C/D motif.
The U3 small nucleolar RNA
(snoRNA)1 is essential for
the processing of the small subunit ribosomal RNA (rRNA) in both
vertebrates and fungi (1-3). This box C/D-type snoRNA functions by
base pairing with the 5'-external transcribed spacer (ETS) of
the pre-rRNA and the 5'-terminal region of the 18 S rRNA
sequence. The latter interaction blocks the formation of a universally
conserved central pseudoknot; a long range interaction essential for
the formation and function of the small subunit rRNA (1, 4). Based on these observations, the U3 snoRNP has been proposed to function as an
RNA chaperone in the assembly of the small subunit of the ribosome.
The U3 snoRNP contains the core proteins common to all box C/D snoRNPs,
namely fibrillarin (Nop1p in yeast), NOP56, NOP58 (Nop5p), and 15.5K
(Snu13p in yeast) (5-12). These proteins are essential for the
biogenesis, stability, and nucleolar localization of the box C/D
snoRNPs. In addition the U3 snoRNA has been shown to be associated with
several U3-specific proteins in yeast, namely Rrp9p (hU3-55K in human),
Imp3p, Imp4p, Mpp10p, Dhr1p, Rcl1p, Sof1p, and Lcp5p (13-19, 22).
However, the purified yeast U3 snoRNP mono-particle was shown to
contain only one U3-specific protein, namely Rrp9p (11), suggesting
that many of the other U3 snoRNA-associated proteins may be part of a
larger processing complex (13, 15, 20, 21). Genetic studies in yeast
have shown that all of the U3 snoRNP-specific proteins are essential for U3 snoRNP function but not for its biogenesis (13-19, 22).
The 3' domain of the U3 snoRNA can be folded into a stem-rich structure
that contains four conserved RNA sequence elements, namely boxes C', B,
C, and D. Boxes C' and D are found opposite each other at the base of
the 3' domain and form the common core box C/D motif (previously termed
the box C'/D motif but referred to here as the box C/D motif for
clarity) (23-25). The box C/D motif is required for the biogenesis of
the box C/D snoRNAs and has been shown to be essential for nucleolar
localization, cap hypermethylation, and accumulation/stability of the
transcript (reviewed in Ref. 26). This motif is also responsible for
the association of the core box C/D proteins (8, 11, 27-30). The U3
snoRNA also contains a U3-specific RNA element, termed the box B/C
motif. This RNA element is a protein-binding site that is essential for
the association of Rrp9p in yeast and U3-55K in vertebrates (19, 31).
However, unlike the box C/D motif, the box B/C motif is not essential
for biogenesis of the U3 snoRNA (32, 33).
The 15.5K protein was initially identified as a U4/U6.U5 tri-snRNP
component that directly binds to the 5' stem-loop structure of the U4
snRNA (34). We have shown recently (11) that the 15.5K protein is
associated with the box C/D snoRNAs in vivo and that it
directly binds to both the box C/D motif and the U3 snoRNA-specific box
B/C motif in vitro. Both the box C/D and the box B/C motifs are capable of forming stem-internal loop-stem structures that are
strikingly similar to the U4 5' stem-loop. The discovery that 15.5K can
bind three similar RNA motifs raises the interesting question of how
15.5K functions in the assembly of these distinct RNP complexes. In
this study we have analyzed the binding of 15.5K and hU3-55K to the box
B/C motif. These two proteins have been shown to require the B/C motif
in the U3 snoRNA for their association (11, 19, 31). To determine how
these two proteins associate with the U3 snoRNA, we made use of a HeLa
nuclear extract assembly system. Our results show that the interaction
between hU3-55K and the U3 snoRNA is dependent on 15.5K binding to the
box B/C motif. Moreover, we show that hU3-55K requires not only the box B/C motif but also sequences flanking this RNA element for its association with the U3 snoRNA. Our data suggest that hU3-55K recognizes structural, rather than sequence-specific, RNA elements within the 15.5K-bound U3 box B/C snoRNA complex.
Generation of U3 snoRNA Constructs--
The U3 construct pSP6U3
(28) was kindly provided by Stuart Maxwell. Constructs encoding human
U3 snoRNA mutants were generated by PCR using pSP6U3 as a substrate,
with the oligonucleotides listed in Table
I. The PCR products, which contain a T7
promoter upstream of the U3 sequence, were cloned into the
EcoRI and BamHI sites of pUC19. Primer sets for
each mutant were as follows: U3BC, oligonucleotides 2 and 3;
U3BC In Vitro Transcription--
For in vitro
transcription all pUC19-U3BC constructs were linearized with
BamHI, whereas pUC19-U3CD was cleaved with KpnI. All transcriptions were performed with T7 RNA polymerase in the presence of [32P]UTP (Amersham Biosciences) as described
previously (17). For cross-linking experiments 4-thio-UTP-labeled RNAs
were generated via in vitro transcription in the presence of
[32P]CTP (Amersham Biosciences) using a final
concentration of 1 mM UTP and 1 mM 4-thio-UTP.
The 4-thio-UTP was a generous gift from Heiko Manninga. The
m7G-capped U3BC and mutB transcripts were generated as
described previously (34).
Construction and in Vitro Translation of Recombinant
Proteins--
The hU3-55K In Vitro Reconstitution Experiments--
The hU3-55K antibodies
were generated by immunizing rabbits with a peptide derived from the
hU3-55K sequence (EEELEETAQEKKLRLAK). The resulting antiserum
specifically recognizes hU3-55K in Western blots and
co-immunoprecipitates only the U3 snoRNA from HeLa total cell extract
(data not shown). HeLa nuclear extract was prepared essentially as
described (34). For each 20-µl reconstitution experiment, 10 µl of
HeLa nuclear extract, 10 µg of tRNA, and 50 fmol of
32P-labeled RNA were incubated in a buffer containing 20 mM HEPES-KOH (pH 7.9), 150 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Triton X-100, 0.5 mM dithiothreitol, and 10% (v/v) glycerol for 30 min at
30 °C. In order to control for the stability of the RNA in nuclear
extract, RNA was extracted from 5% of the assembly reaction. The
reconstituted complex was then immunoprecipitated using anti-hU3-55K
antibodies coupled to protein A-Sepharose in 300 µl of IPP150 (20 mM Tris (pH 7.5), 150 mM NaCl, 0.2% Triton
X-100). The bound material was washed four times in IPP150, and the
co-immunoprecipitated and control RNAs were isolated via
phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.
The recovered RNAs were resolved on an 8% polyacrylamide, 7 M urea gel.
Competition experiments using U4 SL1 and U4 SL17 RNA oligonucleotides
(30, 34) were performed by preincubating nuclear extracts with either
100, 200, 400, or 800 pmol of the competitor for 30 min at 30 °C.
U3BC RNA was added (100 fmol), and the mixture was incubated for
another 30 min at 30 °C. For the add-back experiment shown in Fig.
6C, the U3BCwt RNA was first preincubated with recombinant 15.5K protein (2.5-40 pmol) for 1 h on ice in Buffer A (20 mM HEPES-KOH (pH 7.9), 150 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0), 0.5 mM dithiothreitol, 0.1% Triton X-100).
The in vitro reconstitution experiment shown in Fig.
2D was performed as described previously (34). Basically, 6 pmol of m7G-capped U3BC or m7G-capped mutB RNA
was incubated with 35S-hU3-55K in vitro
translate and increasing concentrations of recombinant 15.5K protein
(0.3, 0.6, and 1.2 pmol) in Buffer A in a final volume of 20 µl.
After 1 h of incubation on ice, 240 µl of Buffer A was added to
the reaction, and the reconstituted complex was immunoprecipitated
using the H20 monoclonal antibody (specific for m2,2,7G and
m7G cap structures (35)). The bound material then separated
on a 12% SDS-polyacrylamide gel.
GST Pull-down Assays--
Recombinant GST15.5K was expressed and
purified as described previously (34). Binding studies with mutant U3BC
RNAs were performed using 1 µg of GST15.5K with 50 fmol of
32P-labeled RNA and 1 µg of yeast total RNA in Buffer A
in a final reaction volume of 10 µl. After 1 h of incubation on
ice, 240 µl of Buffer B (20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0),
0.2% Triton X-100, 150 mM KCl) and 10 µl of
glutathione-Sepharose beads (Amersham Biosciences) were added, and the
samples were rotated for 2 h at 4 °C. The beads were washed
four times with Buffer B. Co-precipitated RNAs were isolated by
phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.
