 |
INTRODUCTION |
The formation of histone mRNA 3'-ends is essential for the
expression of replication-dependent histone genes and is
restricted to the S phase in cultured cell lines (1-5). Formation of
the 3'-end occurs by an endonucleolytic cleavage between a conserved RNA hairpin structure 5' of the cleavage site and a purine-rich spacer
element 3' of the cleavage site (6). The analysis of the molecular
mechanism of histone mRNA 3' processing has been facilitated by the
development of nuclear extracts that promote the cleavage of synthetic
histone RNA in vitro. Three trans-acting factors
are involved in this RNA processing reaction: the U7 small nuclear
ribonucleoprotein particle
(snRNP)1 (7-9), a
heat-labile factor (10), and the hairpin-binding protein (HBP) or
stem-loop binding protein (SLBP) interacting with histone hairpin RNA
(11-13). The RNA moiety in the U7 snRNP, U7 RNA, is required for
histone RNA 3' processing (7, 9, 14-16) and base pairs with the
purine-rich spacer element (8, 17). The protein components of the U7
snRNP are Sm proteins, which are common to all nucleoplasmic snRNPs
(18), and other, as yet uncharacterized, U7-specific proteins (19, 20).
The heat-labile factor, which is present in nuclear extract and
inactivated by incubation at 50 °C (10), was implicated in the cell
cycle regulation of RNA 3' processing (21). HBP was first
identified as a factor binding to the histone hairpin structure (11,
22), an element required for maximal processing efficiency in
vitro (11, 23-25); in vivo, the hairpin element is
most likely essential (26). In processing reactions in
vitro, HBP bound to the hairpin RNA facilitates the formation of
the 3' processing complex composed of histone RNA, U7 snRNP, HBP, and
presumably still other factors (12, 13, 25, 27-29). Interestingly,
Xenopus oocytes contain two hairpin-binding proteins: SLBP1
involved in histone RNA 3' processing and SLBP2, which is thought to be
involved in the translational silencing of histone mRNA in oocytes
(28).
In isolated Xenopus oocytes, histone RNA substrates are
processed either by endogenous U7 snRNPs or, when these are inactivated using antisense oligonucleotides, in a reaction requiring synthetic U7
RNA injected into the nucleus or cytoplasm (15, 30, 31). This
complementation is dependent on the Sm site sequence required for
association of Sm protein with U small nuclear RNAs (snRNAs). Interestingly, U7 Sm OPT RNA with a U2 Sm site, as well as U7 Sm
MUT RNA with a destroyed Sm site (Fig. 1A), are not able to process histone RNA (20). UV cross-linking studies have
demonstrated different associations of proteins with U7 and U7 Sm OPT
RNA, indicating that (i) Sm protein assembly is required, but not
sufficient for processing, and that (ii) the U7 Sm site is necessary
for assembly of U7-specific proteins (20). Within the nucleus, Sm proteins and U7 RNA are concentrated in C-type snurposomes or coiled bodies (32). Interestingly, Xenopus SLBP1 is also
localized in coiled bodies (33), suggesting a common
compartmentalization of histone RNA 3' processing factors. Thus,
Xenopus oocytes contain all of the components of the histone
RNA-processing machinery.
Materials required for organelle biogenesis and cell cycle progression
are stockpiled in Xenopus eggs for use during early development. In addition, the egg is arrested in metaphase and thus
many components are stored in a mitotic, disassembled form. Extracts
derived from eggs can be made to spontaneously enter interphase, and
such extracts are capable of reconstituting nuclei that are
structurally and functionally similar to somatic cell nuclei. Such
reconstituted nuclei contain coiled body-like structures (34) and
undergo semiconservative DNA replication (35). Alternatively, extracts
can be made that are arrested in mitosis, either as a consequence of
the stabilization of endogenous cyclin B/cdc2 kinase, or by the
re-introduction into interphase extracts of indestructible recombinant
cyclin B (35). Thus, extracts derived from Xenopus eggs
provide a unique opportunity for in vitro analysis of the biochemistry and cell biology of nuclear assembly and function, and
also of cell cycle progression (35). This encouraged us to test whether
such extracts can be used as a source of factors for histone RNA 3' processing.
Here we demonstrate for the first time de novo assembly of
U7 snRNP from synthetic RNA and proteins derived from
Xenopus egg extract. We show that histone pre-mRNA
processing can be reconstituted in this cell free system and that
assembly and operation of processing complexes does not require the
prior assembly of an interphase nucleus. As expected, we find
endogenous U7 RNA in extracts, but surprisingly, we show that histone
RNA processing in this system is dependent on de novo U7
snRNP assembly from exogenously added U7 RNA. We show that both the
histone hairpin and SLBP1 have important functions in processing. SLBP1
is a mitotic phosphoprotein, phosphorylated in vitro by the
mitotic kinase cyclin B/cdc2; however SLBP processing function is not
dependent on phosphorylation status. Finally we show that SPH-1, the
Xenopus homologue of human p80-coilin, associates with
snRNAs with a functional Sm site but is not a hallmark of a functional
U7 snRNP.
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EXPERIMENTAL PROCEDURES |
Preparation of Plasmids and Nucleic Acids--
RNAs were
prepared in vitro by Sp6 or T7 RNA polymerase-mediated
transcription from linearized plasmids or partially single-stranded oligonucleotides and purified by denaturing polyacrylamide
electrophoresis. Plasmids encoding U7 RNAs (20) and the histone RNA
fragment derived from the H4-12 gene (36) were described earlier (12/12 RNA (25, 27)). Plasmid pT7-U1 encoding Xenopus U1 RNA was obtained from I. Mattaj (37) and was cleaved with BamHI
prior to RNA synthesis with T7 RNA polymerase. The short histone
hairpin RNA wtHPs RNA (5'-GGACAAAAGGCCCUUUUCAGGGCCACC) was
transcribed from oligonucleotides (note that these RNA molecules are
lacking six nucleotides at the 5'-end present in wtHP RNA used
previously (13, 24)). For the synthesis of uniformly labeled RNA
[
-32P]UTP and for the synthesis of capped RNA
m7GpppG (Roche Molecular Biochemicals) was included
in the transcription reaction. RNA concentrations were determined
either by measuring adsorption at 260 nm (assuming 1 A260 = 40 µg of RNA) or by determining the
amount of [-32P]UTP incorporated into the RNA. RNA was
stored in H2O.
Proteins--
Recombinant cyclin B was as described (38). The
Xenopus SLBP1 cDNA was described (12) and protein was
synthesized in vitro in wheat germ extract (Promega) from a
plasmid kindly provided by Dr. William F. Marzluff. Recombinant
His-tagged human HBP (hHBP) and SLBP1 were produced in SF21
cells using a modified version of the Bac-to-Bac baculovirus expression
system (Life Technologies) for protein expression and purified by
Ni-affinity column chromatography using Ni-NTA resin (Qiagen). Protein
concentrations were determined by the Bradford method (39), using
bovine serum albumin as a standard. Cyclin B/cdc2 kinase complex was
prepared as described (40) with an activity of 15 units/µl (35).
Antibodies--
Anti-Sm antibodies were monoclonal Y12
antibodies (41); control D5 antibodies raised against the yeast protein
Xrn1p were a gift of Dr. W.-D. Heyer. Monoclonal anti-SPH-1 antibody H1
(42) was obtained from J. Gall and concentrated ~5-fold by
ultrafiltration. Sheep anti-SLBP1 antiserum was raised against peptides
RPAPSRWSQGRK and KAVPRHLREPNVHPR of the Xenopus SLBP1. Serum
was concentrated by ultrafiltration to a concentration corresponding to
130 mg/ml IgG and was added to binding reactions at a final
concentration of 37 mg/ml. The antiserum, but not the preimmune serum,
recognizes complexes formed by in vitro translated SLBP1 and
32P-labeled wtHP RNA (not shown). For Western blot
analysis, antibodies were affinity-purified by binding to recombinant
SLBP1 transferred onto nitrocellulose and subsequent elution at low pH.
Binding Assays--
Unless indicated otherwise, 30-60 fmol of
32P-labeled RNA was incubated with extract or protein
(usually 50-60% of reaction volume) in 10 mM Tris-HCl (pH
7.5), 10% glycerol, 1 mM dithiothreitol, 1 mg/ml yeast
tRNA, and 1 unit/µl RNasin (Promega) in 8-20 µl for 20-30 min at
room temperature. Subsequently, the reaction products were analyzed
directly by electrophoretic mobility shift assay (EMSA) as
described (24). Products were visualized by autoradiography.
Histone Pre-mRNA 3'-End Processing Using Xenopus Egg
Extracts--
Unless indicated otherwise, 30 fmol of
32P-labeled or 200 fmol of non-labeled
m7GpppG-capped U7 RNA or a corresponding volume of
H2O were mixed with 6 µl of the indicated extract and
incubated for 30-40 min at room temperature. The mixture was then
adjusted with 20 mM EDTA, 0.3 mg/ml tRNA, and 1 unit/µl
RNasin (in 10 µl) and incubated for further 10 min at room
temperature. Then, unless indicated otherwise, 40 fmol of
m7GpppG-capped 32P-labeled wild-type histone
12/12 pre-mRNA fragments (25, 43) was added (except in the
reactions shown in Fig. 2B, where uncapped 12/12 RNA was
used), and the incubation was continued for 1.5-2 h at room
temperature. The reactions were stopped by addition of 1 volume of
0.5% SDS, 1 mg/ml proteinase K, 10 mM EDTA and incubated
for at least 20 min at room temperature. The samples were then
extracted with phenol/chloroform and concentrated by ethanol
precipitation. The reaction products were separated by 10%
polyacrylamide-7 M urea gel electrophoresis and visualized by autoradiography or by using a Molecular Dynamics PhosphorImager.
Histone Pre-mRNA 3'-End Processing Using K21 Mouse Cell
Nuclear Extracts--
Processing reactions with K21 nuclear extract
and histone RNA fragments were performed as described (44). Reactions
were stopped and analyzed as described above.
Xenopus laevis Egg Extracts--
Xenopus eggs were
lysed by low speed centrifugation and then fractionated by high speed
centrifugation into a cytosolic S200 fraction and a membrane fraction
as described (35). For histone RNA 3' processing and detection of U7
snRNP and Xenopus HBP, substrate RNAs were added to nuclear
reconstitution extract (NRE). Briefly, NRE was composed of 1 volume of
S200 fraction, 1/10 volume membrane fraction, an ATP-regenerating
system (20 mM phosphocreatine, 2 mM ATP, 0.5 mg/ml phosphocreatine kinase), and up to 1500 Xenopus sperm
chromatin per microliter (35). Where indicated, NRE was supplemented
with recombinant cyclin B to produce mitotic extract (ME), or with
EDTA, or microcystin (CalBiochem). Unless stated otherwise, these
mixtures were then incubated for 90-120 min prior to any subsequent
reactions. Alternatively, S200 fraction supplemented with
ATP-regenerating system was used instead of NRE.
Immunodepletion of Xenopus Egg Extracts and
Immunoprecipitations--
Preimmune serum antibodies and anti-SLBP1
antibodies were covalently linked to protein G-Sepharose beads
(Amersham Pharmacia Biotech) using dimethyl pimelimidate (45).
For immunodepletion, 45-50 µl of NRE were mixed with 100 µl of
protein G-Sepharose-coupled antibodies washed five times with 10 mM Hepes-KOH, pH 7.7, 50 mM KCl and mixed on a
wheel for 80-100 min at room temperature. The extract was then cleared
by two subsequent spins (15 s at 12,000 × g), and the
supernatants were used immediately or stored at 
70 °C. For
immunoprecipitations, protein G-Sepharose beads washed with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl (ST) were
incubated with monoclonal Y12 and H1 antibodies for 1 h at room
temperature. Subsequently, unbound antibody was removed by four washes
with ST. 30-90 fmol of 32P-labeled U7 or U1 RNA were
incubated in 30-100 µl of S200 fraction supplemented with an
ATP-regenerating system and 1 unit/µl RNasin (Promega) for 1 h
at room temperature. Subsequently, reaction mixtures were diluted by
addition of 1 volume of ST and then mixed with the antibodies coupled
to protein G-Sepharose. After 1 h of incubation at 4 °C, the
beads were concentrated by centrifugation and the supernatants were
removed. The beads were then washed three times with ST, and
subsequently, the amounts of 32P in pellet and bead
fractions were measured by Cerenkov counting. To recover the RNA, beads
were supplemented with 100 µl of 0.33 M sodium acetate,
50 mg/ml tRNA and 20 µl of 0.5% SDS, 1 mg/ml proteinase K, 10 mM EDTA. Similarly, the supernatants were supplemented with
10-20 µl of 0.5% SDS, 1 mg/ml proteinase K, 10 mM EDTA.
Incubations were done for at least 20 min at room temperature and were
followed by phenol/chloroform extraction and ethanol precipitation.
Protein Modification in Xenopus Egg Extracts--
For the
detection of SLBP1 modification, normally a 1/15 to 1/10 volume
in vitro transcription/translation mixture was added to the
egg extract. After incubations at room temperature, reaction mixtures
were supplemented with at least 2 volumes of SDS-polyacrylamide gel
electrophoresis (PAGE) loading buffer and analyzed by 12% SDS-PAGE.