RNAs were resolved on 8% polyacrylamide, 7 M urea gels.
In order to analyze the binding of mutant hU3-55K in vitro
translates, 10 ng of GST15.5K was incubated with 0.6 pmol of U3BC transcript and the radiolabeled protein in a final reaction volume of
20 µl of Buffer A. After 1 h of incubation on ice, reconstituted complexes were bound to GST beads, washed four times in Buffer B, and
the proteins separated on a 12% SDS polyacrylamide gel.
UV Cross-linking Experiments--
For UV cross-linking
experiments, 500 fmol of modified RNA was used in a standard
reconstitution reaction with a final volume of 50 µl. Samples were
then subjected to UV light (365 nm) for 15 min on ice. For direct
analysis of proteins cross-linked to the RNA (Fig. 6A), 15 µg of RNase T1 was added to the samples followed by a 1-h incubation
at 50 °C, and the resultant cross-links were analyzed by SDS-PAGE.
For immunoprecipitation, reconstitution reactions were first
dissociated by the addition of SDS to 1% (w/v) and incubated at
75 °C for 15 min. Triton X-100 was then added to a final
concentration of 5% (v/v) and the reaction diluted to 350 µl with
IPP150. Immunoprecipitations were then performed as described earlier.
Precipitated complexes were subsequently treated with proteinase K and
the extracted RNAs were resolved on an 8% polyacrylamide, 7 M urea gel.
The Box B/C Is Essential for the Binding of Both 15.5K
and hU3-55K to the B/C Fragment of the U3 snoRNA--
The
15.5K and hU3-55K proteins have both been proposed to bind to the
conserved box B/C region of the U3 snoRNA (11, 31). We therefore used
an in vitro reconstitution assay, using HeLa nuclear
extract, in order to analyze the binding of these two proteins to the
U3 snoRNA. This system has been used previously to study the assembly
of the box C/D and H/ACA snoRNPs (8, 29, 30, 36). In order to analyze
assembly, 32P-labeled U3 snoRNA transcripts were incubated
in nuclear extract, and the association of hU3-55K was determined by
immunoprecipitation with anti-hU3-55K antibodies. The co-precipitated
RNA was then analyzed by PAGE. In order to show that each of the RNAs
used in this investigation was not degraded during our experiments, after incubation in nuclear extract 5% of the material was isolated and also analyzed (Fig. 1B, 5%
I).
The U3 snoRNA was co-precipitated with anti-hU3-55K antibodies (Fig.
1B, lanes 1 and 3) but not with the normal rabbit
serum (lane 2). Under the same conditions no U14 snoRNA was
co-precipitated with anti-hU3-55K antibodies demonstrating the
specificity of our assay (Fig. 1B, lane 6) as hU3-55K is a
U3-specific protein (17, 31). Previous studies (31) have mapped the
sequences required for hU3-55K association to a region of the U3 snoRNA that consists of the box B/C motif and its flanking stems. We could
also show that an RNA comprising just the B/C motif region of the U3
snoRNA (Fig. 1A, U3BC RNA) was effectively
co-precipitated by anti-hU3-55K antibodies after incubation in nuclear
extract (Fig. 1B, lane 9). In contrast, the U3CD region of
the U3 snoRNA (Fig. 1A) was not precipitated by the
anti-hU3-U55K antibodies. This confirmed that all of the essential
sequences necessary for hU3-55K binding are contained within the box
B/C region of the U3 snoRNA. The U3BC RNA was therefore used as the
substrate RNA in our investigations as the analysis of protein binding
to this fragment most likely reflects what is observed with the
full-length snoRNA. In addition, the physical separation of the two
15.5K-binding sites facilitates the direct analysis of 15.5K binding to
the box BC motif in the absence of the box C/D motif.
We have shown previously that the GA dinucleotides present in the U4 5'
stem-loop, box C/D motif, and the box B/C motif are essential for the
binding of 15.5K in vitro (11, 34). Therefore, we next
addressed the role these highly conserved nucleotides play in hU3-55K
binding. Constructs were generated with point mutations in either box B
or box C of the U3BC RNA (Fig. 1A; nucleotides marked by an
asterisk). Mutation of either A113 (mutB) or
G162 (mutC) (numbers refer to the position in the intact U3
snoRNA) in the U3BC RNA abolished the binding of hU3-55K in HeLa
nuclear extract (Fig. 1C, lanes 6, and 8).
However, neither mutation affected the stability of the RNA in nuclear
extract (Fig. 1C, lanes 5 and 7).
We next wanted to address whether the 15.5K protein specifically binds
to the U3BC RNA in HeLa nuclear extract. However, anti-15.5K antibodies
co-precipitated only very low amounts of the U3BC RNA in this assay
(data not shown). The analysis of the interaction of 15.5K with the RNA
by immunoprecipitation was therefore not possible. The 15.5K antibodies
also fail to immunoprecipitate the U4/U6.U5 tri-snRNP suggesting that
in certain complexes this protein is not accessible for antibody
binding (34). As the anti-15.5K antibodies could not be readily used to
study the binding of 15.5K to the U3BC RNA, an alternative means of
analyzing 15.5K binding to the box B/C motif was required. We therefore
analyzed the ability of recombinant GST-tagged 15.5K protein to bind
the mutant and wild-type RNAs. The RNA bound to GST15.5K was
co-precipitated using glutathione-Sepharose beads. The results show
that GST15.5K efficiently bound to the U3BC RNA but not to either of
the mutant transcripts (Fig. 1D, lanes 2-4).
When the GST tag alone was used, no wild-type RNA was co-precipitated
(Fig. 1D, lane 1). These results show that mutations that
inhibit 15.5K binding to the box B/C motif also block the association
of hU3-55K. However, a more direct analysis of the role 15.5K plays in
hU3-55K binding was required before any further conclusions could be
made about the assembly pathway of this RNP complex.
Binding of 15.5K to the B/C Motif Is Essential for the
Association of hU3-55K with the U3 snoRNA--
The fact that the 15.5K
protein can bind the B/C motif in the absence of hU3-55K suggests that
this protein may indeed be the primary RNA-binding protein. Therefore,
we next addressed whether binding of the 15.5K protein to the box B/C
motif was an essential prerequisite for the association of hU3-55K. In
order to investigate this, competition experiments were performed with a chemically synthesized oligonucleotide corresponding to the 5'
stem-loop of the U4 snRNA (Fig. 2A,
U4 SL1) that specifically binds 15.5K (34). The nuclear extract
was preincubated with U4 SL1 oligonucleotide, in order to sequester
15.5K binding, prior to the addition of U3BC RNA, and then assayed for
hU3-55K binding.
As seen in Fig. 2B, the addition of 400 pmol of U4SL1
oligonucleotide inhibited the co-precipitation of the U3BC RNA with anti-hU3-55K antibodies (Fig. 2B, lane 7). As a control,
oligonucleotide U4 SL17 (Fig. 2A), containing a point
mutation that abolishes 15.5K binding (34), was also used. The addition
of up to 800 pmol of the U4 SL17 RNA oligonucleotide had no effect on
the association of hU3-55K with the U3BC RNA (Fig. 2B, lane
13). Therefore, inhibiting the binding of 15.5K to the box B/C
motif effectively blocked the association of hU3-55K with the U3BC RNA
transcript. We next asked whether we could restore hU3-55K binding to
the U3BC RNA, in the 15.5K-sequestered extract, by the addition of
recombinant 15.5K. The 15.5K protein was first pre-bound to U3BC RNA
and then added to the oligonucleotide-inhibited extract. As seen in
Fig. 2C (lanes 6-10) the addition of recombinant
15.5K effectively restored hU3-55K binding. This therefore clearly
demonstrates that 15.5K binding to the box B/C RNA is an essential step
in the hierarchical assembly of the U3 box B/C RNP complex.
It was reported previously (31) that hU3-55K, translated in
vitro in wheat germ extract, binds to the U3 snoRNA in
vitro without the addition of exogenous proteins. We therefore
performed similar experiments in order to determine whether our results are in contradiction to this earlier publication. In vitro
translated hU3-55K was incubated with m7G-capped U3BC RNA
in either the absence or presence of recombinant 15.5K. The complexes
were then isolated using the H20 antibody, which specifically
recognizes the m2,2,7G and m7G cap structures.
The bound protein was then analyzed on an SDS-12% polyacrylamide gel.
Efficient binding of hU3-55K to the U3BC RNA was only observed in the
presence of 15.5K (Fig. 2D). Importantly, this binding was
not observed when the U3BC mutB transcript was used. Therefore, this
confirms that 15.5K binding to the snoRNA is essential for the
subsequent association of hU3-55K. The hU3-55K protein used in the
previously reported experiments was translated in vitro in
wheat germ extract, whereas the protein used in these experiments was
produced in reticulocyte lysate. It is therefore possible that factors
in the wheat germ extract, such as endogenous 15.5K, mediate hU3-55K
binding to the U3 snoRNA.
hU3-55K Requires Additional RNA Elements Flanking the Box
B/C Motif for Its Association with the U3 snoRNA--
We
next decided to analyze whether RNA sequences flanking the box B/C
motif were also necessary for the association of hU3-55K. In order to
achieve this, four deletion mutants were generated in the U3BC RNA
(Fig. 3A). The ability of
15.5K to bind to the various RNAs was determined by pull-down
experiments using GST-tagged 15.5K. As seen in Fig. 3B, each
of the deletion mutants bound 15.5K with approximately the same
efficiency confirming that the binding of this protein to the U3BC RNA
is solely dependent on the central box B/C motif.
The binding of hU3-55K to the mutant transcripts was next analyzed in
nuclear extract by immunoprecipitation with anti-hU3-55K antibodies, as
described above. Importantly, none of the mutations had any affect on
the stability of the RNAs in nuclear extract (Fig. 3C, lanes 1, 3, 5, 7, 9, and 11). Interestingly, the removal of both
stems V and IV (U3BC The Association of hU3-55K with the U3BC RNA Requires an Internal
Loop Structure Adjacent to the Box B/C Motif--
Since the
deletion of stems IV and V abolished the binding of the hU3-55K protein
to the U3BC RNA, we decided to determine the sequence and/or structural
elements in this region that are essential for hU3-55K binding.
Phylogenetic analysis of the U3BC domain from a wide range of
eukaryotes revealed no clear sequence conservation in this region of
the RNA (Fig. 4A, bracketed
area). However, in all cases examined, stem IV is separated from
the flanking helices by small internal loop structures. Indeed, the upper loop between stem IV and stem V is pyrimidine-rich (Fig. 4A). We therefore constructed a series of mutants in order
to define the nucleotides in this region required for hU3-55K
association (Fig. 4B). Since the U3BC
We first analyzed the effect of these mutations on the binding of
GST15.5K in pull-down experiments. This demonstrated that changing the
sequence of the upper internal loop (mutpyrloop), closing this loop to
join stems IV and V (mutloop-stem), or altering the sequence of a
section of stem IV (mutstem IV) had no noticeable effect on 15.5K
binding (Fig. 4C, lanes 3-5). In contrast, disruption of
stem IV (mutGG-AA) or disruption of the AU base pair in stem III
(mutAU-AA) abolished the binding of 15.5K (Fig. 4C, lanes 6 and 8). This effect is likely due to the destabilization of the secondary structure in this region of the RNA inhibiting the correct folding of the box B/C motif for 15.5K binding. This hypothesis is enforced by the fact that mutations that stabilize the structure of
this region of the RNA, i.e. by either closing the internal loop joining stem III and IV (mutloopA-GG) or the exchange of a AU base
pair in stem III for a GC base pair (mutstem III), significantly enhanced the binding of 15.5K (Fig. 4C, lanes 7 and
9).
We next analyzed the effect of these mutations on hU3-55K binding in
nuclear extract. Importantly, none of the mutations affected the
stability of the RNA in nuclear extract (Fig. 4D, lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17). Consistent with the fact
that 15.5K binding is essential for the association of the U3-specific protein, mutations that block 15.5K binding by destabilizing stem III
(mutAU-AA) or IV (mutG-AA) also inhibited the association of hU3-55K
(Fig. 4D, lanes 12 and 16). Interestingly, the
A-GG mutation, which closes the internal loop between stem III and IV,
completely blocked the binding of hU3-55K (Fig. 4D, lane
14). This point is even more striking considering the fact that
this mutation significantly enhanced the binding of 15.5K (Fig.
4C, lane 7). In contrast, mutations that altered the upper
internal loop (mutpyrloop and mutloop-stem) or that changed the
sequence of stem IV (mutstem IV) had no visible effect on hU3-55K
binding (Fig. 4D, lanes 6, 8, and 10). In
addition, the exchange of the AU base pair for a GC base pair in stem
III (mutstem III) did not noticeably affect the binding of hU3-55K
(Fig. 4D, lane 18). Taken together, these data clearly show
that the internal loop structure joining stem III and stem IV is
absolutely essential for the binding of hU3-55K and probably helps to
provide the structural platform necessary for the binding of this protein.
The Structure but Not the Sequence of Stem II in the U3BC RNA Is
Essential for the Association of Both hU3-55K and 15.5K--
The
crystal structure of the U4 5' stem-loop bound to 15.5K demonstrated
that this protein primarily contacts the internal loop and makes
minimal contact to the two stem structures (37). Our data suggest that
15.5K binds the box B/C motif in a manner analogous to the U4 snRNA and
primarily contacts the internal-loop nucleotides (11, 34, 37). The
nucleotides of stem II in the U3BC RNA that are part of the conserved
box B/C motif sequence are likely not bound by 15.5K and could
therefore provide a sequence-specific binding site for U3-specific
proteins such as hU3-55K. In order to confirm the role of stem II in
15.5K binding, as well as to determine the role of the conserved
nucleotides in U3 snoRNP assembly, a series of mutations were generated
in stem II (Fig. 5A) and analyzed for 15.5K and hU3-55K binding as described above.
We first analyzed the effect of these mutations on the binding of
GST15.5K. As can be seen in Fig. 5B, all of the mutations that disrupted the base pairing in stem II, whether conserved or
non-conserved nucleotides, either severely reduced or abolished 15.5K
binding to the U3BC RNA (Fig. 5B, lanes 3, 4, 7, and
8). In contrast, all mutations that altered the sequence of
stem II but retained the base-pairing potential (Fig. 5B,
stemCG and stemCCGCGG, lanes 5 and 9,
respectively) bound 15.5K as well as the wild-type RNA. This strongly
supports the fact that stem II exists in the U3 snoRNA and confirms its
involvement in the formation of the stem-internal loop-stem structure
necessary for 15.5K binding. Interestingly, substitution of the UU pair
with a GC base pair (mutUU-GC) reduced the association of 15.5K at
least 2-fold (Fig. 5B, lane 6). This is somewhat surprising
considering that a GC base pair is present at the equivalent position
in the U4 5' stem-loop. However, it is entirely possible that some
differences exist in the binding of 15.5K between the box B/C motif and
the U4 5' stem-loop and that in the context of the U3 snoRNA the UU
pair is more favorable for protein binding.
We next analyzed the effect of these mutations on the binding of
hU3-55K in nuclear extract. Consistent with previous data, none of the
mutations affected RNA stability in nuclear extract (Fig. 5C,
lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17). As seen in Fig. 5C, mutations that inhibiting 15.5K binding, either by
disrupting stem II or mutating the potential UU base pair, also inhibit
the association of hU3-55K to an equivalent degree (Fig. 5C,
lanes 8, 10, 14, and 16). In contrast, mutations
that change the sequence but do not disrupt the structure of stem II,
thus retaining 15.5K binding, do not effect hU3-55K association
(lanes 12 and 18). Therefore, the structure but
not the sequence of stem II is important for both 15.5K and hU3-55K
association. This means that the conserved sequence of stem II is not
essential for 15.5K and hU3-55K association and may therefore function
in the recruitment of other U3-associated proteins (see
"Discussion").
hU3-55K Directly Binds the U3BC RNA--
The association of
hU3-55K with the box B/C motif is dependent on the binding of 15.5K as
well as the conserved structure of the flanking RNA. This implies that
hU3-55K, or an additional protein other than 15.5K, directly contacts
the U3BC RNA. We therefore performed UV cross-linking experiments in
order to determine whether hU3-55K indeed directly contacts the U3
snoRNA. To achieve this we used U3BC wild-type and U3BC mutB
transcripts (Fig. 1C) synthesized in the presence of
4-thio-UTP. Following incubation of the RNA in nuclear extract, the
reaction was UV-irradiated and subsequently digested with RNase T1 (see
"Experimental Procedures"). Cross-linked proteins were then
analyzed by SDS-PAGE.