Detection of protein was achieved using the PhosphorImager.
In Vitro Phosphorylation--
Reactions were done in 50 mM Tris-HCl (pH 7.5), 10% glycerol, 10 mM
MgCl2, 10 mM dithiothreitol, 10 µM ATP 6 nM [32P]ATP (3000 Ci/mmol), 0.14 mg/ml SLBP1 (or histone H1), and 0.5 µl of cyclin
B/cdc2 kinase in a volume of 5 µl. Incubations were done at
30 °C.
| |
RESULTS |
U7 Content of Xenopus Egg Extracts--
Xenopus egg
extracts are rich in components used for the first rapid cell divisions
during embryonic development. Fractionation of Xenopus eggs
produces a membrane fraction and a cytosolic S200 fraction (35). These
can be mixed to form a nuclear reconstitution extract (NRE), which, in
the presence of DNA or chromatin, is able to assemble nuclei that
undergo semiconservative DNA replication in vitro (35).
First, we tested whether endogenous U7 snRNPs, which are functional in
oocytes (20), are able to process synthetic histone mRNA. Because
we did not detect any processing (data not shown and see Figs. 4-6
below), we wondered whether this was due to the absence of the U7 snRNA
from the Xenopus egg extract preparation used. We therefore
prepared RNA from oocytes and from various stages of the
Xenopus egg extract preparation and estimated the amount of
U7 RNA present by detecting U7 RNA by primer extension from U7
RNA-specific primers. The efficiency of primer extension was then
compared with primer extension in control reactions with known amounts
of synthetic Xenopus U7 RNA. This comparison indicated that
the amounts of U7 RNA in oocytes, eggs, and in NRE were very similar,
corresponding to the proportion of primer extended with 0.3, 0.2, and
0.3 pmol of synthetic U7 RNA, respectively (data not shown). U7 was
present in both the cytosolic S200 and in the membrane fraction used
for reconstitution at a ratio of ~2:1 (data not shown). No
significant loss of U7 RNA occurred at the different stages of the
preparation. This suggests that the absence of processing in our
reactions was not caused by the absence of U7 snRNP. Attempts to
further investigate this lack of processing activity, which included
testing whether the Xenopus U7 RNA 5'-end was accessible for
hybridization to oligonucleotides, were not conclusive. We therefore
decided to test whether addition of synthetic U7 snRNA would restore
processing activity in these extracts.
Assembly of U7 snRNPs with Synthetic U7 RNA in Egg
Extracts--
To test whether these extracts contain the factors
required for assembly of U7 snRNP, we added three different labeled
synthetic U7 RNAs into NRE. The three RNAs vary in the Sm site required for the binding of the snRNP Sm core proteins (illustrated in Fig.
1A). The complexes resulting
from this incubation were analyzed by EMSA. Incubation of wild-type
mouse U7 RNA in NRE led to the formation of a well-defined complex with
low mobility (Fig. 2, lane 6,
indicated snRNP). A distinct complex was formed with U7 Sm OPT RNA,
which contains the Sm site of U2 snRNA (46). In contrast, the addition
of U7 Sm MUT RNA, a molecule where the Sm site was abolished by
mutagenesis, did not form a distinct low mobility complex (lane
4) (indicating that the presence of a Sm sequence was essential
for the formation of the slowly migrating complex). Complex formation
with wild-type U7 RNA was sensitive to the presence of excess unlabeled
wild-type U7 RNA competitor but not U7 Sm MUT competitor (lanes
7-10), indicating that complex formation was sequence-specific.
Additional retardation of the complex with wild-type U7 RNA was
observed in incubations containing the Y12 monoclonal antibody specific
for Sm proteins (41) (lane 11) but not with an irrelevant
control antibody (lane 12). Taken together, we conclude that
incubation of U7 RNA in Xenopus egg extract leads to the
assembly of a U7 snRNP particle.
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Fig. 1.
Histone RNA processing substrate and U7 RNA
sequence. A, synthetic mouse U7 RNA sequence was
derived from the mouse U7 RNA (9). Variations in the Sm site are
indicated, and the sequence complementary to histone RNA is
underlined (20, 46). In U7 Sm OPT RNA, the Sm site is
changed to the Sm site of U2, and in U7 Sm MUT, this site is destroyed
(20, 46). B, the histone RNA fragment is derived from the
mouse histone H4-12/12 gene (12/12 RNA) (25, 43, 62). The additional
non-histone 5' sequences is 5'-GAAUACACGGAAUUCGAGCU. The
arrows mark the major and minor cleavage sites in the
histone RNA fragment, and the histone RNA spacer element complementary
to U7 RNA is underlined.
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Fig. 2.
Assembly of U7 snRNP in Xenopus
egg extracts. NRE (lanes 1-12), ME (lanes
13-15), or S200 fraction (lanes 16-18) were prepared
as described under "Experimental Procedures." Binding assays were
performed with 4 µl of the indicated extract and
32P-labeled wild-type U7 RNA (w), U7 Sm MUT RNA
(m), or U7 Sm OPT RNA (o) in 8 µl for 1 h
as described under "Experimental Procedures." U7 RNAs alone are
shown in lanes 1-3. For competition experiments, either 450 fmol or 2.2 pmol of U7 RNA (U7, lanes 7 and
8) or U7 Sm MUT RNA (U7 Sm MUT, lanes
9 and 10) were mixed with the 32P-labeled
U7 RNA before addition of NRE. 7.5 µg of Y12 anti-Sm antibodies
(anti-Sm Ab; lane 11) or 7.5 µg of D5 control
antibody (control Ab; lane 12) were included in
the reaction mixture before addition of proteins. Products were
analyzed by EMSA and visualized by autoradiography as described under
"Experimental Procedures." snRNP marks the position of
ribonucleoprotein particles formed with U7 and U7 Sm OPT RNAs.
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Reassembled nuclei formed in Xenopus egg extract have been
shown to contain coiled body-like structures (34). These structures contain small nuclear RNAs (snoRNAs), spliceosomal snRNAs, and U7 snRNA and have been proposed to be sites of assembly of functional processing complexes (34, 47). Many subnuclear bodies undergo significant alterations as cells enter mitosis, and the timing and
mechanism of relocalization following the re-establishment of the
nucleus in interphase are increasingly studied (48). We wished to
determine whether postmitotic nuclear reassembly is a prerequisite for
U7 snRNP assembly. In our first approach, U7 RNA was incubated with the
S200 fraction only, thus omitting the membrane fraction and chromatin
from the mixture. Under these conditions, events such as nuclear
envelope assembly and DNA synthesis cannot take place (35). U7 snRNPs
were formed with the same sequence specificity in the cytosolic S200
fraction as in NRE (lanes 16-18). This indicates that
nuclear reassembly is not required for U7 snRNP assembly. In our second
approach we examined U7 snRNP assembly in mitotic extract (ME)
generated by addition of recombinant indestructible cyclin B into NRE.
This leads to chromosome condensation, depolymerization of the nuclear
lamina, and dispersal of the nuclear envelope (35). Addition of
synthetic U7 RNAs into ME still led to snRNP assembly (lanes
13-15), indicating that mitotic extracts do not bring about
changes in U7 snRNP assembly that can be detected in our assay. Taken
together, these results indicate that U7 snRNP assembly is not
dependent on the prior assembly of a nucleus and appears unaffected by
the cell cycle status in this system.
Assembled U7 snRNPs Are Functional--
To test whether the
assembled U7 snRNPs were functional in histone RNA 3' processing, we
included the short 86-nucleotide synthetic 12/12 RNA fragment derived
from the mouse histone H4-12 gene (36) in the NRE. This RNA molecule
encompasses all the sequence elements required for 3' processing
(illustrated in Fig. 1B). In previous experiments, it was
processed in mouse nuclear extract (25, 27, 43) and upon injection into
Xenopus oocytes (20). Inclusion of 32P-labeled
synthetic mouse U7 RNA together with 32P-labeled 12/12 RNA
into the reaction led to the formation of a faster migrating RNA
species (Fig. 3A, lane
3). These molecules comigrated with the 5' product of a histone
processing reaction in K21 cell nuclear extract (compare lanes
1 and 3). They were absent in a reaction containing
only 32P-labeled U7 RNA (lane 6) but were formed
in a reaction containing 32P-labeled histone RNA and
non-labeled U7 RNA (lane 7), indicating that they derived
from the histone RNA fragment. Furthermore, these products were not
formed when wild-type U7 RNA was replaced by U7 Sm MUT RNA or by U7 Sm
OPT RNA (lanes 4 and 5) (20). All these
observations indicate that this RNA species is the product of a
bona fide histone RNA 3' processing reaction.
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Fig. 3.
U7 RNA-dependent histone RNA 3'
processing in Xenopus egg extracts. A,
processing reactions contained 6 µl of NRE aliquots and either
32P-labeled wild-type U7 RNA (32P-U7;
lanes 3 and 6), mutant U7 RNAs
(32P-U7 Sm OPT, lane 4;
32P-U7 Sm MUT, lane 5), or
non-radiolabeled wild-type U7 RNA (U7, lane 7)
and non-capped 32P-labeled 12/12 RNA (lanes 3,
4, 5, and 7). Processing was done, and
the products were analyzed and visualized by autoradiography as
described under "Experimental Procedures." Products of histone RNA
processing in K21 nuclear extract (nxt, lane 1)
are shown for comparison. 5' product marks the position of
the major 5' processing product. B, 32P-labeled
U7 wild-type or U7 Sm MUT RNAs were mixed with either 6 µl of NRE
(lanes 3 and 4) or 6 µl of S200 fraction
(lanes 5 and 6) prepared as described under
"Experimental Procedures." In this case, the
32P-labeled 12/12 RNA was m7GpppG-capped at the
5'-end. Marker M is pBR322 DNA cleaved with HpaII
and 32P end-labeled.
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Because U7 snRNPs were also formed in incubations with the S200
fraction only (Fig. 2, lane 18), we wished to establish
whether the S200 fraction alone would support histone RNA 3'
processing. Fig. 3B demonstrates the appearance of a similar
product under these conditions as in a processing reaction with NRE
(compare lanes 3 and 5), indicating that this
fraction contains all the factors necessary for processing.
As described above, the presence of U7 RNA in NRE led us to expect that
endogenous U7 snRNPs would participate in processing as occurs with
nuclear extracts of somatic cells. We wished to exclude the possibility
that processing observed following the addition of exogenous U7 RNA was
not the consequence of spurious activation of the endogenous molecules
and to confirm that it was the consequence of de novo
assembly of functional U7 snRNPs. To do this, we utilized the
observation that histone pre-mRNA processing is dependent on base
pairing between U7 RNA and the spacer element of histone pre-mRNA
(17, 49). We utilized all combinations of wild-type and mutant histone
RNAs with wild-type and mutant U7 RNAs for processing reactions. In the
mutant histone 12/st3.5 RNA, the sequence 10-14 nucleotides away from
the 3'-end was changed from CACUU to GGGAA, thus changing a 5-base
region complementary to mouse U7 RNA (43) (Fig.
4A). To restore histone RNA-U7
RNA base pairing potential in this region, U7 RNA was changed at the
5'-end to generate the U7sup3.5 mutant RNA. In incubations with 12/12
RNA, only reactions containing wild-type U7, but not the mutant
U7sup3.5 RNA, led to the formation of detectable amounts of processing
products (Fig. 4B, compare lanes 3 and
4). On the other hand, in incubations with mutant 12/st3.5
histone RNA, only reactions with mutant, but not wild-type U7 RNA,
produced detectable amounts of product (lanes 7 and
6, respectively). These results clearly show that newly
assembled U7 snRNPs are directly participating in the processing
reaction.
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Fig. 4.
Complementarity between U7 RNA and histone
RNA is required for processing in Xenopus egg
extracts. A, alignment of wild-type U7 and 12/12 RNA
(top) and of histone RNA mutated in the spacer region
(12/st3.5 RNA) with U7 RNA mutated to restore complementarity
(U7sup3.5; bottom) (43). The sequences changes introduced
into the mutated histone and U7 RNA are boxed. B,
85 µl of NRE was incubated as described under "Experimental
Procedures" for 145 min. Then, 6-µl aliquots were supplemented with
32P-labeled U7 RNA (U7), mutant U7 RNA
(U7sup3.5), or H2O. After 50 min of incubation,
processing reactions with 32P-labeled histone RNA fragments
were started and incubated as described under "Experimental
Procedures" for 100 min with wild-type U7 RNA (lanes 3 and
6) or for 180 min for reactions with U7sup3.5 RNA
(lanes 4 and 7). Reactions containing histone
RNAs or U7 RNAs only (lanes 2 and 5 or
lanes 8 and 9) were incubated for 180 min. Reaction products were analyzed and visualized by autoradiography
as described under "Experimental Procedures."
Marker M (lanes 1 and 10) was as in
Fig. 3.
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We have also tested other histone RNA substrates such as a H4-wt RNA
fragment (11) and a fragment from the 3'-end of the mouse H2A.614 gene
(50) used for in vitro processing in nuclear extracts
prepared from mammalian cells. Although we were able to observe
processing, background cleavage in the absence of U7 RNA was for
unknown reasons higher with these RNAs, making an interpretation more
difficult. We therefore decided to use the 12/12 RNA substrate for our
RNA 3' processing reactions. We also noticed that nuclease activities
in the egg extracts varied from batch to batch; however, the use of
good quality eggs and quick processing of the extract kept these
activities low in general.