Analysis of the cross-linked products revealed that one specific
product of about 65 kDa was observed when U3BC RNA was used as a
substrate (Fig. 6A). This
product was dependent on both UV irradiation and the presence of
4-thio-UTP in the transcript. Importantly, this cross-link was not
observed when the mutB RNA (Fig. 1C) was used, suggesting
that the interaction of this protein with the U3BC RNA is specific and
dependent on 15.5K binding to the box B/C motif. We next analyzed the
cross-link products by immunoprecipitation in order to determine
whether hU3-55K is specifically cross-linked to the U3BC RNA. To
achieve this, the complexes were immunoprecipitated under
semi-denaturing conditions with anti-hU3-55K antibodies (see
"Experimental Procedures"). Under these conditions, complexes
present within the binding reaction are disrupted, and therefore only
RNA covalently attached to the hU3-55K protein, by an UV-induced
cross-link, will be co-precipitated by the anti-hU3-55K antibodies.
Precipitated complexes were subsequently treated with proteinase K, and
the extracted RNAs were resolved on an 8% polyacrylamide, 7 M urea gel. As seen in Fig. 6B, hU3-55K
specifically cross-links to the U3BC RNA. The co-precipitation of the
U3BC RNA was only observed with the wild-type RNA and not with the box
B mutant (Fig. 6B, lanes 5 and 6). In addition,
this signal was dependent on UV irradiation and the presence of
4-thio-UTP in the transcript (Fig. 6B, lanes 1-4). This
therefore clearly demonstrates that the WD40 protein, hU3-55K,
specifically binds the U3BC RNA.
The WD40 Repeats Alone Are Sufficient for the
15.5K-dependent Binding of hU3-55K to the U3BC RNA--
It
was initially reported that hU3-55K contained six WD40 repeats, located
between amino acids 144 and 405 (31). In re-examining its amino acid
sequence, we have recently observed a seventh WD40 repeat located
between amino acids 419 and 461 in the C terminus of the protein. An
alignment of the seven WD40 repeats is shown in Fig.
7A. It was demonstrated
previously (31) that deletions in the region of amino acids 140-460,
the seven WD40 repeats, inhibited the association of hU3-55K with the
U3 snoRNA. We therefore decided to re-examine the role the WD40 repeats
play in 15.5K-dependent binding of hU3-55K to the U3BC
RNA.
Radiolabeled hU3-55K or one of the mutant proteins was incubated with
U3BC RNA and recombinant GST-tagged 15.5K. The resulting complex was
isolated using GST beads, and the association of the in
vitro translated protein was analyzed by SDS-PAGE. As seen in Fig.
7C, the binding of hU3-55K to the GST 15.5K requires the presence of the U3BC RNA (lanes 1 and 2).
Analysis of the previously published hU3-55K deletion mutants clarifies
that, as with the previous experiments, mutations in the WD40 domain
abolish binding to the 15.5K-bound U3BC RNA, whereas mutations in the
N-terminal domain have little or no effect (Fig. 7C, lanes 3 and 4 and 6-10) (17, 31). In order to refine
this analysis further, we generated a new mutant, in which the complete
N terminus of this protein is deleted ( The 15.5K Protein Is Essential for the Binding of hU3-55K to the U3
snoRNA--
In this article we have shown that the binding of 15.5K to
the box B/C motif is essential for the subsequent association of hU3-55K in either nuclear extract or using in vitro
translated protein. This demonstrates a hierarchical assembly of the U3
snoRNP in which 15.5K binds first to the box B/C motif in order to
recruit hU3-55K. The binding of 15.5K to the box B/C motif functions to direct the specific association of hU3-55K to the U3 snoRNA. The association of hU3-55K is also dependent on the RNA elements
surrounding the box B/C motif. This therefore suggests that 15.5K
binding to the box B/C motif results in the creation of a highly
specific binding site for hU3-55K. The crystal structure of 15.5K bound to the U4 5' stem-loop clearly showed that the binding of this protein
has a dramatic effect on the folding of the RNA (37). The data
presented in this paper strongly suggest that the box B/C motif
probably folds into a stem-internal loop-stem structure very similar to
that of the U4 5' stem-loop. It is therefore likely that 15.5K binds
the box B/C motif in a similar manner. The binding of this protein to
the snoRNA will have a profound effect on the structure of this region
of the U3 snoRNA (see below). Thus, we believe that 15.5K binding to
the box B/C motif functions to nucleate the assembly of the U3 box B/C
motif RNP by the specific creation of the hU3-55K-binding site.
The 15.5K protein has now been shown to function as a nucleation factor
in the hierarchical assembly of three distinct RNP complexes, namely
the U3-specific box B/C RNP (this work), the box C/D core snoRNP (30),
and the spliceosomal U4/U6 snRNP (38). In the box C/D core complex,
15.5K binding is essential for the association of NOP56, NOP58,
fibrillarin, TIP48, and TIP49. In addition, in the U4/U6 snRNP 15.5K
binding is essential for the association of 61K (hPrp31) and the
heteromeric 20/60/90K complex. Interestingly, hU3-55K and the
60K protein are both WD40 repeat proteins suggesting that there may be
some similarity in the way they bind their respective 15.5K-RNA complexes.
hU3-55K Is a WD40 Protein That Directly Binds RNA--
The results
presented in this paper clearly show that there are two equally
important elements required for the binding of hU3-55K to the U3
snoRNA. These are the 15.5K protein bound to the box B/C motif as well
as the flanking RNA structures, in particular an internal loop
structure (nucleotides 115-118/155-157) adjacent to the box B/C
motif. Indeed, disruption of the secondary structure of the loop
joining stem III and stem IV specifically disrupted the association of
hU3-55K. The structure, but not the primary sequence of the U3 snoRNA
elements important for hU3-55K association, appears to be conserved
among the various U3 snoRNAs. This suggests that the specific
structural arrangement of the U3 snoRNA, after the binding of 15.5K, is
the essential requirement for hU3-55K binding. It has been shown
previously in yeast that the stem-loops surrounding the box B/C motif
are not essential for U3 function. Although the data presented in this
paper would predict, in the case of the human U3 snoRNP, that hU3-55K
binding would be abolished, it is entirely possible that the
association of the yeast protein is not affected by these changes or
compensated by protein-protein interactions.
Due to the solubility problems experienced expressing hU3-55K in
E. coli, we were not able to study the direct interaction between hU3-55K and the 15.5K-U3BC RNA complex using recombinant proteins. However, several points suggest that the hU3-55K interaction with the 15.5K-bound U3B C RNA complex is direct and not mediated by an
additional protein(s). First, the binding of in vitro
translated hU3-55K to the U3 snoRNA is completely dependent on the
presence of recombinant 15.5K (Fig. 2D). Second, the
purified yeast U3 snoRNP monoparticle contained just one U3-specific
protein, namely Rrp9p (yeast homologue of hU3-55K (11, 19)). This
suggests that, at least in yeast, no other U3-specific protein is
required for the stable association of Rrp9p with the U3 snoRNA. Third, the hU3-55K protein specifically cross-links to the U3BC RNA. Finally,
we have partially purified the U3BC RNP complex assembled in nuclear
extract. This complex was shown, by Western blot of the recovered
material, not to contain the human homologues of the U3-specific
proteins Mpp10, Sof1, and Imp4. In addition, the core box C/D proteins
NOP58, NOP56, and fibrillarin were also not associated with this RNA
(data not shown). It was shown previously (39) that an intact box C, of
the box B/C motif, was important for fibrillarin binding to the U3
snoRNA in HeLa total cell extracts. We did not observe fibrillarin
binding to the U3BC RNA in our HeLa nuclear extract; however, under the
same conditions we could observe fibrillarin binding to the box C/D
motif (30). Furthermore, it has since been shown that the box B/C motif
is not required for fibrillarin association with either the yeast or
human U3 snoRNAs in vivo
(40).2 We therefore believe
that the hU3-55K interacts directly with the 15.5K-bound U3BC RNA complex.
The main structural feature of the hU3-55K protein is the presence of
seven WD40 repeats (this work and Refs. 17 and 31) that are generally
thought to play a role in protein-protein interactions. Cross-link
experiments demonstrated that the hU3-55K protein directly contacts the
U3 snoRNA in our in vitro assembly reaction. Furthermore, we
have shown that the WD40 repeats of hU3-55K are sufficient for the
RNA-dependent interaction with the 15.5K-bound U3BC RNA. There was no observable interaction between hU3-55K and 15.5K in the
absence of RNA suggesting that protein-RNA contacts play a significant
role in the binding of the WD40 repeats to the U3BC RNA. To our
knowledge, we believe that this is the first example of a WD40 repeat
protein directly interacting with RNA, and it is likely that the
hU3-55K interaction with the U3 snoRNA is mediated by one or more of
the WD40 repeats.
The U3 snoRNP Contains Two Independent 15.5K Containing RNP
Complexes--
The 3' domain of the U3 snoRNA contains two
15.5K-binding sites, namely the C/D and B/C motifs. Several studies
have shown that in the U3 snoRNA, the box B/C and box C/D motifs are
functionally distinct. Mutations in box B/C motif appear not to affect
the stability of the RNA in vivo, whereas mutations in the
box C/D motif render the RNA unstable2 (32). The box B/C
motif alone was found to be sufficient for targeting the RNA to Cajal
bodies, whereas the box C/D motif is essential for nucleolar
localization (41). Furthermore, in contrast to the box B/C, the box C/D
is essential for 5' cap hypermethylation (42). This functional
distinctiveness between the box B/C and C/D motifs is likely due to the
distinct sets of proteins that bind these two RNA elements.
The B/C motif is associated with hU3-55K and 15.5K, and the core box
C/D motif is associated with 15.5K, NOP56, NOP58, and fibrillarin. How
do these two RNA motifs, which are very similar in structure and both
bound by 15.5K, direct the specific assembly of two distinct RNP
complexes? In this paper, we show that a specific structural element,
present between boxes B and C in the primary sequence, is essential for
the recruitment of hU3-55K to the 15.5K-bound U3BC RNA. In contrast,
recent work (30) has shown that the association of the core snoRNP
proteins with the box C/D snoRNA-15.5K complex is dependent on the
conserved sequence of stem II in the box C/D motif (corresponds to stem
II in the U3BC RNA). Interestingly, the sequence of this stem is
different in the B/C motif explaining why the core box C/D snoRNP
proteins do not associate with the U3BC RNA. In addition, the structure
but not the sequence of this stem in the box B/C motif is essential for
both 15.5K and hU3-55K protein binding. Likewise, the conserved
structural element found between boxes B and C in the U3 snoRNA is not
present in the core box C/D motif. Therefore, the distinct flanking
sequences/structures, surrounding the highly similar 15.5K-binding
sites, provide the specificity for the recruitment of the additional,
complex-specific proteins.
The Structure of the 3' Domain of the U3 snoRNA--
The results
presented in this paper provide a significant insight into the
structure of the 3' domain of the U3 snoRNA. All the work presented
here was performed with a sub-fragment of the U3 snoRNA; however,
the U3BC RNA contained all of the sequences necessary for the binding
of hU3-55K. Therefore, we believe that this reflects what occurs in the
full-length RNA. This approach has been used previously (31, 33, 41,
42) in the systematic analysis of the U3 snoRNA in vitro and
in vivo. Furthermore, since there are two 15.5K-binding
sites in the U3 snoRNA, it is essential to physically separate these
two motifs in order to enable the clear analysis of the binding
requirements of this protein in the BC region of the U3 snoRNA.
The mutational analysis of the U3BC RNA has not only resulted in the
definition of the binding sites for the 15.5K and hU3-55K proteins, it
has also provided convincing functional evidence for our model of the
U3 snoRNA. In this model, we have brought together the boxes B and C to
form stems II and III in the 3' domain of the U3 snoRNA. Stem III is
only 2 bp in length; however, this interaction is feasible in all U3
snoRNAs examined even though in some cases only one potential base pair
is found. Our mutational analysis has shown the importance of base
pairing in stem II for 15.5K binding. Furthermore, stem IV appears to
be necessary to assist in the formation or stabilization of stem III.
This point is emphasized by the dramatic increase in 15.5K binding that
was observed upon closing the small internal loop between stems III and
IV. These data are therefore highly suggestive that the short stem III,
which is probably stable only upon 15.5K binding, is found in the U3
snoRNP. The short nature of this stem is probably necessary to form the
correct structure for hU3-55K binding. Base pairing in stem II of the
B/C RNA is also required for 15.5K binding. This stem contains a G-C
base pair that is found in all of the available U3 snoRNA
sequences. Although the structure, but not the sequence, of this base
pair is essential for 15.5K binding, it is possible that this conserved
element may play a role, perhaps along with the hU3-55K protein, in the
sequence-specific recruitment of additional U3-specific proteins.
We thank Stuart Maxwell for the U14 and U3
plasmids; Hossein Kohansal and Peter Kempkes for excellent technical
assistance; Heiko Manninga for providing 4-thio-UTP and help with
cross-linking; Claudia Schneider for critically reading the manuscript;
and Sylviane Muller for generating the anti-hU3-55K antibodies.
*
This work was supported by Deutsche Forschungsgemeinschaft
Grants Lu294/12-1 and Fonds der Chemischen Industrie (to R. L.), the
Council for Chemical Sciences of the Netherlands Organization for
Scientific Research (NWO-CW) Grant 97-033 (to W.J.vV.), and by an EMBO
Short Term Fellowship ASTF 9781 (to S. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed:
Max-Planck-Institut für Biophysikalische Chemie, Abteilung
Zelluläre Biochemie, Am Faßberg 11, D-37077, Göttingen,
Germany. Tel.: 49-551-201-1413; Fax: 49 551-201-1197; E-mail:
nwatkin1@gwdg.de.
Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M206631200
2
S. Granneman, N. J. Watkins, J. Vogelzangs,
R. Lührmann, W. J. van Venrooij, and G. J. M. Pruijn, manuscript in preparation.
The abbreviations used are:
snoRNA, small
nucleolar RNA;
snoRNP, small nucleolar ribonucleoprotein;
snRNA, small
nuclear RNA;
snRNP, small nuclear ribonucleoprotein;
GST, glutathione
S-transferase.
The hU3-55K Protein Requires 15.5K Binding to the Box B/C Motif
as Well as Flanking RNA Elements for Its Association with the U3 Small
Nucleolar RNA in Vitro*
,,,, 161 Department of Biochemistry, University
of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
and § Max-Planck-Institute of Biophysical Chemistry, Am
Fassberg 11, D-37077 Göttingen, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
stem V, 2 and 6; U3BCstem IV+V, 7 and 8; U3BCstem VI, 2 and 9; U3BCstem I+VI, 10 and 11; U3BCmutB, 3 and 4; U3BCmutC, 2 and
5; U3BCstemC, 24 and 3; U3BCstemG, 2 and 25; U3BCstemCG, 24 and 25;
U3BCstemCCG, 26 and 3; U3BCstemCGG, 2 and 27; U3BCstemCCGCGG, 26 and
27; U3BCmutUU-GC, 22 and 23; U3BCmutloopA-GG, 2 and 28. The U3 snoRNA
mutants U3BCmutstem III and U3BCmutAU-AA were generated using the
pUC19-U3BCstem VI construct as template with oligonucleotides 14 and
15, and 1 and 12, respectively. U3BCmutGG-AA and U3BCmutloop-stem were
generated via a two-step PCR method using pUC19-U3BCstem VI as
template. In the first round of PCR, oligonucleotides 16 and 19 and 13 and 19 were used for U3BCmutGG-AA and U3BCmutloop-stem, respectively.
PCR products were gel-purified and used as primers for a second PCR
amplification in combination with oligonucleotide 1. To generate the
U3BCmutpyrloop and U3BCmutstem IV constructs, oligonucleotides 17 and
19 were used as a templates, respectively, and amplified using primers 1 and 19 in a PCR. RNA encoded by U3CD PCR product, generated using
oligonucleotides 21 and 22, contains a tetraloop (UUCG) in order to
join the two sides of the U3 snoRNA. This PCR product was cloned into
the KpnI and EcoRI sites of pUC19. The integrity of each construct was verified by DNA sequencing.