Efficient Histone RNA 3' Processing Is Dependent on the Hairpin
Structure and Hairpin-binding Proteins--
To determine whether the
conserved histone hairpin element is important for histone RNA 3'
processing in Xenopus egg extracts, we compared processing
of wild-type 12/12 RNA to processing of B/12 RNA carrying a mutant
hairpin element (Fig. 5A). As
in previous experiments (25), both RNA molecules were processed in K21
mouse cell nuclear extract, but processing with the mutant
RNA was significantly less efficient (Fig. 5B, compare
lanes 2 and 4; 18% of RNA processed instead of
34%). In fact, in four experiments processing efficiency of B/12 RNA
in K21 nuclear extract was at 45-59% of processing of 12/12 RNA,
lower than reported earlier (25), reflecting probably batch-to-batch
variations of nuclear extract preparations and emphasizing the
importance of the hairpin element for processing. In NRE, product
formation with the B/12 substrate was about three times lower then
product formation with the 12/12 substrate (lanes 6 and
8, 3.8% of RNA processed instead of 10.9%). This indicates
that in NRE, similar to nuclear extract, the hairpin RNA sequence is
important, but not essential for processing histone RNA.
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Fig. 5.
Histone RNA 3' processing in
Xenopus egg extracts is stimulated by the presence of
a wild-type histone hairpin RNA sequence and by hairpin-binding
proteins. A, wild-type (12/12) and mutant (B/12)
hairpin RNA sequences inserted between A-28 and A-45 of the histone RNA
shown in Fig. 1B. B, 12 µl of NRE was
supplemented with either 1.2 µl of H2O or 1.2 µl of 200 nM U7 RNA and incubated at room temperature for 2 h.
Subsequently, 5-µl aliquots of each mixture were used in processing
reactions with either 32P-labeled 12/12 RNA (lanes
7 and 8) or 32P-labeled B/12 RNA
(lanes 5 and 6). The reactions were performed and
analyzed as described under "Experimental Procedures" and
visualized using a Molecular Dynamics PhosphorImager. Processing
reactions with K21 nuclear extract (lane 1-4) were done as
described under "Experimental Procedures." C, NRE was
treated with either preimmune serum or anti-SLBP1 serum coupled to
protein G-Sepharose as described. Aliquots (3 µl) of NRE (lane
2) NRE treated with preimmune serum (mock-depleted NRE, lane
3) or NRE treated with the antiserum ( SLBP1 NRE, lane
4) were mixed with 32P-labeled wtHPs RNA as described,
and complexes were analyzed by EMSA. D, for processing
reactions, aliquots (6 µl) of NRE or of SLBP1-depleted NRE were
supplemented with U7 RNA and for reactions shown in lanes 3 and 4, with 1 µl of SLBP1 or hHBP (0.2 mg/ml). EDTA, tRNA,
and finally 32P-labeled 12/12 histone RNA were added
subsequently as described. After 3-h incubation, the reactions were
stopped and the products analyzed as described above.
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To determine whether the hairpin is a binding site for factors, we
performed binding experiments with various short hairpin RNAs and
detected a factor binding, specifically, the wild-type histone hairpin
RNA (Fig. 5C) but not the mutant hairpin (data not shown).
To determine whether this factor was the Xenopus SLBP1 (12),
we raised antibodies against this protein and used the antibodies to
deplete NRE of SLBP1. Fig. 5C shows that the formation of a
major RNA-protein complex detected in NRE and also in mock-depleted NRE
was prevented when SLBP1 was depleted from the extract (lanes 2, 3, and 4, respectively). This indicates
that this complex was formed by SLBP1 and that extract could be
effectively depleted of SLBP1. The SLBP1-depleted extract was then used
for processing reactions. SLBP1 depletion essentially abolished
processing (Fig. 5D, compare lanes 1 and
2), and this was rescued by the addition of either
recombinant Xenopus SLBP1 (lane 3) or recombinant
human HBP (lane 4), at least in part. These results strongly
suggest that removal of SLBP results in the loss of an essential
function in the processing reaction and rescue by recombinant protein
indicates that depletion of SLBP1 does not remove any other essential
factor required for processing. We thus conclude that SLBP1
participates in histone mRNA 3'-end formation and is a true
homologue of hHBP.
SLBP1 Modification and Histone RNA Processing in Mitotic
Extracts--
During mitosis, the nucleus and many of the
subcompartments contained within, including the nucleolus and coiled
bodies, undergo disassembly or dramatic rearrangement (51). In
addition, many fundamental cellular processes are inhibited in M phase.
Entry into mitosis is driven by the highly conserved mitotic protein kinase cyclin B/cdc2, and subsequently, the cyclin subunit undergoes proteolytic degradation for the cell to exit M phase. In Chinese hamster ovary cells that have been synchronized by mitotic shake-off, histone mRNA levels in G1 are dramatically reduced
compared with cells in S phase, and this change is due in part to
significant up-regulation of processing at the G1/S
boundary (52). At the end of S phase, the decrease in histone RNA
levels is principally due to decreased stability of the mRNA,
raising the question of when in the cell cycle the processing machinery
is down-regulated and whether processing components per se
undergo cell cycle-regulated control.
Xenopus egg extracts retain the core cell cycle components
necessary to recapitulate in vitro a simplified cell cycle
that operates in vivo during the early embryonic cell
cycles, oscillating between S phase and mitosis (35). This system is
thus biochemically tractable and amenable for determining whether
specific cellular elements are subjected to cell
cycle-dependent translational or post-translational
control. Even cell cycle-regulated events such as mitotic repression of
RNA polymerase III transcription or replication checkpoint control,
which do not ordinarily operate in early embryonic cell cycles, can be
reconstituted in appropriate circumstances (38, 51). Because depletion
of SLBP1 from extracts abolished processing (Fig. 5D), we
wished to determine whether SLBP1 might be a target for mitotic
regulation. As for the experiment described in Fig. 2, lanes
13-15, we prepared ME by inclusion of indestructible cyclin B
into the extract. First, we established whether SLBP1 was affected by
this treatment. The comparison of SLBP1 in NRE and ME by Western
blotting revealed that the mobility of SLBP1 was reduced in ME (Fig.
6A). To further investigate
this change in Xenopus SLBP1 mobility, we prepared
35S-labeled Xenopus SLBP1 in vitro
and added this protein into NRE. 35S-SLBP1 migrated as a
single polypeptide during SDS-PAGE (Fig. 6B,
lane 1), and incubation in NRE did not affect this mobility (lanes 2 and 5). However, in ME as well as in
extract supplemented with the protein phosphatase inhibitor
microcystin, the protein quantitatively underwent band shifts
characteristic of phosphorylation (lanes 3 and
6, and lane 4, respectively). This change in
mobility suggests that SLBP1 may be phosphorylated in mitotic extracts, and may also be a target for phosphorylation in NRE by a kinase other
then cyclin B/cdc2. This latter phosphorylation, however, appears
normally to be subject to immediate dephosphorylation by endogenous
protein phosphatase(s).
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Fig. 6.
SLBP1 modification and histone RNA 3'
processing in ME. A, SLBP1 in NRE or ME was detected by
SDS-PAGE followed by Western blotting with anti-SLBP1 antibodies.
B, 35S-labeled Xenopus SLBP1
(lane 1) was added to 15 µl of NRE (lane 2), 15 µl of ME (lane 3), or 15 µl of NRE supplemented with 1 µM microcystin (lane 4) and incubated for
2 h before analysis by 12% SDS-PAGE. Lanes
5-9, 35S-labeled Xenopus HBP was
incubated in 10 µl of NRE (lane 5) or in 45 µl of ME
(lanes 6-9) for 2 h. Subsequently, the reaction in NRE
and a 10-µl aliquot of the reaction with ME were stopped (lane
6), while three other 10 µl-aliquots of the incubation with ME
were supplemented with either 5 mM EDTA (lane
7); 5 mM EDTA and PP2A (lane 8); or 5 mM EDTA, PP2A, and 10 µM microcystin
(lane 9). The incubation of these reactions was continued
for 90 min before analysis by 12% SDS-PAGE. C, 3.5 µl of
histone H1 (0.2 mg/ml) (lanes 1 and 2) or
recombinant SLBP1 (0.2 mg/ml) (lanes 3 and 4)
were incubated in the presence or absence of 0.5 µl of cyclin B/cdc2
kinase for 90 min at 30 °C in 5 µl as described under
"Experimental Procedures." Reactions were stopped by addition of
loading buffer and products were separated by 12% SDS-PAGE and
visualized by autoradiography. D, processing reactions
contained 6 µl of NRE (lanes 1 and 2) or 6 µl
of ME (lanes 3 and 4), wild-type U7 RNA
(U7, lanes 1 and 3), and
32P-labeled 12/12 RNA. Reactions and product analysis were
carried out as described in Fig. 3. Marker M is pBR322 DNA
cleaved with HpaII and 32P end-labeled.
E, time course of processing in the absence or presence of
cyclin B. 100-µl processing reactions were prepared as described
either with or without cyclin B. Processing was started by the addition
of 12/12 RNA, and 15-µl aliquots of the reactions were stopped as
described at the indicated times. Subsequent analysis was as described,
using a PhosphorImager to determine processing efficiency.
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To confirm that the bandshift in ME was due to phosphorylation, labeled
protein was incubated for 2 h in ME. Subsequently, the reaction
mixture was split into three aliquots that were treated with either
EDTA (acting as kinase inhibitor, thus allowing endogenous phosphatases
1 and 2A to dephosphorylate proteins); EDTA and additional exogenous
protein phosphatase 2A (PP2A); or with EDTA, PP2A, and the protein
phosphatase inhibitor microcystin (lanes 7-9) for 90 min.
Incubations in EDTA (lane 7) partially reversed the
bandshift induced in mitotic extract and the presence of additional
PP2A (lane 8) reduced most of SLBP1 to a mobility
corresponding to that observed in NRE. These results indicate that most
of the cyclin B/cdc2-induced modification was caused by
threonine/serine phosphorylation. Finally, addition of microcystin
inhibited PP2A action (lane 9), confirming that the
difference of mobility observed between lanes 7 and
8 was caused by dephosphorylation by PP2A. This indicates
that SLBP1 is a mitotic phosphoprotein. SLBP1 has two consensus
cdk phosphorylation sites (Thr-169 and Thr-228) as well as a
non-consensus site (Thr-60), and it is possible that in the extract,
SLBP1 is a direct target of this kinase. To test this in
vitro, recombinant SLBP1 was incubated with recombinant cyclin
B/cdc2 kinase in the presence of [
-32P]ATP (Fig.
6C). Transfer of the radiolabeled phosphate group by the
kinase was then detected by gel electrophoresis followed by
autoradiography. This experiment demonstrates that SLBP1 is a substrate
for recombinant cyclin B/cdc2 kinase and may well be directly
phosphorylated by this kinase in mitosis.
We tested different SLBP1 functions for possible effects of
phosphorylation. Phosphorylation in mitotic egg extract or by cyclin
B/cdc2 kinase does not abolish hairpin RNA binding (data not shown). We
then wished to establish whether SLBP1 phosphorylation affects histone
mRNA 3' processing. We tested processing in ME, and, as illustrated
in Fig. 6D, this did not prevent 3'-end formation (compare
lanes 1 and 3) and there was no significant
difference between time courses of processing in ME versus
NRE (Fig. 6E). In summary, in these and other experiments,
we were not able to detect any significant difference in processing
between mitotic and S phase extracts.
Interaction of the Xenopus p80-coilin Homologue SPH-1 with U7
snRNA--
In Xenopus oocytes, U7 snRNA was found to be
localized in coiled bodies, which are also referred to as C snurposomes
or Cajal bodies in the oocyte nucleus (32, 53, 54). In oocytes, U7 snRNP can associate with Xenopus SPH1 protein, the homologue
of human p80-coilin characteristic for coiled bodies (42, 55). To
determine whether this association is a hallmark of a U7 snRNP functional in histone RNA 3'-end processing, we tested whether SPH-1
was interacting with U7 RNA in the S200 fraction, our minimal system
allowing for U7 snRNP assembly (Fig. 2) and histone mRNA 3'
processing (Fig. 3). Radiolabeled U7 RNA was incubated in the S200
fraction and then mixed with either the monoclonal anti-Sm antibody Y12
or the monoclonal antibody H1 recognizing SPH-1 (42) bound to protein
G-Sepharose beads. The fraction of U7 snRNA associated with the beads
after several washes was then determined. Table I summarizes the results of six
independent experiments. Precipitations with Y12 and H1 antibodies with
U7 Sm MUT RNA (1.6-6.4%) were close to background levels determined
with U7 and U1 RNAs (only 1.7-5.3%). This indicates that no specific
interaction between either Sm proteins or SPH-1 and U7 Sm MUT was
detected. On the other hand, both U7 RNA and U7 Sm OPT RNAs were
enriched in precipitations with Y12 antibodies as well as with H1
antibodies. In these reactions, precipitations with U7 Sm OPT RNA were
slightly more efficient than precipitations with U7 RNA, and, for each
of these RNAs, precipitations with Y12 antibodies were more efficient
than precipitations with H1 antibodies. Similar results were obtained
in additional experiments where the integrity of the RNA isolated from
supernatants and pellet fractions was confirmed by denaturing gel
electrophoresis (data not shown). We conclude from these experiments
that the Sm sequence is important for the interaction with Sm proteins and SPH-1.
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Table I
Association of SPH-1, the Xenopus p80-coilin homologue, with snRNAs in
Xenopus egg extract
The indicated RNAs were incubated in S200 fraction and subsequently
precipitated with either anti-Sm (Y12) or anti-SPH-1 (H1) antibodies as
described under "Experimental Procedures."
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An interesting further question is then whether the association of
SPH-1 is specific for U7 RNA. To test this we produced synthetic U1 RNA
and added this RNA into Xenopus egg extracts. A second set
of experiments was then performed with Y12 and H1 antibodies as above.
The data summarized in Table I demonstrate that, similar to U7 RNA, U1
RNA is precipitated by Y12 and H1 antibodies, indicating that it
becomes associated with Sm proteins and also with SPH-1. These results
indicate that, in these extracts, SPH-1 does not serve as a hallmark
for a functional U7 snRNP but appears to associate with RNA-protein
complexes containing Sm proteins.
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DISCUSSION |
In this paper we report the characterization of a novel in
vitro system for the analysis of histone mRNA 3' processing.
We have utilized extracts derived from Xenopus eggs as a
source of trans-acting factors required for the processing
reaction. Four distinct lines of evidence presented in Figs. 3 and 4
indicate that the observed modification of the model substrate reflects a bona fide processing reaction: First, the processed
reaction product comigrated with the product obtained from previously
characterized K21 cell nuclear extract (25, 43). Second, processing was dependent on the addition of U7 RNA. Third, processing was only supported by U7 RNA containing a wild-type Sm site required for snRNP
assembly but not by two U7 RNAs with mutations in this sequence. And
finally, processing, which was inhibited by the introduction of
mutations into the histone RNA, could be restored by the introduction of complementary mutations within the U7 RNA, indicating that the newly
assembled snRNPs participate directly in the processing reaction. We
conclude that xenopus egg extracts support bona
fide U7-dependent snRNP assembly and histone 3' processing.
Previous work involving microinjection in Xenopus oocytes
indicated that oocytes support the efficient processing of histone mRNA in a reaction dependent either on endogenous U7 RNA or, after its destruction, on the injection of an appropriate synthetic U7 RNA
(20, 31). Such processing presumably reflects the requirement of the
oocyte to stockpile histone mRNA and protein needed to provide for
the histones necessary for the condensation of the exponentially
increasing amount of chromatin synthesized in the early preblastula
cell cycles, during which all transcription is inhibited (56-58). The
detection of processed products in extracts derived from
metaphase-arrested eggs strongly suggests that the protein components
involved in histone 3' processing are retained following oocyte
maturation and fertilization. They may be stored as a relatively
abundant maternal pool to be used at the onset of zygotic histone gene
expression, and their assembly is not dependent on the prior assembly
of an interphase nucleus.
In contrast to the retention of functional protein components, we were
surprised to find that reconstitution of processing in egg extracts
required the addition of U7 RNA. Xenopus U7 RNA is clearly
functional in oocytes that support processing of sea urchin,
Xenopus, and mammalian histone pre-mRNA, including the 12/12 RNA used here, in the absence of any exogenous U7 RNA (20, 31,
59). In addition, U7 RNA has been shown, by hybridization with labeled
complementary probes, to be enriched in coiled bodies in the oocyte
nucleus as well as in similar structures in nuclei reconstituted in
cell free extracts (32, 34). The function(s) of these subnuclear
structures, which have been proposed to disassemble in M phase and
reassemble in interphase, remain(s) elusive, and suggestions range from
storage sites to essential processing locations for elements involved
in RNA processing (discussed in Ref. 34). Our observation that
processing occurs in both interphase S200 and mitotic extract, both
unable to form reconstituted nuclei (for different reasons), indicates
that nuclear reassembly is not a prerequisite for the assembly of
functional processing complexes. This is in sharp contrast to the
assembly of functional DNA replication complexes in the extract, which
are absolutely dependent on nuclear formation (35). In addition, our
results suggest that the in vitro reassembly of coiled
bodies is not an essential step for the formation of functional
processing complexes.
We found endogenous U7 levels in extracts to be very similar to those
observed in oocytes, excluding the possibility that U7 RNA is
specifically lost during oocyte maturation. The inability of endogenous
U7 to support a significant degree of mRNA processing in this study
may reflect a functional inactivation of U7 RNA during early
development. Interestingly, both sense and antisense probes to U7 RNA
have been reported to localize to the same coiled body-like structures
in nuclear reconstitution extracts but not in oocytes (34), raising the
intriguing possibility that, during Xenopus oocyte
maturation, U7 snRNPs may be sequestered by a complementary nucleic
acid. Alternatively, U7 RNA may be limiting in G2-arrested oocytes, and the disassembly of the oocyte germinal vesicle, which occurs on maturation (35), may result in the dilution of essential factors throughout the cytoplasm such that in vitro
processing cannot be detected by current assays unless exogenous U7 is
added. Whatever the reason for the lack of detectable processing by the endogenous U7 RNA, Xenopus egg extracts have provided us
with the first cell-free system allowing the direct analysis of the interactions between the two RNAs involved in the processing reaction. Processing in this system was in general less efficient than that observed in somatic cell nuclear extracts, although it is clear that
products accumulated linearly as a function of time (Fig. 6E). The difference in efficiency is likely due to the fact
that, in the system described here, total cytoplasm, and not simply nuclear extract, is the starting source for the protein factors involved.
In addition to NRE composed of membrane and cytosolic fractions, U7
snRNPs were also formed in incubations with only the cytosolic S200
fraction (Fig. 2, lane 18). Thus, we wished to establish whether the S200 fraction alone would support histone RNA 3'
processing. Fig. 3B demonstrates the appearance of a similar
product under these conditions as in a processing reaction with NRE
(compare lanes 3 and 5), indicating that this
fraction contains all the factors necessary for processing.
A system allowing the direct analysis of the interactions between the
two RNAs involved in the processing reaction is of particular interest.
Despite reports of the recovery of polypeptides specifically associated
with U7 RNAs (19), their molecular identity remains unknown, and to
date a system for the recovery and molecular characterization of
functional U7 snRNPs has been lacking. Interestingly, snRNP assembly
with U7 snRNA is not sufficient for processing, as is demonstrated by
the example of U7 Sm OPT RNA, which has an Sm site similar to U2 snRNA
and assembles into a non-functional snRNP (Figs. 2 and 3). This is in
agreement with earlier observations, where U7 Sm OPT was found to
associate with Sm proteins in Xenopus oocytes but was not
able to process histone RNA, and probably reflects a requirement for
further U7-specific proteins (20).
When we were testing different substrates for processing, we found that
processing efficiency decreased when we exchanged the hairpin in the
12/12 histone RNA with a hairpin that was not bound by hairpin-binding
proteins (Fig. 5 and data not shown). This was not surprising, because
similar observations were made with this substrate in other cell free
systems (25). Xenopus SLBP1 is the major activity
binding to hairpin RNA in these extracts. Immunoprecipitation of SLBP1,
however, reduced RNA processing activity to background levels. This was
reversed by the re-addition of either SLBP1 or human HBP. Thus the
presence of a mutant hairpin, which cannot bind SLBP1, results in
suboptimal processing, whereas removal of SLBP1 makes processing
undetectable. Taken together, these results suggest that SLBP1-depleted
extracts lack an essential function in processing that is independent
of SLBP1 hairpin binding function. Recovery of processing was achieved
by the re-addition of either SLBP1 or human HBP. This strongly suggests
that no other essential protein was removed in the immunoprecipitation.
The reason for only partial reversal is unknown. Protein is in excess over histone RNA in these reactions, but it is possible that
recombinant protein lacks full functionality. Alternatively, it is
possible that we have removed a stimulatory factor, and the analysis of the immunoprecipitate will be of great interest. A role for SLBP1 in
histone RNA processing was also observed by Marzluff and coworkers in
Xenopus oocytes (28). In some extract preparations, traces of a second hairpin-RNA binding activity, probably the
Xenopus SLBP2 (28) involved in silencing of maternal histone
mRNA in oocytes, were observed.
Addition of non-destructible cyclin B converts an interphase extract
(NRE) into a mitotic extract (ME). Interestingly, SLBP1 was
phosphorylated in ME and also directly by cyclin B/cdc2 protein kinase
(Fig. 6). SLBP1 has three potential cdk phosphorylation sites,
Thr-60, Thr-169, and Thr-228. Preliminary results indicate that, in ME
and in incubations with cyclin B/cdc2 kinase, Thr-60 is the major
phosphorylation site.2 This
modification is clearly of interest. However, we did not detect any
differences between histone RNA processing in NRE and ME, indicating
that the SLBP1 modification had no effect on this reaction. Other
possible functions, which will be addressed in future experiments, are
for example in regulating translation or protein stability.
As mentioned above, the protein composition of the U7 snRNP is not well
characterized. As a first step toward the molecular characterization of
the U7 snRNP, we investigated the significance of the association of
SPH-1 protein with U7 snRNPs in our system. In Xenopus
oocytes, wild-type U7 RNA was found to be enriched in coiled bodies and
associated with SPH-1 protein, the Xenopus homologue of
p80-coilin associated with coiled bodies (32, 53, 55). In addition,
injected U7 RNA has been shown to induce the formation of coiled
body-like structures in oocytes (60), and endogenous U7 RNA was
enriched in structures similar to coiled bodies in nuclei reconstituted
in cell free extracts (34). These latter coiled body-like structures
also contained spliceosomal snRNAs and snoRNAs (34). The S200 fraction
contains all the protein components for histone mRNA 3' processing
but, in the absence of chromatin and membrane fraction, does not form
nuclear structures (35). We used immunoprecipitation with H1 and, as a
control, Y12 antibodies to test for interactions of SPH-1 and Sm
proteins with U RNAs upon incubation in the S200 fraction. Although the fraction of RNA precipitated with, e.g. U7 Sm
OPT did vary between experiments, probably reflecting a batch-to-batch variation between extracts, the results obtained were consistent. They
confirmed the interaction between Sm proteins and U7 and U7 Sm OPT
(Fig. 2 and Ref. 20) and between U1 RNA and Sm proteins. In addition,
we also detected interactions between U1, U7, and U7 Sm OPT and SPH-1.
The data in Table I suggest that these interactions are weaker than
with Sm proteins. This may be due to a less efficient precipitation,
caused either by poorer assembly or poorer interaction between antibody
and epitope in the immunoprecipitation. Our preferred interpretation of
this observation is, however, that in egg extract, SPH-1 associates
with U-RNA·Sm protein complexes but not with RNA directly. Thus
immunoprecipitation would depend on Sm protein assembly and the
interaction between SPH-1 and U RNA Sm protein complex. Both of these
interactions may be inefficient in our system, thus leading to the poor
but reproducible precipitation of U1, U7 Sm OPT, and U7 RNAs with H1
antibodies (Table I). This indicates that association of the
Xenopus p80-coilin homologue with U7 snRNP is not a hallmark
of a functional snRNP.
Gall and coworkers (32, 55) made apparently contradictory observations
when the interaction between SPH-1 and U7 RNA was tested upon injection
of U7 RNA into oocytes RNA. In these experiments, an interaction was
observed between SPH-1 and U7 RNA or a U7 mutant similar to U7 Sm OPT
but not with U1 RNA. It is likely that this is related to the different
localization of spliceosomal U RNAs (as illustrated at the
example of U2 RNA) and U7 RNA within subnuclear structures in oocytes
and the colocalization of SPH-1 and U7 RNA (32, 47, 55). However, the
same authors reported that in Xenopus egg extracts,
endogenous spliceosomal snRNAs, snoRNAs, U7 RNA, and SPH-1 colocalize
to the same structures, also referred to as coiled bodies (34). SPH-1
is essential for this localization of Sm proteins, and therefore
presumably also for the localization of spliceosomal RNA and U7 RNA
(61). Although no quantitative interaction between SPH-1 and endogenous
spliceosomal snRNAs or U7 RNA was detected in these extracts (61), this
suggests that the Xenopus coilin homologue and Sm snRNPs
must somehow interact for a correct localization. The results of our
experiments with synthetic RNAs and S200 fraction (where nuclear
assembly does not take place), indicate that a direct interaction
between U RNAs and SPH-1 that is dependent on the association of Sm
proteins with U RNA is possible. This interaction may be instrumental
for the localization observed.
In conclusion, we have identified a system that allows histone RNA
processing in a reaction dependent on the addition of U7 snRNA. Our
observations indicate that the requirements for other factors are
similar to processing in Xenopus oocytes or nuclear extract,
the other commonly used in vitro systems to study histone RNA processing. We have also more closely investigated the association of one particular protein, the coilin homologue SPH-1, with U7 snRNPs
and found that SPH1 associates with both functional and non-functional snRNPs.
§¶,
TOP
ABSTRACT
INTRODUCTION