Oligonucleotides used in this study
1-136 deletion mutation was generated by
PCR using pCI-neo VSVhU3-55K as a substrate (17) with oligonucleotides 55Kdelta1-136For and 55Krev (Table I). The remaining pCI-neo VSVhU3-55K deletion mutants were described previously (17, 31). 35S-Labeled proteins were generated using the
TNT reticulate lysate-coupled transcription/translation kit
(Promega) according to the manufacturer's directions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
The box BC fragment of the U3 snoRNA
associates with 15.5K and hU3-55K in vitro. A,
sequence and proposed secondary structure of the U3 box B/C and C/D
motifs in the 3' domain of the U3 snoRNA. This model includes the new
structural organization of the box B/C motif (43). The secondary
structure of the box C/D motif (containing the C' and D sequences) was
drawn as described previously (25, 43). The highly conserved
nucleotides in either the box C/D or box B/C motifs are depicted in
white on a black background. The dotted
line indicates non-Watson-Crick base pair interactions predicted
from the crystal structure of the U4 5' stem-loop bound by 15.5K (37).
The dashed line dissecting the 3' domain of the U3 snoRNA
indicates how this region of the RNA was sub-divided to generate the BC
(upper part) and CD (lower part) fragments of the
U3 snoRNA. In the U3CD RNA, the BC fragment is replaced by the
tetra-loop sequence UUGA. The asterisks in the U3BC fragment
mark the nucleotides mutated in box C and box B in order to generate
mutC and mutB, respectively. B, the U3BC RNA is sufficient
for the association of hU3-55K. In vitro reconstitution
assays were performed using 32P-radiolabeled RNAs incubated
in HeLa nuclear extract. RNP complexes formed during this reaction were
then immunoprecipitated, and the co-purifying RNAs were analyzed on an
8% polyacrylamide, 7 M urea gel. The RNA used is indicated
above each panel. NRS, normal rabbit serum;
55K, anti-hU3-55K antibodies; 5% I, 5% of
the input material after incubation in nuclear extract; 10%
I, 10% of the input material after incubation in nuclear extract.
C, binding of hU3-55K to the U3BC RNA is dependent on an
intact box B and box C. In vitro reconstitution experiments
using HeLa nuclear extract were performed as described in B
using the mutB and mutC RNAs. The RNA used is indicated at the
top, and the antibodies used are as described in
B. D, binding of 15.5K to the U3BC RNA is
dependent on an intact box B and box C. GST pull-down experiments,
using either recombinant GST or GST15.5K, with the U3BC, mutB, and mutC
RNAs, were performed as described under "Experimental Procedures."
The bound RNAs were then resolved on an 8% polyacrylamide, 7 M urea gel (indicated by Pellet). The use of GST
or GST15.5K is indicated. The RNA used is indicated above
each lane. Input represents 10% of the starting
material.
View larger version (29K):
[in a new window]
Fig. 2.
The 15.5K protein is required for the binding
of hU3-55K to the U3 snoRNA. A, sequence and structure of
the two U4 5' stem-loop oligonucleotides used to block 15.5K binding.
The conserved internal loop nucleotides are shown in white on a
black background. U4 SL1 is the wild-type sequence, whereas U4
SL17 contains a point mutation in the internal loop that inhibits 15.5K
binding (34). B, an excess of a U4 5' stem-loop
oligonucleotide can specifically block the assembly of the box C/D
snoRNP. Radiolabeled U3BC RNA was incubated in HeLa nuclear extract
that had been pre-incubated with increasing amounts of either U4 SL1 or
U4 SL17 RNA oligonucleotides. The binding of hU3-55K was then assayed
by immunoprecipitation with anti-hU3-55K antibodies. Bound RNA was
recovered and then separated on an 8% polyacrylamide, 7 M
urea gel. The amount of oligonucleotide used was as follows: no
oligonucleotide in lanes 1-4 and 9; 100 pmol in
lanes 5 and 10; 200 pmol in lanes 6 and 11; 400 pmol in lanes 7 and 12;
800 pmol in lanes 8 and 13. NRS,
normal rabbit serum; 55K, positive immunoprecipitation
using anti-hU3-55K antibodies in the absence of U4 oligonucleotides;
5% I, 5% of the input material after incubation in nuclear
extract. The identity of the RNA oligonucleotide used is indicated at
the top of the panel. C, rescue of U4
SL1-oligonucleotide blocked extract by the addition of recombinant
15.5K. HeLa nuclear extract was pre-incubated with either 400 pmol of
U4 SL1 RNA oligonucleotide (lane 5) or buffer (lanes
2-4). Radiolabeled U3BC RNA was subsequently added in the absence
or presence of increasing amounts of recombinant 15.5K (lanes
6-10). The inclusion of the U4 SL1 oligonucleotide and the amount
of recombinant 15.5K used is indicated above each lane. The
binding of hU3-55K was determined as described in B. D, the 15.5K protein is required for the binding of in
vitro translated hU3-55K to the U3BC RNA.
35S-Labeled-hU3-55K was incubated with 6 pmol of wild-type
m7G-capped U3BC RNA (lanes 2-5) or 6 pmol of
m7G-capped U3BC mutB RNA (lanes 6-9) in the
presence or absence of recombinant 15.5K. Bound radiolabeled proteins
were precipitated with anti-cap antibodies (H20) and analyzed on an
SDS-13% polyacrylamide gel. The amount of recombinant 15.5K used is
indicated above each lane. 10%I, 10% of the
input in vitro translated hU3-55K protein.
View larger version (43K):
[in a new window]
Fig. 3.
RNA elements flanking the box B/C motif are
involved in the binding of hU3-55K. A, schematic
representation of U3BC RNA deletion mutants. The conserved nucleotides
of the box B/C motif are represented as in Fig. 1A.
B, binding of GST15.5K to U3BC deletion mutants. GST
pull-down experiments were performed with either recombinant GST or
GST15.5K using the RNAs outlined in A. The bound RNAs were
then resolved on an 8% polyacrylamide, 7 M urea gel
(indicated by Pellet). The use of GST or GST15.5K is
indicated. The RNA used is indicated above each lane.
Input represents 10% of the material used for binding.
C, in vitro reconstitution experiments performed
in HeLa nuclear extract with the U3BC RNA and the deletion mutants. The
U3BC as well as the mutant transcripts outlined in A were
incubated in HeLa nuclear extract, and the binding of hU3-55K was
assayed by immunoprecipitation with anti-hU3-55K antibodies
( 55K) with the bound RNAs being separated on an 8%
polyacrylamide, 7 M urea gel. The RNAs used are indicated
above the respective lanes. NRS, normal rabbit
serum; 5% I, 5% of the input material after incubation in
nuclear extract.
stem IV+V) abolished the association of hU3-55K
(Fig. 3C, lane 8), whereas deletion of stem I and stem-loop VI (U3BCstem I+VI) or deletion of stem V (U3BCstem V) only
reduced the binding of hU3-55K (Fig. 3C, lanes 6 and
12). In contrast, deletion of stem VI (U3BCstem VI) did
not affect the association of hU3-55K (Fig. 3C, lane 10).
These results show that in addition to the box B/C motif, other regions
of the U3BC RNA, most notably stems IV and V, are important for the
stable association of hU3-55K with the U3 snoRNA.
stem VI mutant bound
hU3-55K as well as the full-length U3BC, we decided to use the
U3BCstem VI as template for this analysis.
View larger version (37K):
[in a new window]
Fig. 4.
A short internal loop between stem III and
stem IV of the U3BC RNA is essential for hU3-55K binding to the U3
snoRNA. A, the proposed secondary structure of U3BC RNA
fragments derived from the Homo sapiens (Hs. BC),
Rattus norvegicus (Rn. BC), Xenopus
laevis (Xl. BC), Dictyostelium discoideum
(Dd. BC), Bombyx mori (Bm. BC), and
Saccharomyces cerevisiae (Sc. BC). The conserved
nucleotides of the box B/C motif are represented as in Fig.
1A. The bracket indicates the conserved stem
structures found above the box B/C motif that are essential for the
binding of hU3-55K to human U3 snoRNA. B, schematic
representation of the mutations generated in the upper region of the
U3BC RNA mutant U3BC stem VI. The mutations and their likely effect
on the secondary structure of the RNA are depicted. The conserved
nucleotides of the box B/C motif are represented as in Fig. 1A.
C, binding of GST15.5K to mutations in U3BCstem VI. GST
pull-down experiments were performed with either recombinant GST or
GST15.5K using the RNAs outlined in B. The bound RNAs were
then resolved on an 8% polyacrylamide, 7 M urea gel
(indicated by Pellet). The use of GST or GST15.5K is
indicated. The RNA used is indicated above each lane.
Input represents 10% of the starting material.
D, in vitro reconstitution experiments performed
in HeLa nuclear extract with the U3BCstem VI RNA and mutants. The
U3BCstem VI as well as the mutant transcripts outlined in
B were incubated in HeLa nuclear extract, and the binding of
hU3-55K was assayed by immunoprecipitation with the bound RNAs being
separated on an 8% polyacrylamide, 7 M urea gel. The RNA
mutants used are indicated above the respective lanes.
NRS, normal rabbit serum; 55K, anti-hU3-55K
antibodies; 5% I, 5% of the input material after
incubation in nuclear extract.
View larger version (33K):
[in a new window]
Fig. 5.
The structure but not the sequence of stem II
in the box B/C motif is essential for both 15.5K and hU3-55K binding to
the U3 snoRNA. A, schematic representation of the U3BC RNA
stem II mutants. Conserved nucleotides of the box B/C motif are
represented as in Fig. 1A. B, binding of GST15.5K to U3BC
stem II mutants. GST pull-down experiments were performed with either
recombinant GST or GST15.5K using the RNAs outlined in A.
The bound RNAs were then resolved on an 8% polyacrylamide, 7 M urea gel (indicated by Pellet). The use of GST
or GST15.5K is indicated. The RNA used is indicated above
each lane. Input represents 10% of the starting material.
C, in vitro reconstitution experiments performed
in HeLa nuclear extract with the U3BC stem II mutants. The U3BC as well
as the mutant transcripts outlined in A were incubated in
HeLa nuclear extract, and the binding of hU3-55K was assayed by
immunoprecipitation. The bound RNAs were then separated on an 8%
polyacrylamide, 7 M urea gel. The RNA mutants used are
indicated above the respective lanes. NRS, normal
rabbit serum; 55K, anti-hU3-55K antibodies; 5%
I, 5% of the input material after incubation in nuclear
extract.
View larger version (30K):
[in a new window]
Fig. 6.
A direct contact between hU3-55K and the U3BC
RNA. A, single protein specifically cross-links to the U3BC
RNA in HeLa nuclear extract. In vitro reconstitution
experiments were performed in HeLa nuclear extracts using 4-thio-UTP
containing 32P-labeled U3BC transcripts. The extracts were
then UV-irradiated, RNase T1-digested, and then resolved on an SDS-13%
polyacrylamide gel. The RNA used, the inclusion of 4-thio-UTP in the
transcript, and whether the sample was UV-irradiated is indicated
above each lane. NRS, normal rabbit serum;
55K, anti-hU3-55K antibodies. B, hU3-55K
specifically cross-links to the U3BC RNA. In vitro
reconstitution and UV cross-linking experiments were performed as in
A. After UV irradiation, the samples were immunoprecipitated
either under semi-denaturing conditions (lanes 1-6) or
normal native conditions (lanes 7-9) with either
anti-hU3-55K antibodies (55K) or normal rabbit serum
(NRS). The bound RNA was then separated on an 8%
polyacrylamide, 7 M urea gel. The RNA used, the inclusion
of 4-thio-UTP in the transcript, and whether the sample was
UV-irradiated is indicated above each lane.
View larger version (25K):
[in a new window]
Fig. 7.
The WD40 domain of hU3-55K is necessary and
sufficient for the interaction with the 15.5K-U3BC RNA complex.
A, alignment of the seven putative WD40 repeats identified
in hU3-55K. By using a motif search program
(www.embl-heidelberg.de/~andrade/papers/rep/search.html), the seven
WD40 repeats were identified in the amino acid sequence of hU3-55K.
Highly conserved amino acids are shown in white on a
black background. Conserved amino acids are represented in
white on a gray background and are grouped
according to Ref. 44. The WD40 repeat profiles used for these studies
have been described (45)
(bmerc-www.bu.edu/bioinformatics/wdrepeat.html). B,
schematic representation of the hU3-55K protein. The putative nuclear
localization signal (NLS) and the glutamic acid stretch
(E-stretch, gray box) have been described
previously (17, 31). The WD40 repeats are represented as black
boxes. The amino acid positions relevant to the deletions used in
C are indicated. C, in vitro
translated wild-type or mutant 35S labeled hU3-55K protein
was incubated with GST 15.5K in the absence or presence of U3BC RNA.
Reconstituted complexes were precipitated using glutathione-Sepharose
beads and the co-precipitated proteins resolved on an SDS-13%
polyacrylamide gel. The identity of the in vitro translated
protein used is indicated above each lane. The designation
of the mutants indicates which regions of hU3-55K were deleted, as
described previously (17, 31). Ten percent of the input for each
35S-labeled protein is shown in the lower panel.
D, the WD40 domain of hU3-55K requires RNA elements flanking
the 15.5K-binding site for its association with the U3BC RNA. The
binding of the hU3-55K mutant 1-136 to the GST 15.5K-RNA complex
was performed as described above using either the U3BC wild-type or
mutloopA-GG RNAs (see Fig. 4B). The RNA transcript used is
indicated above each lane. Lane 1, 10% of the
in vitro translated protein used in each
reconstitution.
1-136), effectively leaving
just the seven WD40 repeats of hU3-55K. This mutation still efficiently
binds the 15.5K-U3BC complex (Fig. 7C, lane 5) demonstrating
that the WD40 motifs alone are sufficient for this interaction. We next
analyzed whether the binding of 1-136 is dependent on the flanking
RNA sequences. The use of the U3BC RNA mutation, which blocks the
binding of hU3-55K in nuclear extract, instead of the wild-type RNA,
completely inhibits the binding of the in vitro translated
WD40 domain of hU3-55K (Fig. 7D) although this mutant still
binds the 15.5K protein. Therefore, these data are highly suggestive
that the WD40 domain of hU3-55K makes specific contacts to the U3BC RNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Hughes, J. M.
(1996)
J. Mol. Biol.
259,
645-654[CrossRef][Medline]
[Order article via Infotrieve]
2.
Kass, S.,
Tyc, K.,
Steitz, J. A.,
and Sollner-Webb, B.
(1990)
Cell
60,
897-908[CrossRef][Medline]
[Order article via Infotrieve]
3.
Savino, R.,
and Gerbi, S. A.
(1991)
Biochimie (Paris)
73,
805-812
4.
Sharma, K.,
and Tollervey, D.
(1999)
Mol. Cell. Biol.
19,
6012-6019 5.
Lafontaine, D. L.,
and Tollervey, D.
(1999)
RNA (New York)
5,
455-467
6.
Lafontaine, D. L.,
and Tollervey, D.
(2000)
Mol. Cell. Biol.
20,
2650-2659 7.
Lyman, S. K.,
Gerace, L.,
and Baserga, S. J.
(1999)
RNA (New York)
5,
1597-1604
8.
Newman, D. R.,
Kuhn, J. F.,
Shanab, G. M.,
and Maxwell, E. S.
(2000)
RNA (New York)
6,
861-879
9.
Schimmang, T.,
Tollervey, D.,
Kern, H.,
Frank, R.,
and Hurt, E. C.
(1989)
EMBO J.
8,
4015-4024[Medline]
[Order article via Infotrieve]
10.
Tyc, K.,
and Steitz, J. A.
(1989)
EMBO J.
8,
3113-3119[Medline]
[Order article via Infotrieve]
11.
Watkins, N. J.,
Segault, V.,
Charpentier, B.,
Nottrott, S.,
Fabrizio, P.,
Bachi, A.,
Wilm, M.,
Rosbash, M.,
Branlant, C.,
and Lührmann, R.
(2000)
Cell
103,
457-466[CrossRef][Medline]
[Order article via Infotrieve]
12.
Wu, P.,
Brockenbrough, J. S.,
Metcalfe, A. C.,
Chen, S.,
and Aris, J. P.
(1998)
J. Biol. Chem.
273,
16453-16463 13.
Billy, E.,
Wegierski, T.,
Nasr, F.,
and Filipowicz, W.
(2000)
EMBO J.
19,
2115-2126[CrossRef][Medline]
[Order article via Infotrieve]
14.
Colley, A.,
Beggs, J. D.,
Tollervey, D.,
and Lafontaine, D. L.
(2000)
Mol. Cell. Biol.
20,
7238-7246 15.
Dragon, F.,
Gallagher, J. E.,
Compagnone-Post, P. A.,
Mitchell, B. M.,
Porwancher, K. A.,
Wehner, K. A.,
Wormsley, S.,
Settlage, R. E.,
Shabanowitz, J.,
Osheim, Y.,
Beyer, A. L.,
Hunt, D. F.,
and Baserga, S. J.
(2002)
Nature
417,
967-970[CrossRef][Medline]
[Order article via Infotrieve]
16.
Dunbar, D. A.,
Wormsley, S.,
Agentis, T. M.,
and Baserga, S. J.
(1997)
Mol. Cell. Biol.
17,
5803-5812[Abstract]
17.
Pluk, H.,
Soffner, J.,
Lührmann, R.,
and van Venrooij, W. J.
(1998)
Mol. Cell. Biol.
18,
488-498 18.
Venema, J.,
and Tollervey, D.
(1999)
Annu. Rev. Genet.
33,
261-311[CrossRef][Medline]
[Order article via Infotrieve]
19.
Venema, J.,
Vos, H. R.,
Faber, A. W.,
van Venrooij, W. J.,
and Raue, H. A.
(2000)
RNA (New York)
6,
1660-1671
20.
Fatica, A.,
and Tollervey, D.
(2002)
Curr. Opin. Cell Biol.
14,
313-318[CrossRef][Medline]
[Order article via Infotrieve]
21.
Grandi, P.,
Rybin, V.,
Bassler, J.,
Petfalski, E.,
Strauss, D.,
Marzioch, M.,
Schafer, T.,
Kuster, B.,
Tschochner, H.,
Tollervey, D.,
Gavin, A. C.,
and Hurt, E.
(2002)
Mol. Cell
10,
105-115[CrossRef][Medline]
[Order article via Infotrieve]
22.
Lee, S. J.,
and Baserga, S. J.
(1999)
Mol. Cell. Biol.
19,
5441-5452 23.
Jeppesen, C.,
Stebbins-Boaz, B.,
and Gerbi, S. A.
(1988)
Nucleic Acids Res.
16,
2127-2148 24.
Méreau, A.,
Fournier, R.,
Gregoire, A.,
Mougin, A.,
Fabrizio, P.,
Luhrmann, R.,
and Branlant, C.
(1997)
J. Mol. Biol.
273,
552-571[CrossRef][Medline]
[Order article via Infotrieve]
25.
Parker, K. A.,
and Steitz, J. A.
(1987)
Mol. Cell. Biol.
7,
2899-2913 26.
Terns, M. P.,
and Terns, R. M.
(2002)
Gene Expr.
10,
17-39[Medline]
[Order article via Infotrieve]
27.
Caffarelli, E.,
Losito, M.,
Giorgi, C.,
Fatica, A.,
and Bozzoni, I.
(1998)
Mol. Cell. Biol.
18,
1023-1028 28.
Watkins, N. J.,
Leverette, R. D.,
Xia, L.,
Andrews, M. T.,
and Maxwell, E. S.
(1996)
RNA (New York)
2,
118-133
29.
Watkins, N. J.,
Newman, D. R.,
Kuhn, J. F.,
and Maxwell, E. S.
(1998)
RNA (New York)
4,
582-593
30.
Watkins, N. J.,
Dickmanns, A.,
and Lührmann, R.
(2002)
Mol. Cell. Biol.
22,
8342-8352 31.
Lukowiak, A. A.,
Granneman, S.,
Mattox, S. A.,
Speckmann, W. A.,
Jones, K.,
Pluk, H.,
Venrooij, W. J.,
Terns, R. M.,
and Terns, M. P.
(2000)
Nucleic Acids Res.
28,
3462-3471 32.
Samarsky, D. A.,
and Fournier, M. J.
(1998)
Mol. Cell. Biol.
18,
3431-3444 33.
Speckmann, W.,
Narayanan, A.,
Terns, R.,
and Terns, M. P.
(1999)
Mol. Cell. Biol.
19,
8412-8421 34.
Nottrott, S.,
Hartmuth, K.,
Fabrizio, P.,
Urlaub, H.,
Vidovic, I.,
Ficner, R.,
and Lührmann, R.
(1999)
EMBO J.
18,
6119-6133[CrossRef][Medline]
[Order article via Infotrieve]
35.
Bochnig, P.,
Reuter, R.,
Bringmann, P.,
and Lührmann, R.
(1987)
Eur. J. Biochem.
168,
461-467[Medline]
[Order article via Infotrieve]
36.
Dragon, F.,
Pogacic, V.,
and Filipowicz, W.
(2000)
Mol. Cell. Biol.
20,
3037-3048 37.
Vidovic, I.,
Nottrott, S.,
Hartmuth, K.,
Lührmann, R.,
and Ficner, R.
(2000)
Mol. Cell
6,
1331-1342[CrossRef][Medline]
[Order article via Infotrieve]
38.
Nottrott, S.,
Urlaub, H.,
and Lührmann, R.
(2002)
EMBO J.
21,
5527-5538[CrossRef][Medline]
[Order article via Infotrieve]
39.
Baserga, S. J.,
Yang, X. D.,
and Steitz, J. A.
(1991)
EMBO J.
10,
2645-2651[Medline]
[Order article via Infotrieve]
40.
Wormsley, S.,
Samarsky, D. A.,
Fournier, M. J.,
and Baserga, S. J.
(2001)
RNA (New York)
7,
904-919
41.
Narayanan, A.,
Speckmann, W.,
Terns, R.,
and Terns, M. P.
(1999)
Mol. Biol. Cell
10,
2131-2147 42.
Speckmann, W. A.,
Terns, R. M.,
and Terns, M. P.
(2000)
Nucleic Acids Res.
28,
4467-4473 43.
Wang, H.,
Boisvert, D.,
Kim, K. K.,
Kim, R.,
and Kim, S. H.
(2000)
EMBO J.
19,
317-323[CrossRef][Medline]
[Order article via Infotrieve]
44.
Schulz, G. E.,
and Schirmer, R. H.
(1979)
in
Principles of Protein Structure
(Cantor, C. R., ed)
, pp. 1-16, Springer-Verlag Inc., New York
45.
Smith, T. F.,
Gaitatzes, C.,
Saxena, K.,
and Neer, E. J.
(1999)
Trends Biochem. Sci.
24,
181-185[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. J. Turner, A. A. Knox, J.-L. Prieto, B. McStay, and N. J. Watkins A Novel Small-Subunit Processome Assembly Intermediate That Contains the U3 snoRNP, Nucleolin, RRP5, and DBP4 Mol. Cell. Biol., June 1, 2009; 29(11): 3007 - 3017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Boulon, N. Marmier-Gourrier, B. Pradet-Balade, L. Wurth, C. Verheggen, B. E. Jady, B. Rothe, C. Pescia, M.-C. Robert, T. Kiss, et al. The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery J. Cell Biol., February 6, 2008; 180(3): 579 - 595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Clery, V. Bourguignon-Igel, C. Allmang, A. Krol, and C. Branlant An improved definition of the RNA-binding specificity of SECIS-binding protein 2, an essential component of the selenocysteine incorporation machinery Nucleic Acids Res., March 19, 2007; 35(6): 1868 - 1884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Clery, V. Senty-Segault, F. Leclerc, H. A. Raue, and C. Branlant Analysis of Sequence and Structural Features That Identify the B/C Motif of U3 Small Nucleolar RNA as the Recognition Site for the Snu13p-Rrp9p Protein Pair Mol. Cell. Biol., February 15, 2007; 27(4): 1191 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schultz, S. Nottrott, N. J. Watkins, and R. Luhrmann Protein-Protein and Protein-RNA Contacts both Contribute to the 15.5K-Mediated Assembly of the U4/U6 snRNP and the Box C/D snoRNPs. Mol. Cell. Biol., July 1, 2006; 26(13): 5146 - 5154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Granneman, J. Vogelzangs, R. Luhrmann, W. J. van Venrooij, G. J. M. Pruijn, and N. J. Watkins Role of Pre-rRNA Base Pairing and 80S Complex Formation in Subnucleolar Localization of the U3 snoRNP Mol. Cell. Biol., October 1, 2004; 24(19): 8600 - 8610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. DOBBYN and R. T. O'KEEFE Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA RNA, February 1, 2004; 10(2): 308 - 320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. MARMIER-GOURRIER, A. CLERY, V. SENTY-SEGAULT, B. CHARPENTIER, F. SCHLOTTER, F. LECLERC, R. FOURNIER, and C. BRANLANT A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA RNA, July 1, 2003; 9(7): 821 - 838. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |