Previous Article | Next Article 
Molecular and Cellular Biology, March 2001, p. 2008-2017, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2008-2017.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutations in the RNA Binding Domain of Stem-Loop Binding Protein
Define Separable Requirements for RNA Binding and for Histone
Pre-mRNA Processing
Zbigniew
Dominski,
Judith A.
Erkmann,
John A.
Greenland, and
William F.
Marzluff*
Department of Biochemistry and Biophysics,
Program in Molecular Biology and Biotechnology, University of North
Carolina, Chapel Hill, North Carolina 27599
Received 18 September 2000/Returned for modification 13 November
2000/Accepted 13 December 2000
 |
ABSTRACT |
Expression of replication-dependent histone genes at the
posttranscriptional level is controlled by stem-loop binding
protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3' untranslated region of histone pre-mRNAs and facilitate 3' end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial loss of SLBP binding activity. We
also identify residues in the RBD which do not contribute to binding to
the stem-loop RNA but instead are required for efficient recruitment of
U7 snRNP to histone pre-mRNA. Recruitment of the U7 snRNP to the
pre-mRNA also depends on the 20-amino-acid region located immediately
downstream of the RBD. A critical region of the RBD contains the
sequence YDRY. The tyrosines are required for RNA binding, and the DR
dipeptide is essential for processing but not for RNA binding. It is
likely that the RBD of SLBP interacts directly with both the stem-loop
RNA and other processing factor(s), most likely the U7 snRNP, to
facilitate histone pre-mRNA processing.
| |
INTRODUCTION |
Replication-dependent histone genes
comprise a unique group of genes whose expression is coordinately
regulated with DNA synthesis (15). Expression of these
genes peaks during S phase of the cell cycle and rapidly declines upon
completion of DNA replication at the end of S phase (10,
30). In contrast to all other mRNAs, replication-dependent
histone mRNAs are not polyadenylated and instead terminate with a
highly conserved stem-loop structure (15). The stem-loop
structure, consisting of six base pairs and a four nucleotide loop,
associates with a protein termed the stem-loop binding protein (SLBP)
or the hairpin binding protein (14, 29). Mammalian SLBP is
a 30-kDa protein containing 270 amino acids and can be divided into
three domains: the centrally located RNA binding domain (RBD) and the
flanking N-terminal and C-terminal domains. The RBD of SLBP does not
resemble any previously identified motifs involved in RNA recognition
(29).
Replication-dependent histone mRNAs are formed from longer pre-mRNA
transcripts by an endonucleolytic cleavage (3, 8). The
processing reaction depends on two sequence elements in the histone
pre-mRNA: the stem-loop structure (27) and a purine-rich element, termed the histone downstream element (HDE), located 10 to 15 nucleotides further downstream (2). The stem-loop is
recognized by SLBP, whereas the HDE associates with the U7 snRNP,
containing the 60-nucleotide U7 snRNA and associated proteins (20, 22). Binding of U7 snRNP to the pre-mRNA occurs via
base pairing between the HDE and the 5' end of U7 snRNA (2,
18) and is likely also strengthened by interactions between a
U7-specific protein(s) and the SLBP-stem-loop (SLBP/SL) complex
(5). This additional interaction is especially important
in processing of histone pre-mRNAs containing HDEs which can form only
a weak duplex with the U7 snRNA and thus are unable to efficiently
recruit U7 snRNP to the pre-mRNA (5, 17, 23). Stable
binding of SLBP and U7 snRNP to their respective targets in the
pre-mRNA leads to a subsequent association of additional
trans-acting factors, including a poorly characterized
heat-labile factor (9), followed by cleavage of the
pre-mRNA four to five nucleotides downstream from the stem-loop. After
3' end processing, SLBP remains associated with the terminal stem-loop
and assists the mature histone mRNA to the cytoplasm, where it likely
plays an important role in histone mRNA translation and stability
(7, 25, 31). SLBP is cell cycle regulated and therefore
may be a key factor responsible for cell cycle regulation of histone
mRNA levels (30).
Two proteins that bind the stem-loop structure at the 3' end of histone
mRNA have been isolated from Xenopus laevis oocytes (28). One protein, referred to as xSLBP1, is homologous to
mammalian SLBPs and is also involved in 3' end processing of histone
pre-mRNAs. A second SLBP found in Xenopus oocytes,
designated xSLBP2, does not participate in 3' end processing and
probably functions in storage of histone mRNA during oogenesis
(28). xSLBP2 is similar to xSLBP1 only in the central RBD.
Replacement of the RBD of xSLBP1 with the corresponding domain of
xSLBP2 resulted in a chimeric protein that retained high affinity for
the stem-loop. Surprisingly, this chimeric protein was inactive in
processing although it contains the C-terminal region from xSLBP1
required for processing (11). Thus, binding to the
stem-loop of histone pre-mRNA is not the only function of the RBD in 3'
end processing. Here we identify residues in the RBD of human SLBP that
are not required for binding to the RNA target and instead play another
role in processing. Our experiments indicate that these residues
together with the 20-amino-acid region in the C terminus are each
important for efficient recruitment of the U7 snRNP to the histone
pre-mRNA. In addition, by aligning RBD sequences of various SLBPs, we
identified absolutely conserved amino acids and demonstrated that these
residues are required for binding to the stem-loop RNA. Changes in some of these residues results in a reduction in affinity for the stem-loop, accompanied by a decrease in processing activity, demonstrating the
importance of high-affinity binding of SLBP to the stem-loop for
efficient 3' end processing.
| |
MATERIALS AND METHODS |
Construction of the mutant and chimeric SLBP clones.
Substitutions of the amino acid residues within the RBD of SLBP were
carried out in the pGEM3 vector containing the human SLBP cDNA spanning
from the initiation codon to the MscI site in the 3'
untranslated region, 57 nucleotides downstream from the stop codon.
Substitution of these residues was facilitated by the presence of the
following unique restriction sites located within the cDNA region
encoding the RBD: NgoMIV, MfeI, BsmI,
SalI, and BamHI. The appropriate restriction
fragments in the wild-type SLBP cDNA were replaced by double-stranded
oligonucleotides terminating with compatible sticky ends and containing
the desired mutations. The following combinations of restriction
enzymes were used during mutagenesis: NgoMIV and
MfeI (to insert mutations located between amino acids 1 and
21), MfeI and BsmI (to insert mutations located between amino acids 22 and 39), BsmI and SalI (to
insert mutations located between amino acids 40 and 52), and
SalI and BamHI (to insert mutations located
between amino acids 53 and 72). To facilitate identification of the
desired clones, where possible, oligonucleotides were designed so that
one of the two restriction sites was disrupted upon ligation of the
insert into the pGEM3 vector, without changing the amino acid
sequence of the RBD. The double mutants QPF, RHLF, QPQVA, and
RHLQVA were constructed by recombining appropriate fragments containing
the individual mutations. Details of each construct and sequences of
the oligonucleotides are available on request. For in vitro protein
synthesis, the SLBP cDNAs containing appropriate mutations were
subcloned into the pSP64T vector. This vector was modified from the
pSP64 Poly(A) vector (Promega) by introducing the 5' and 3'
untranslated regions from the rabbit 
-globin gene and a fragment
encoding the poly(A) tail (12). The mutant SLBP cDNAs were
inserted into the pSP64T vector downstream from the 5' untranslated
region using NcoI and XbaI restriction sites. For
expression of the mutant forms of SLBP in Sf9 insect cells using the
Bac-TO-Bac expression system, mutant cDNAs were cloned into
one of the pFastBac vectors (Gibco-BRL).
Synthesis of SLBP by in vitro translation.
The in vitro
coupled transcription and translation reaction was carried out in the
rabbit reticulocyte extract using a Promega TnT kit according to the
manufacturer's protocol. Each reaction (total volume of 25 µl)
contained 12.5 µl of the extract, 5 U of SP6 RNA polymerase
(Promega), and 1.0 µg of the DNA template encoding either the
wild-type or mutant version of the human SLBP subcloned into the pSP64T
vector. To determine the amount of SLBP made during the TnT reactions,
the samples were supplemented with [35S]methionine (NEN)
and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.
Expression and purification of SLBP using the baculovirus
system.
The wild-type and mutant forms of SLBP were expressed in
Sf9 insect cells using the Bac-TO-Bac baculovirus
expression system (Gibco-BRL) as recommended by the manufacturer. The
His-tagged proteins were purified by affinity chromatography on
Ni-nitrilotriacetic acid agarose (Qiagen).
Mobility shift assay.
The ability to bind the histone
stem-loop RNA by the mutant SLBP proteins synthesized either in vitro
using the TnT kit or in vivo using the baculovirus system was
determined by band shift assay as previously described (5,
11). Routinely, 2.5 µl of the TnT reaction or 0.1 µg of the
protein expressed in the baculovirus-infected cells was mixed on ice in
a total volume of 10 µl with 100 fmol of the 5'-labeled stem-loop
RNA, 20 mM EDTA, and 2.5 µl of buffer D used for dialysis of the
nuclear extract (20 mM HEPES-KOH [pH 7.9], 100 mM KCl, 0.5 mM
dithiothreitol, 0.2 mM EDTA [pH 8.0], 20% glycerol). The samples
were immediately resolved on a 7.5% native polyacrylamide gel (37.5 parts of acrylamide to 1 part of bisacrylamide) containing
Tris-borate-EDTA buffer. The gel was dried, and radioactive bands were
detected by autoradiography and/or by PhosphorImager.
Preparation of RNA.
In vitro 3' end processing was carried
out using 86-nucleotide pre-mRNAs containing the stem-loop structure
and the U7 binding site from either the H2a-614 or H1t pre-mRNA. A
30-nucleotide RNA containing the wild-type stem-loop structure and the
5-nucleotide flanks was used in the band shift assay. Sequences of all
three RNA species and methods for their synthesis and labeling were described previously (5). The U7 snRNA was detected by
hybridization with the anti-U7 RNA probe, synthesized, and labeled as
described elsewhere (5).
Preparation of nuclear extract and 3' end processing.
Preparation of the nuclear extract from mouse myeloma cells,
immunodepletion of the SLBP, and complementation of the depleted extract with the recombinant SLBP were performed according to protocols
described previously (4, 5, 16). Each processing reaction
contained 5 µl of the nuclear extract corresponding to approximately
50 µg of total protein, 180 fmol of the pre-mRNA substrate labeled at
the 5' end with [
-32P]ATP, and 20 mM EDTA. Samples
were incubated at 32°C for 1 h and processed as previously
described (5).
Western blots.
Nuclear protein (50 µg) was resolved on an
SDS-12% polyacrylamide gel and transferred to a nitrocellulose
membrane. SLBP was detected with an antibody raised against the
C-terminal 13 amino acids of the protein using an enhanced
chemiluminescence system.
Immunoprecipitation of processing complexes.
Processing
complexes containing the U7 snRNP were formed and subsequently
precipitated by anti-SLBP essentially as described elsewhere
(5). Each reaction contained 50 ng of unlabeled pre-mRNA substrate, 50 µl of the nuclear extract, and 20 mM EDTA (pH 8) in a
total volume of 100 µl and was supplemented with 0.01 to 0.1 ng of
the substrate labeled at the 5' end with
[-32PO4]ATP (500 to 5,000 cpm
respectively). The radiolabeled pre-mRNA was used to monitor the
overall efficiency of immunoprecipitation of the pre-mRNA substrate by
the SLBP antibody. In some experiments, the SLBP-depleted nuclear
extract supplemented with different recombinant SLBPs was used instead
of the undepleted nuclear extract. In all cases, the reaction samples
were prepared on ice followed by a short (3- to 5-min) incubation at
22°C to allow formation of the processing complexes. The
affinity-purified SLBP antibody (10 µl at 1.0 µg/µl) was
subsequently added to each reaction, and the samples were rotated at
4°C for 1.5 h and then transferred to a new tube containing 15 µl
of protein A-agarose beads (Gibco-BRL). Prior to use, the protein
A-agarose beads were incubated for 2 h in an extract from sea
urchin blastula nuclei (containing a U7 snRNA which does not
cross-react with the anti-mouse U7 snRNA probe), to reduce nonspecific
binding of components of the mouse nuclear extract. Subsequent steps in
the immunoprecipitation procedure and Northern blot analysis of U7
snRNA were performed as previously described (5).
| |
RESULTS |
Amino acids conserved in the RBDs of all SLBPs are involved in RNA
recognition.
In Xenopus oocytes there are two different
SLBPs, xSLBP1 and xSLBP2, which bind the stem-loop structure at the 3'
end of replication-dependent histone mRNAs with the same affinity
(28). xSLBP1 is a homologue of human and mouse SLBPs and
is involved in processing of histone pre-mRNAs in the nucleus. Similar
to mammalian SLBPs, xSLBP1 is also found in the cytoplasm, where it may
function in regulating translation of histone mRNA. xSLBP2 is found
only in the cytoplasm and is inactive in 3' end processing of histone
pre-mRNA both in vivo and in vitro. Expression of xSLBP2 is restricted
to oogenesis and the protein is degraded during oocyte maturation,
indicating that xSLBP2 may function in translational repression of
histone mRNA (28). This protein shares amino acid
similarity with processing-specific SLBPs only within the central RBD.
Figure 1A presents a sequence alignment
of the 73-amino-acid RBD from human SLBP, both Xenopus
SLBPs, and Drosophila SLBP (24). In the four
SLBPs, 36 of the 73 amino acids of the RBD are identical and 13 other
amino acids are replaced by similar residues. The strong evolutionary
conservation of these amino acids suggested that they are all involved
in recognition of the common target for each protein; the stem-loop
structure at the 3' end of histone mRNAs. We tested this hypothesis by
substituting some of the conserved amino acids with either similar,
neutral, or biochemically different residues (Fig. 1B) and by
determining the relative binding affinity of mutant proteins in the
band shift assay. All mutant proteins were expressed in the presence of
[35S]methionine using the TnT system (Promega) and
resolved by SDS-polyacrylamide gel electrophoresis (Fig.
2, top row of each panel). The gels were
used for autoradiography and PhosphorImager analysis, allowing precise
monitoring of both the quality of SLBP synthesized in vitro (e.g.,
presence of possible prematurely terminated or improperly initiated
products) and the quantity of the full-length protein subsequently used
for the band shift assay. All mutant SLBPs were generated in vitro at
relatively low concentrations and are likely to be properly folded in
the reticulocyte extract. This approach ensured that any changes in the
ability to shift the RNA probe likely resulted from differences in the
binding affinity between the tested mutant proteins. All amino acids
subjected to mutagenesis, with the exception of the tryptophan at
position 56, which is replaced by phenylalanine in Drosophila
melanogaster SLBP, are absolutely conserved in the four SLBPs
shown in Fig. 1A.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 1.
Conserved amino acids within the RBD of the SLBP. (A)
Schematic of the vertebrate SLBPs, with the position of the RBD
indicated. The RBDs of the human, two X. laevis, and
Drosophila (Fly) SLBPs were aligned. The amino acids
conserved in all four proteins are highlighted and the residues
subjected to mutagenesis are underlined. N-ter and C-ter, N and C
termini. (B) Amino acid substitutions made within the RBD of the human
SLBP and their relative effect on binding to the RNA stem-loop
structure. The binding affinity of each mutant SLBP is reported as a
percentage of wild-type (WT) binding affinity (100%). The values
represent the range of affinities obtained from at least two
independent experiments.
|
|
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 2.
The conserved amino acids in the RBD are required for
efficient binding of the protein to the stem-loop structure. Wild-type
(WT) and mutant forms of the human SLBP containing various amino acid
substitutions of the conserved residues, as indicated above each lane
and listed in Fig. 1, were synthesized in vitro using the rabbit
reticulocyte TnT system (Promega) and analyzed by electrophoresis in
SDS-10% polyacrylamide gels (top). Each mutant protein was
subsequently tested for binding efficiency in the band shift assay
using the 30-nucleotide stem-loop RNA labeled at the 5' end as a probe
(bottom). Lane 1 of panels A and C represents a negative control in
which the TnT reaction was carried out in the absence of exogenous
DNA.
|
|
The wild-type human SLBP expressed in vitro using the TnT system
efficiently binds to the stem-loop RNA probe. As shown in
Fig.
2, 10%
of the entire TnT reaction consistently shifted between
75 and 90% of
the RNA probe. The complex formed by the SLBP expressed
in the
reticulocyte lysate has a mobility identical to that of
the complex
formed in nuclear extracts (
29) or with baculovirus
protein (not shown). As determined by the same assay, replacement
of
the two conserved arginines at positions 10 and 11 of the RBD
with
lysines (RR/KK mutant) completely abolished binding to the
stem-loop
RNA probe (SL probe) (Fig.
2A, lane 3). No band corresponding
to the
SLBP/SL complex was detected even after a long exposure
of the film
(not shown). Arginines 10 and 11 are adjacent to a
conserved QKQ
tripeptide at positions 12 to 14 of the RBD. Substitution
of this
tripeptide with alanines resulted in a protein (QKQ mutant)
that was
unable to bind the stem-loop RNA (Fig.
2A, lane 4). Lysine
19 is
absolutely conserved in all known SLBPs, including those
of sea urchin,
Caenorhabditis elegans (
13), and
Chlamydomonas (unpublished results). Interestingly, changing
this amino acid
to arginine did not reduce binding to the stem-loop RNA
(K19R
mutant) (Fig.
2A, lane 5; Fig.
2B, lane 3). However, replacing
lysine 19 with a neutral residue, alanine, resulted in a protein
severely impaired in stem-loop RNA binding (Fig.
2B, lane
2).
In addition to changing the basic residues and the QKQ tripeptide, we
mutated two sets of conserved aromatic residues, the
two tryptophans at
positions 56 and 63 and the two tyrosines at
positions 24 and 27. As
determined by the band shift assay, replacement
of the two
tryptophans with isoleucines produced an SLBP mutant
unable to bind
the stem-loop structure (WW/II mutant) (Fig.
2A,
lane 7). A very
conservative mutation replacing the two tyrosines
at position 24 and 27 with phenylalanines (YY/FF mutant) resulted
in a protein that retains
only 5 to 10% of the wild-type binding
affinity (Fig.
2A, lane 6; Fig.
2C, lane 3). Replacement of the
same tyrosines with either serines
(YY/SS mutant) or threonines
(YY/TT mutant), other amino acids
containing an OH group, abolished
binding (Fig.
2C, lane 4 or 5, respectively). We next made two
single mutations by individually
substituting each tyrosine with
phenylalanine. The tyrosine at position
24 is more important for
RNA recognition, since replacement of this
residue with phenylalanine
(Y24F mutant) resulted in a more severe
decrease in binding than
the same replacement of the tyrosine at
position 27 (Y27F mutant).
The Y24F and Y27F SLBPs retained less than
10 and approximately
50%, respectively, of the wild-type binding
efficiency (Fig.
2C,
lanes 6 and 7). Binding of SLBP to the stem-loop
RNA was also
dramatically reduced by replacing histidine at position 41 with
phenylalanine (H41F mutant) (Fig.
2C, lane 8). The effect of this
mutation on binding was similar to that of the Y24F
mutation.
Complementation of the SLBP-depleted nuclear extract with mutant
SLBPs.
We and others have previously shown that high-affinity
binding of SLBP to the stem-loop structure in histone pre-mRNA is
critically important for efficient 3' end processing of histone
pre-mRNA (5, 17, 19, 27). Mutations of the stem-loop
structure that reduced SLBP binding more than 10-fold resulted in a
complete inhibition of processing. Other changes in the stem-loop
reducing affinity to SLBP 5- to 10-fold had a less drastic effect on
the efficiency of 3' end processing (5, 19). Here we
address whether processing efficiency is affected in the same fashion by changes in SLBP that decrease its affinity for histone pre-mRNA. Using the baculovirus expression system, we expressed three mutant SLBPs that had reduced binding activity and evaluated their ability to
rescue processing activity of the SLBP-depleted nuclear extract. As
determined by Western blotting, immunodepletion of the nuclear extract
with the SLBP antibody resulted in complete removal of the protein
(Fig. 3A, lane 2). We used a histone
pre-mRNA containing the HDE from the H1t gene, since processing of this
pre-mRNA is completely dependent on SLBP (5). Incubation
of H1t pre-mRNA in the nuclear extract resulted in processing of 75%
of the input pre-mRNA, and removal of SLBP from the extract abolished
processing (Fig. 3B, top, lanes 1 and 2, respectively). Addition of 100 ng of recombinant wild-type SLBP restored processing of the H1t
substrate to the initial level (Fig. 3B, top, lane 3). A similar
increase in processing efficiency was achieved upon addition of the
Y27F mutant SLBP, which binds the stem-loop about 50% as efficiently as the wild-type protein (Fig. 3B, top, lane 5). Addition of the same
amount of the weakly binding Y24F mutant protein did not stimulate
processing, and addition of the H41F mutant protein, which has similar
stem-loop binding activity, resulted in accumulation of only a trace
amount of the cleavage product (Fig. 3B, top, lanes 4 and 6, respectively). These results are consistent with our earlier studies in
which weakening the interaction between SLBP and the RNA by mutations
in the stem-loop structure resulted in a decrease in processing
efficiency both in vivo (19) and in vitro
(5). The fact that the Y27F mutant SLBP does not
significantly affect the processing efficiency of SLBP in spite of a
twofold reduction in RNA binding suggests that there is a threshold
level of affinity above which processing occurs with maximal
efficiency.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 3.
High-affinity stem-loop binding is required for
efficient processing of histone H1t pre-mRNA but not H2a pre-mRNA. (A)
The nuclear extract (NE) was immunodepleted of SLBP using the SLBP
antibody, and the efficiency of depletion was determined by Western
blotting carried out with the same antibody (lane 2). Lane 1 represents
an undepleted control nuclear extract. The asterisk indicates a protein
that cross-reacts with the SLBP antibody when denatured but not in the
nuclear extract and serves as a control for specificity of depletion.
The position of a 52.5-kDa marker is indicated. (B) Ability of
wild-type (WT) and mutant forms of SLBP with reduced binding activity
to support processing of the H1t (top) and H2a (bottom) pre-mRNAs. The
indicated mutant proteins were expressed in the baculovirus system and
used to complement the SLBP-deleted nuclear extract. The processing
activity of the nuclear extract before and after depletion is shown in
lanes 1 and 2, respectively. The uncut band corresponds to the input
histone pre-mRNA, and the cut band corresponds to the mature product of
3' end processing.
|
|
We tested the same proteins for their ability to restore processing of
the H2a-614 pre-mRNA, which contains an HDE capable
of forming a
relatively strong duplex (12 base pairs interrupted
by only one
mismatch) with the 5' end of U7 snRNA. Depending on
the batch of
nuclear extract used, this processing substrate is
cleaved in vitro
with 10 to 20% efficiency even in the absence
of SLBP (Fig.
3B,
bottom, lane 2; references
4 and
29). The
basal level of processing was elevated to virtually 100% by addition
of either the wild-type SLBP or the Y27F mutant protein (Fig.
3B,
bottom, lanes 3 and 5, respectively). The Y24F mutant protein
did not
stimulate processing of the H2a pre-mRNA substrate (Fig.
3B, lane 4).
Surprisingly, the H41F SLBP increased processing
of the H2a-614
pre-mRNA as much as fivefold (Fig.
3B, bottom,
lane 6), although it has
similar affinity for the stem-loop as
Y24F SLBP. It is likely that the
region of SLBP around tyrosine
24 may be involved in both RNA binding
and other interactions
essential for processing (see
below).
Residues in the RBD essential for processing.
We have
previously shown that replacement of the RBD from xSLBP2 in xSLBP1
resulted in a protein, 1-2-1, that binds the stem-loop with the same
affinity as xSLBP1 but is inactive in processing, both in frog oocytes
and in vitro (11). Thus, the RBD must have other functions
in histone pre-mRNA processing in addition to binding the pre-mRNA.
Here we define residues in the RBD that are important for efficient
histone pre-mRNA processing in vitro. The RBDs of SLBP from more
distantly related metazoans, C. elegans, and D. melanogaster, also do not substitute for the mammalian RBD in
histone pre-mRNA processing (unpublished results). This is likely due
to incompatibility of the vertebrate and invertebrate processing
machineries as has been shown for Xenopus and sea urchins (6). Thus, the SLBP sequences from C. elegans
and D. melanogaster were not informative in identification
of amino acids required for processing.
There are 15 identical and 2 similar amino acids (highlighted in Fig.
4A) in the human SLBPs and xSLBP1 which
are different
in xSLBP2. Of the 15 identical residues, 10 are clustered
between
amino acids 22 and 38 of the RBD, while 3 of the remaining 5 are
in the C-terminal portion of the RBD. The absence of all or some
of
these amino acids in the chimeric protein 1-2-1 must be responsible
for
its inability to function in 3' end processing in spite of
efficient
binding to the histone pre-mRNA. To determine which
of these 17 amino
acids are critical for 3' end processing, we
systematically mutated
most of them either individually or in
small groups in the human SLBP
and tested the ability of the mutant
proteins to bind RNA and to
restore processing to an SLBP-depleted
extract. We replaced the amino
acids in human SLBP with the amino
acids found in the same position in
xSLBP2, since these residues
are likely to be neutral for RNA binding
(Fig.
4). In the first
round of this scanning mutagenesis, we altered
nine amino acids
near the center of the RBD (9aaR mutation). In
addition, we replaced
the phenylalanine at position 48 with serine
(F48S mutation) and
the QVA in the C-terminal part of the RBD with MRD
(QVA mutation).
As determined by band shift assay, all of the mutant
proteins
expressed in baculovirus-infected insect cells efficiently
bound
to the stem-loop RNA (Fig.
5A).
Subsequently, the same preparation
of each mutant SLBP was tested for
the ability to restore in vitro
processing activity to the
SLBP-depleted nuclear extract using
the H1t histone pre-mRNA substrate.
In each experiment, approximately
100 ng of the baculovirus-expressed
SLBP was added to the depleted
extract. This amount of protein is
sufficient to bind the vast
majority of the input substrate (not
shown). The 9aaR mutant protein
was completely inactive in processing
of the H1t histone pre-mRNA
(Fig.
5B, lane 4), demonstrating that the
14-residue region from
amino acids 25 to 38 of the human RBD is
critical for processing.
The F48S and QVA mutant proteins complemented
the depleted extract
as efficiently as the wild-type SLBP (Fig.
5B,
lanes 5 and 6,
respectively). We next prepared a second set of
mutations by altering
a small number of the amino acids within the
14-amino-acid cluster.
The DR dipeptide at positions 25 and 26 that is
flanked by the
two tyrosines critical for RNA recognition was replaced
with QC
found in xSLBP2 (DR/QC mutation). This substitution reduced the
processing activity on the H1t substrate by more than 95% (Fig.
5C,
lane 3), although it did not affect the ability of the SLBP
to form a
stable complex with the stem-loop (Fig.
5A, lane7).
There was
approximately a 50% reduction in processing activity
as a result of
the IK/LQ mutation, replacing the IK dipeptide
(amino acids 28 and 29)
with LQ found in xSLBP2 (Fig.
5C, lane
4). The DRIK mutant protein in
which all four amino acids were
mutated had even less processing
activity than the DR mutant,
consistent with an additive effect of the
DR and IK mutations
(Fig.
5C, lane 5). Both IK/LQ (not shown) and DRIK
(Fig.
5A, lane
8) efficiently bound the stem-loop RNA in the band shift
assay.
Partial or complete alteration of the RHLQP region revealed that
these five remaining amino acids of the original 9aaR mutation
do not
play a major role in the processing activity of human SLBP.
The RHL and
QP/KS mutant proteins (Fig.
5C, lanes 8 and 9, respectively)
as well as
the RHLQP mutant protein (data not shown and Fig.
4B)
were as active in
processing as the wild-type SLBP.
View larger version (32K):
[in this window]
[in a new window]
|
FIG. 4.
Amino acids conserved in the RBD of human SLBP and
xSLBP1 but not in xSLBP2. (A) Alignment of the RBDs from the three
different SLBPs. Amino acids conserved or similar between the human
SLBP and the xSLBP1 that differ from the amino acids in xSLBP2 are
highlighted. The residues subjected to mutagenesis are underlined. (B)
Amino acid substitutions made within the RBD of human SLBP and their
effect on the activity of the protein in the 3' end processing of
histone H1t pre-mRNA. The processing efficiency of each mutant SLBP is
reported as a percentage of the processing efficiency of wild-type (WT)
SLBP (100%). The values represent the approximate efficiencies
obtained from at least two independent experiments. *, data not
shown.
|
|
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 5.
Substitution of nine amino acids within the RBD of human
SLBP abolishes processing of H1t pre-mRNA but not binding activity of
the protein. The RBD of human SLBP was mutated in three different
regions by replacing the amino acids (underlined in Fig. 4) with the
corresponding amino acids found in xSLBP2. (A) Ability of wild-type
(WT) and mutant (indicated above each lane) SLBPs to bind the RNA
stem-loop probe, determined by band shift assay. The probe is shown in
lanes 1 and 6. (B and C) Activity of the nuclear extract (NE) before
(panel B, lane 1) and after (panel B, lane 2; panel C, lanes 1 and 6)
depletion of SLBP in processing of the H1t pre-mRNA. The effect of
addition of 100 ng of the different SLBPs (indicated above each lane)
to the SLBP-depleted extract on 3' end processing is shown in panel B,
lanes 3 to 6, and panel C, lanes 2 to 5 and 7 to 9.
|
|
We combined mutations that alone resulted in no reduction in processing
activity (RHL, QP/KS, QVA, and F48S). The F48S mutation
in conjunction
with either the QP/KS or RHL mutation resulted
in about a 50%
reduction in processing activity (Fig.
4B). A similar
reduction of SLBP
processing activity was also observed when the
QP/KS or RHL mutation
was combined with the other neutral mutation,
QVA (Fig.
4B).
Activity of mutant SLBPs in processing is substrate dependent.
We have previously reported that there is a region of 20 amino acids in
human SLBP immediately downstream of the RBD that contains residues
required for efficient processing of the histone H1t pre-mRNA
(5). Replacing this region with an unrelated sequence of
20 amino acids found in the same place in xSLBP2 abolished the ability
of human SLBP to support in vitro processing (5). This
mutant was originally designated 20aa but here is referred to as 20aaC.
We have thus identified two relatively short regions of SLBP which are
absolutely required for its activity in processing of H1t pre-mRNA and
not for RNA binding: a cluster of amino acids in the amino-terminal
half of the RBD including the DR dipeptide, and the first 20 amino
acids of the C-terminal domain.
We tested the ability of the 9aaR and the 20aaC mutant proteins, which
were inactive in processing of the H1t pre-mRNA (Fig.
6, top), to process the H2a-614 pre-mRNA.
As discussed above,
the SLBP-depleted extract itself can cleave between
10 and 20%
of the H2a-614 pre-mRNA (Fig.
6, bottom, lane 2),
consistent with
only partial dependence of processing of this substrate
on SLBP
(
4,
5,
23,
29). Addition of the wild-type SLBP to
the
depleted extract elevated this basal level of processing to more
than 95% (Fig.
6, bottom, lane 3), restoring the initial processing
efficiency of the nuclear extract (Fig.
6, bottom, lane 1). In
the same
assay, the 9aaR (Fig.
6, bottom, lane 4) and 20aaC (Fig.
6, bottom,
lane 5) mutant proteins increased processing efficiency
to
approximately 90% and 80%, respectively. The 20aaC mutant protein
was
reproducibly less active than the 9aaR protein in complementing
the
SLBP-depleted nuclear extract. Under the conditions of these
assays,
virtually all of the substrate added to the nuclear extract
is stably
associated with the mutant proteins, as determined by
using either the
SLBP antibody or Ni-nitrilotriacetic acid agarose
beads to isolate the
SLBP-pre-mRNA complex (not shown).
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 6.
Activity of 9aaR and 20aaC mutant SLBPs in 3' end
processing is substrate dependent. Processing activity of the nuclear
extract (NE) using the H1t (top) and H2a-614 (bottom) histone pre-mRNAs
before and after depletion of SLBP (lanes 1 and 2, respectively) and
effect of addition of 100 ng of the different SLBPs (indicated
above each lane) to the SLBP-depleted extract on 3' end
processing (lanes 3 to 5). WT, wild type.
|
|
We also tested the effect of addition of excess 9aaR and 20aaC mutant
proteins to a standard nuclear extract on processing
of the histone H1t
pre-mRNA. The nuclear extract that had not
been depleted of SLBP
processed about 40% of the H1t pre-mRNA
(Fig.
7, lane 1). Supplementing this extract
with the exogenous
wild-type SLBP (100 ng) increased the efficiency of
processing
to over 60% (Fig.
7, lane 2), while addition of the same
amount
of the 9aaR protein resulted in reduction of processing
efficiency
to less than 10% (Fig.
7, lane 3). A consistently stronger
inhibition
was caused by addition of the 20aaC mutant protein (Fig.
7,
lane
4), which reduced processing efficiency to approximately 5%. This
dominant negative effect is similar to that seen when xSLBP2 or
1-2-1 is added to the processing extract (
11) and almost
certainly
results from interaction of the substrate with the excess
mutant
SLBP rather than with the endogenous SLBP in the extract.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 7.
Mutant proteins 9aaR and 20aaC inhibit H1t pre-mRNA
processing when added to a nuclear extract. Lane 1 shows the processing
activity of the nuclear extract (NE) using the H1t pre-mRNA as a
substrate; in lanes 2 to 4, 250 ng of each protein was added to the
nuclear extract, and processing of the H1t pre-mRNA was analyzed. WT,
wild type.
|
|
The processing-deficient mutant SLBPs inefficiently recruit U7
snRNP to the processing complexes.
Binding of U7 snRNP to the
substrate pre-mRNA occurs primarily by base pairing between the 5' end
of U7 snRNA and the HDE, located approximately 10 nucleotides
downstream from the cleavage site. A major, if not the only, function
of SLBP in 3' end processing is stabilization of the interaction
between U7 snRNP and the pre-mRNA substrate (5, 17). SLBP
bound to the stem-loop structure may interact with one of the protein
components of the U7 snRNP providing an additional support in anchoring
the particle to the pre-mRNA. The stabilizing role of SLBP is
critically important in processing of pre-mRNA substrates, including
the H1t pre-mRNA, which contain an HDE that can form relatively few
base pairs with U7 snRNA (5).
We have previously shown using anti-SLBP that less U7 snRNP
coimmunoprecipitates with H1t pre-mRNA than with H2a-614 pre-mRNA,
which contains a nearly perfect HDE (
5). To confirm that
this
difference did not result from variability in the efficiency of
immunoprecipitation of the pre-mRNA-SLBP complexes between samples,
we
carried out the following experiment. The same amounts (50
ng) of
either the H1t or the H2a-614 histone pre-mRNA were mixed
with an
undepleted nuclear extract and incubated for 5 min at
22°C, allowing
formation of the processing complexes, but not
cleavage of the
substrate to mature mRNA. A small amount of labeled
RNA substrate (100 pg) was added simultaneously with the unlabeled
substrate to monitor
the efficiency of immunoprecipitation (control
1 bands in Fig.
8A and
B). The reaction mixtures were
subsequently
incubated with the SLBP antibody at 4°C, and complexes
assembled
on the pre-mRNA were isolated on protein A-agarose beads. RNA
was prepared from the immunoprecipitates and then analyzed for
the
presence of U7 snRNA by Northern blotting (
5). During
recovery
of the RNA from agarose beads, a small amount of synthetic
unlabeled
U7 snRNA (containing additional nucleotides at the 5' and 3'
ends
and therefore migrating slower than the endogenous U7 snRNA) was
added to each sample (control 2 bands in Fig.
8A and B) to monitor
the
efficiency of ethanol precipitation and hybridization with
the anti-U7
probe. In the presence of the small amount of radioactive
pre-mRNA,
only a trace amount of U7 snRNA was recovered from the
agarose beads
(Fig.
8A, lane 1). In this reaction, nearly 100%
of the radioactive
pre-mRNA was immunoprecipitated, since SLBP
is in molar excess of the
pre-mRNA (control 1 band, Fig.
8A, lane
1). After addition of 50 ng of
unlabeled H1t or H2a-614 pre-RNA,
the efficiency of immunoprecipitation
of the radioactive pre-mRNA
was reduced to approximately 40% (Fig.
8A,
lane 2) or 30% (Fig.
8A, lane 3), respectively, due to limiting
amounts of the endogenous
SLBP. Significantly, in spite of similar
efficiency of pre-mRNA
immunoprecipitation, the H2a-614 pre-mRNA bound
four- to fivefold
more U7 snRNP than the H1t pre mRNA (Fig.
8A, lanes 2 and 3).
We conclude that in agreement with our previous results
(
5),
these differences are due to the decreased ability of
the U7 snRNP
to form a stable complex with the H1t pre-mRNA.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 8.
9aaR and 20aaC mutant SLBPs have significantly reduced
ability to recruit the U7 snRNP to the histone pre-mRNA. (A) A nuclear
extract was incubated in the absence of unlabeled substrate (lane 1) or
in the presence of 50 ng of either the H1t (lane 2) or H2a-614 (lane 3)
histone pre-mRNA. Anti-SLBP was added, and RNA prepared from the
immunoprecipitate was resolved on an 8% polyacrylamide-7M urea gel.
U7 snRNA was detected by Northern blotting using an antisense U7 snRNA
probe as described in Materials and Methods. Control 1 is the H2a-614
histone pre-mRNA radiolabeled at the 5' end, which was added to each
reaction (100 pg) to monitor the efficiency of immunoprecipitation of
the unlabeled substrate RNA. Control 2 (1 ng) represents a synthetic
unlabeled U7 snRNA which contains additional nucleotides at both 5' and
3' flanks and therefore migrates slower than the endogenous U7 snRNA.
This control RNA is added to each sample prior to ethanol precipitation
and allows monitoring of the efficiency of the total RNA recovery and
hybridization. (B) A nuclear extract (NE) was depleted of SLBP (lane 1)
and then supplemented with the indicated baculovirus-expressed proteins
(lanes 2 to 4); 50 ng of unlabeled H1t histone pre-mRNA was added to
each extract together with 10 pg of the labeled H1t substrate (control
1). Anti-SLBP was added, and the immunoprecipitates were processed for
detection of U7 snRNA as in panel A; 0.1 ng of control 2 RNA was used
as a control for precipitation of the RNA. WT, wild type. (C) Mobility
shift assay to determine the binding activity in the control extract
(lane 2), the SLBP-depleted nuclear extract (lane 3), and the
SLBP-depleted nuclear extract supplemented with the
baculovirus-expressed SLBPs (lanes 4 to 6), indicated above each lane.
Lane 1 contains the probe alone.
|
|
The most likely explanation for the inability of both the 9aaR and
20aaC mutant proteins to function in H1t pre-mRNA processing
was that
each mutation disrupts the interaction of SLBP with U7
snRNP and thus
causes inefficient recruitment of the U7 snRNP
to the pre-mRNA
substrate. We tested this possibility by directly
measuring the ability
of the 9aaR and 20aaC mutant proteins to
recruit U7 snRNP to processing
complexes assembled on the H1t
pre-mRNA (Fig.
8B). H2a pre-mRNA was not
used in this assay due
to the high level of SLBP-independent
recruitment of U7 snRNA
to this substrate. Approximately 50 ng of
unlabeled H1t pre-mRNA
was mixed with the SLBP-depleted nuclear extract
alone or supplemented
with either the wild-type or mutant SLBPs. The
amounts of both
control RNAs used in this experiment, i.e., control 1 RNA (10
pg of the H1t substrate labeled at the 5' end) and control 2 RNA
(0.1 ng of cold synthetic U7 snRNA), were reduced 10 times compared
to the previous experiment (Fig.
8A) to avoid possible interference
of
a strong radioactive signal generated by the control bands
with the
ability to detect the small amounts of U7 snRNA recruited
by the H1t
pre-mRNA. Incubation of the SLBP-depleted nuclear extract
containing
both labeled and unlabeled H1t pre-mRNA with the anti-SLBP
did not
result in immunoprecipitation of detectable amounts of
the pre-mRNA
(control 1) or the U7 snRNA (Fig.
8B, lane 1), demonstrating
that the
sample was free of SLBP. The essentially complete removal
of SLBP from
the nuclear extract was confirmed by band shift assay
(Fig.
8C, lane 3)
and Western blotting with the SLBP antibody
(Fig.
3A). The synthetic U7
snRNA (control 2) was detected in
this sample at the expected level,
indicating that any immunoprecipitated
RNA was quantitatively recovered
during ethanol precipitation
(Fig.
8B, lane 1). Addition of 1 µg of
the baculovirus-expressed
wild-type SLBP to the SLBP-depleted extract
restored binding activity
to the extract (Fig.
8C, lane 4) and allowed
immunoprecipitation
of a significant amount of processing complexes
containing U7
snRNP (Fig.
8B, lane 2). The same amount of the 9aaR
mutant protein,
although as efficient as the wild-type SLBP in
restoring binding
activity to the depleted extract (Fig.
8C, lane 5)
and in immunoprecipitating
the substrate RNA, recruited only about 40%
of the amount of U7
snRNA recruited by the wild-type protein (Fig.
8B,
lane 3). The
20aaC mutant SLBP was even more impaired in recruiting U7
snRNP
to the H1t pre-mRNA and allowed immunoprecipitation of 25% of
the control amount of U7 snRNP (Fig.
8B, lane 4). Again, addition
of
the 20aaC protein to the SLBP-depleted extract restored binding
activity to the regular level (Fig.
8C, lane 6) and resulted in
efficient precipitation of the H1t pre-mRNA (Fig.
8B, lane 4).
Thus,
reduction in the amount of U7 snRNP immunoprecipitated in
the presence
of the 20aaC protein did not result from lower affinity
of the mutant
SLBP for the substrate
RNA.
| |
DISCUSSION |
Human SLBP and one of the two SLBPs isolated from
Xenopus, xSLBP1, are closely related to each other and are
involved in 3' end processing of replication-dependent histone pre-mRNA
in the nucleus. The second form of SLBP in Xenopus, xSLBP2,
does not function in processing and is similar to the other SLBPs only within the RBD (28). Although it binds to the same RNA
target, the RBD of xSLBP2 cannot substitute for the RBD in the
processing-specific SLBPs (11).
Residues of the RBD required for RNA recognition.
A minimal
RBD required for efficient binding to the stem-loop structure was
mapped in human SLBP to a 73-amino-acid region in the center of the
protein, between amino acids 126 and 198 (29). There are
two regions in the RBD highly conserved among different SLBPs, one
located between amino acids 10 and 21 and the other located between
amino acids 41 and 54. In addition, the RBD contains a number of
conserved aromatic residues scattered throughout the domain,
reminiscent of the conserved aromatic residues present in the RNA
recognition motif (RRM) (26). Many of these residues in
the RRM make contacts with the single-stranded regions of the RNA and
may be involved in stacking interactions with the bases of the RNA
target. A number of the highly conserved residues in the SLBP RBD are
basic and are always either lysines or arginines. In several RNA
binding proteins, arginine-rich regions in particular are important in
binding and the arginines cannot be substituted with lysines
(1). Basic residues in RNA binding proteins are also
frequently involved in electrostatic interactions with the phosphate
groups of the recognized RNA sequences. Some of these residues can be
either lysine or arginine, but they cannot be changed to alanine
without a major reduction in binding affinity.
Since SLBP has a novel, previously unknown RBD, we tested whether the
same types of rules apply for binding of the stem-loop
RNA by SLBP.
Martin et al. showed that changing the two highly
conserved arginines
at position 10 and 11 of the RBD to alanines
resulted in complete loss
of binding activity (
13), suggesting
that the positive
charge at both positions was important. We demonstrated
here that
changing the same arginines to lysines (RR/KK mutant)
also abolished
binding activity of SLBP. The guanidinium group
of the arginines is
likely to be involved in specific interactions
with the phosphodiester
backbone and cannot be replaced by the
amino group of the lysine
(
1). In addition to the RR/KK mutation,
we made a K19R
mutation by replacing the absolutely conserved
lysine at position 19 (also present in SLBPs of the sea urchin,
Drosophila and
Chlamydomonas [unpublished results]) with arginine.
In
contrast to the RR/KK mutation, replacement of lysine 19 with
arginine
did not reduce binding affinity of the protein. However,
replacing
lysine 19 with alanine resulted in virtually complete
disruption of
SLBP binding, suggesting that the presence of a
positively charged
residue in this position is essential for RNA
binding. Changing the
tyrosines at positions 24 and 27 of the
RBD to phenylalanines greatly
reduced binding affinity, suggesting
that there is a major role for the
hydroxyl group as well as for
the aromatic ring in RNA binding. Perhaps
the ring of tyrosine
stacks against bases of the RNA target and the
hydroxyl group
is involved in formation of specific hydrogen bonds. In
addition,
in most SLBPs there are two tryptophans (residues 56 and 63)
near
the carboxyl end of the RBD. Replacement of these tryptophans
with
isoleucines completely abolished binding of SLBP to the stem-loop
structure, confirming the importance of the conserved aromatic
residues
in sequence specific recognition of the RNA
target.
Based on these and other studies (
13), it seems likely
that binding of SLBP to the stem-loop structure involves a set of
interactions similar to those identified in other RNA-protein
complexes. However, details of these interactions will be known
only
after determining the structure of the SLBP-RNA complex by
X-ray
crystallography or nuclear magnetic resonance
spectroscopy.
Role of the RBD and the C-terminal domain in histone pre-mRNA
processing.
The RBDs of vertebrate processing-specific SLBPs,
including mouse and human SLBPs and xSLBP1 from Xenopus,
contain a number of residues that are not conserved in the
processing-deficient xSLBP2. Systematic substitution of these amino
acids in the RBD of human SLBP with the amino acids found in the same
place in xSLBP2 allowed identification of a region of the RBD that
plays an important role in processing. This region includes two key amino acids, an aspartic acid at position 25 (D25) and arginine at
position 26 (R26), mutation of which makes SLBP almost completely inactive in H1t pre-mRNA processing. Also important is the nearby IK
dipeptide, mutation of which results in a twofold reduction in
processing efficiency. The remaining residues conserved in mammalian
SLBPs and xSLBP1 but not in xSLBP2 make a much smaller contribution to
the overall processing activity of SLBP.
The DR dipeptide is flanked by two tyrosines critical for RNA binding,
Y24 and Y27. The proximity of these residues may be
functionally
important. It is likely that SLBP interacts with
another component of
the processing machinery only when it is
bound to the histone pre-mRNA.
Binding of SLBP to its RNA target,
partially mediated by the two
tyrosines, could for example trigger
some conformational changes in the
RBD, exposing the DR dipeptide
thus facilitating its direct involvement
in processing. The Y24F
mutant is completely processing deficient,
although it retains
the same residual binding activity as the H41F
protein that is
capable of supporting substantial processing of the H2a
pre-mRNA.
This observation suggests that the Y24F mutation, adjacent to
the DR dipeptide, affects both RNA binding and
processing.
Immunoprecipitation experiments with the 9aaR mutant protein support
the notion that the DR dipeptide and the other amino
acids of the RBD
conserved only in processing-specific SLBPs are
involved in
stabilization of the U7 snRNP on the pre-mRNA. The
same role seems to
be played by residues in the 20-amino-acid
region immediately C
terminal to the RBD. The 20aaC and 9aaR mutants
recruit 4- and
2.5-fold, respectively, less U7 snRNP to the H1t
pre-mRNA than the
wild-type SLBP. Consistent with this result,
the 20aaC mutant protein
is also less efficient in rescuing processing
of the H2a pre-mRNA in
the SLBP-depleted nuclear extract and has
a stronger dominant negative
effect on histone H1t pre-mRNA processing
in the presence of the
endogenous SLBP. Combining both the 9aaR
and the 20aaC mutations did
not result in further reduction of
the amount of immunoprecipitated U7
snRNP nor in further reduction
of SLBP activity in H2a pre-mRNA
processing (not shown). We conclude
that both regions of SLBP
participate in the same interaction
that results in stabilizing U7
snRNP on the pre-mRNA. The 20aaC
mutation results in more severe
disruption of this interaction.
It is likely that the residual
processing activity of the 20aaC
mutant SLBP on H2a-614 pre-mRNA is due
to the high affinity of
U7 snRNP for the H2a-614 HDE and remaining weak
interactions between
the mutant SLBP and U7
snRNP.
Surprisingly, although both 9aaR and 20aaC mutant SLBPs are inactive in
processing of the H1t pre-mRNA, they can still recruit
a significant
amount of the U7 snRNP to this substrate at 4°C.
A likely explanation
for this observation is the lower temperature
of the
immunoprecipitation assay (22° followed by 4°C) than of
the
processing assay (32°C). It is possible that under these conditions,
the HDE of the H1t pre-mRNA can form a relatively strong duplex
with
the U7 snRNA, which is not stable at 32°C. This interpretation
is
supported by previous studies which demonstrated that lowering
the
temperature of incubation favors stronger interaction between
the
pre-mRNA and U7 snRNA and decreases the SLBP dependence of
in vitro
processing (
21).
Requirements for formation of a stable processing complex.
Although the mutant SLBP protein 9aaR containing nine amino acids of
the RBD replaced with the xSLBP2 sequence was completely inactive in
processing of H1t pre-mRNA, it showed significant activity when the
H2a-614 pre-mRNA substrate was used. A similar result was obtained with
the 20aaC mutant protein. These mutants bind the pre-mRNA with the same
affinity as the wild-type SLBP but are impaired in recruitment of U7
snRNP. The same variable effect on processing of the two substrates was
observed with the H41F mutant SLBP which binds the pre-mRNA weakly.
Thus, strong interaction (either direct or indirect) between the SLBP
and U7 snRNP as well as high-affinity binding of the SLBP to the
histone pre-mRNA are critical for processing of histone pre-mRNAs that form relatively few base pairs with the U7 snRNA (e.g., the H1t pre-mRNA) and are less important for processing of pre-mRNAs that contain a strong HDE (e.g., the H2a pre-mRNA). Taken together, our
results indicate that assembly of a stable processing complex on the
histone pre-mRNA depends on the additive strength of at least three
interactions: RNA-RNA interactions between U7 snRNA and the HDE,
binding of SLBP to the stem-loop, and interaction of the SLBP-stem-loop
complex with U7 snRNP. A similar requirement for multiple interactions
to stabilize an RNA processing complex is seen in recognition of the 3'
splice site by U2aF65 and U2aF35 which interact
with the polypyrimidine tract and the AG dinucleotide, respectively.
The presence of a long and uninterrupted polypyrimidine tract tightly
bound by U2aF65 diminishes the requirement for binding of
U2aF35, which is indispensable for cleavage of the 3'
splice sites containing weak polypyrimidine tracts (32,
33).
SLBP does not interact with the U7 snRNP in the absence of the pre-mRNA
substrate and, as judged by gel filtration chromatography,
is not found
in a complex with other nuclear proteins (unpublished
results). It is
likely that the first step in histone pre-mRNA
processing is binding of
SLBP to the stem-loop structure followed
by stabilization of the U7
snRNP on the HDE. The stabilization
of the U7 snRNP may be achieved by
direct interaction of the SLBP-RNA
complex with one of the proteins of
the U7 snRNP. However, it
is possible that there is a yet unknown
component of the processing
machinery which interacts with both SLBP
and U7 snRNP and provides
a bridging link between the two processing
factors, resulting
in stable association of the U7 snRNP with the
pre-mRNA. One approach
to identifying this unknown component may be to
screen for proteins
that interact with the SLBP/SL complex using a
modification of
the yeast two-hybrid system. These studies are in
progress.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant GM29832 to W.F.M.
We thank Francesca Perez for help in constructing some of the clones
and Xiaocui Yang for technical assistance. We are grateful to Tom
Ingledue for the Xenopus SLBP constructs and members of the
Marzluff laboratory for helpful discussions.
Z.D. and J.A.E. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Program in
Molecular Biology and Biotechnology, CB #7100, University of North
Carolina, Chapel Hill, NC 27599. Phone: (919) 962-8920. Fax: (919)
966-6821. E-mail: marzluff{at}med.unc.edu.
| |
REFERENCES |
| 1.
|
Calnan, B. J.,
S. Biancalana,
D. Hudson, and A. D. Frankel.
1991.
Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition.
Genes Dev.
5:201-210[Abstract/Free Full Text].
|
| 2.
|
Cotten, M.,
O. Gick,
A. Vasserot,
G. Schaffner, and M. L. Birnstiel.
1988.
Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro RNA processing reaction.
EMBO J.
7:801-808[Medline].
|
| 3.
|
Dominski, Z., and W. F. Marzluff.
1999.
Formation of the 3' end of histone mRNA.
Gene
239:1-14[CrossRef][Medline].
|
| 4.
|
Dominski, Z.,
J. Sumerel,
R. J. Hanson, and W. F. Marzluff.
1995.
The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.
RNA
1:915-923[Abstract].
|
| 5.
|
Dominski, Z.,
L.-X. Zheng,
R. Sanchez, and W. F. Marzluff.
1999.
The stem-loop binding protein facilitates 3' end formation by stabilizing U7 snRNP binding to the histone pre-mRNA.
Mol. Cell. Biol.
19:3561-3570[Abstract/Free Full Text].
|
| 6.
|
Galli, G.,
H. Hofstetter,
H. G. Stunnenberg, and M. L. Birnstiel.
1983.
Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini.
Cell
34:823-828[CrossRef][Medline].
|
| 7.
|
Gallie, D. R.,
N. J. Lewis, and W. F. Marzluff.
1996.
The histone 3'-terminal stem-loop is necessary for translation in Chinese hamster ovary cells.
Nucleic Acids Res.
24:1954-1962[Abstract/Free Full Text].
|
| 8.
|
Gick, O.,
A. Krämer,
W. Keller, and M. L. Birnstiel.
1986.
Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction.
EMBO J.
5:1319-1326[Medline].
|
| 9.
|
Gick, O.,
A. Krämer,
A. Vasserot, and M. L. Birnstiel.
1987.
Heat-labile regulatory factor is required for 3' processing of histone precursor mRNAs.
Proc. Natl. Acad. Sci. USA
84:8937-8940[Abstract/Free Full Text].
|
| 10.
|
Harris, M. E.,
R. Böhni,
M. H. Schneiderman,
L. Ramamurthy,
D. Schümperli, and W. F. Marzluff.
1991.
Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.
Mol. Cell. Biol.
11:2416-2424[Abstract/Free Full Text].
|
| 11.
|
Ingledue, T. C.,
Z. Dominski,
R. Sanchez,
J. A. Erkmann, and W. F. Marzluff.
2000.
Dual role for the RNA-binding domain of Xenopus laevis SLBP1 in histone pre-mRNA processing.
RNA
6:1635-1648[Abstract].
|
| 12.
|
Krieg, P. A., and D. A. Melton.
1984.
Formation of the 3' end of histone mRNA by post-transcriptional processing.
Nature
308:203-206[CrossRef][Medline].
|
| 13.
|
Martin, F.,
F. Michel,
D. Zenklusen,
B. Müller, and D. Schümperli.
2000.
Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system.
Nucleic Acids Res.
28:1594-1603[Abstract/Free Full Text].
|
| 14.
|
Martin, F.,
A. Schaller,
S. Eglite,
D. Schümperli, and B. Müller.
1997.
The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.
EMBO J.
16:769-778[CrossRef][Medline].
|
| 15.
|
Marzluff, W. F.
1992.
Histone 3' ends: essential and regulatory functions.
Gene Expr.
2:93-97[Medline].
|
| 16.
|
Marzluff, W. F.,
M. L. Whitfield,
Z. Dominski, and Z.-F. Wang.
1997.
Identification of the protein that interacts with the 3' end of histone mRNA, p. 163-193.
In
J. D. Richter (ed.), mRNA formation and function. Academic Press, New York, N.Y.
|
| 17.
|
Melin, L.,
D. Soldati,
R. Mital,
A. Streit, and D. Schümperli.
1992.
Biochemical demonstration of complex formation of histone pre- mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors.
EMBO J.
11:691-697[Medline].
|
| 18.
|
Mowry, K. L., and J. A. Steitz.
1987.
Identification of the human U7 snRNP as one of several factors involved in the 3' end maturation of histone premessenger RNA's.
Science
238:1682-1687[Abstract/Free Full Text].
|
| 19.
|
Pandey, N. B.,
A. S. Williams,
J.-H. Sun,
V. D. Brown,
U. Bond, and W. F. Marzluff.
1994.
Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.
Mol. Cell. Biol.
14:1709-1720[Abstract/Free Full Text].
|
| 20.
|
Smith, H. O.,
K. Tabiti,
G. Schaffner,
D. Soldati,
U. Albrecht, and M. L. Birnstiel.
1991.
Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides.
Proc. Natl. Acad. Sci. USA
88:9784-9788[Abstract/Free Full Text].
|
| 21.
|
Spycher, C.,
A. Streit,
B. Stefanovic,
D. Albrecht,
T. H. W. Koning, and D. Schümperli.
1994.
3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.
Nucleic Acids Res.
22:4023-4030[Abstract/Free Full Text].
|
| 22.
|
Stefanovic, B.,
W. Hackl,
R. Lührmann, and D. Schümperli.
1995.
Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.
Nucleic Acids Res.
23:3141-3151[Abstract/Free Full Text].
|
| 23.
|
Streit, A.,
T. W. Koning,
D. Soldati,
L. Melin, and D. Schümperli.
1993.
Variable effects of the conserved RNA hairpin element upon 3' end processing of histone pre-mRNA in vitro.
Nucleic Acids Res.
21:1569-1575[Abstract/Free Full Text].
|
| 24.
|
Sullivan, E.,
C. Santiago,
E. D. Parker,
Z. Dominski,
X. Yang,
D. J. Lanzotti,
T. C. Ingledue,
W. F. Marzluff, and R. J. Duronio.
2001.
Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression.
Genes Dev.
15:173-181[Abstract/Free Full Text].
|
| 25.
|
Sun, J.-H.,
D. R. Pilch, and W. F. Marzluff.
1992.
The histone mRNA 3' end is required for localization of histone mRNA to polyribosomes.
Nucleic Acids Res.
20:6057-6066[Abstract/Free Full Text].
|
| 26.
|
Varani, G., and K. Nagai.
1998.
RNA recognition by RNP proteins during RNA processing.
Annu. Rev. Biophys. Biomol. Struct.
27:407-445[CrossRef][Medline].
|
| 27.
|
Vasserot, A. P.,
F. J. Schaufele, and M. L. Birnstiel.
1989.
Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor.
Proc. Natl. Acad. Sci. USA
86:4345-4349[Abstract/Free Full Text].
|
| 28.
|
Wang, Z.-F.,
T. C. Ingledue,
Z. Dominski,
R. Sanchez, and W. F. Marzluff.
1999.
Two Xenopus proteins that bind the 3' end of histone mRNA: implications for translational control of histone synthesis during oogenesis.
Mol. Cell. Biol.
19:835-845[Abstract/Free Full Text].
|
| 29.
|
Wang, Z.-F.,
M. L. Whitfield,
T. I. Ingledue,
Z. Dominski, and W. F. Marzluff.
1996.
The protein which binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.
Genes Dev.
10:3028-3040[Abstract/Free Full Text].
|
| 30.
|
Whitfield, M. L.,
L.-X. Zheng,
A. Baldwin,
T. Ohta,
M. M. Hurt, and W. F. Marzluff.
2000.
Stem-loop binding protein, the protein that binds the 3' end of histone mRNA, is cell cycle regulated by both translational and posttranslational mechanisms.
Mol. Cell. Biol.
20:4188-4198[Abstract/Free Full Text].
|
| 31.
|
Williams, A. S.,
T. C. Ingledue,
B. K. Kay, and W. F. Marzluff.
1994.
Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.
Nucleic Acids Res.
22:4660-4666[Abstract/Free Full Text].
|
| 32.
|
Wu, S. P.,
C. M. Romfo,
T. W. Nilsen, and M. R. Green.
1999.
Functional recognition of the 3' splice site AG by the splicing factor U2AF35.
Nature
402:832-835[CrossRef][Medline].
|
| 33.
|
Zorio, D. A. R., and T. Blumenthal.
1999.
Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans.
Nature
402:835-838[CrossRef][Medline].
|
Molecular and Cellular Biology, March 2001, p. 2008-2017, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2008-2017.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Yang, X.-c., Torres, M. P., Marzluff, W. F., Dominski, Z.
(2009). Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3'-End Processing in Mammalian Histone Pre-mRNA. Mol. Cell. Biol.
29: 4045-4056
[Abstract]
[Full Text]
-
Sullivan, K. D., Mullen, T. E., Marzluff, W. F., Wagner, E. J.
(2009). Knockdown of SLBP results in nuclear retention of histone mRNA. RNA
15: 459-472
[Abstract]
[Full Text]
-
Erkmann, J. A., Wagner, E. J., Dong, J., Zhang, Y., Kutay, U., Marzluff, W. F.
(2005). Nuclear Import of the Stem-Loop Binding Protein and Localization during the Cell Cycle. Mol. Biol. Cell
16: 2960-2971
[Abstract]
[Full Text]
-
Lanzotti, D. J., Kupsco, J. M., Yang, X.-C., Dominski, Z., Marzluff, W. F., Duronio, R. J.
(2004). Drosophila Stem-Loop Binding Protein Intracellular Localization Is Mediated by Phosphorylation and Is Required for Cell Cycle-regulated Histone mRNA Expression. Mol. Biol. Cell
15: 1112-1123
[Abstract]
[Full Text]
-
Robertson, A. J., Howard, J. T., Dominski, Z., Schnackenberg, B. J., Sumerel, J. L., McCarthy, J. J., Coffman, J. A., Marzluff, W. F.
(2004). The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA. Nucleic Acids Res
32: 811-818
[Abstract]
[Full Text]
-
Ling, J., Morley, S. J., Pain, V. M., Marzluff, W. F., Gallie, D. R.
(2002). The Histone 3'-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3. Mol. Cell. Biol.
22: 7853-7867
[Abstract]
[Full Text]
-
Nelson, D. M., Ye, X., Hall, C., Santos, H., Ma, T., Kao, G. D., Yen, T. J., Harper, J. W., Adams, P. D.
(2002). Coupling of DNA Synthesis and Histone Synthesis in S Phase Independent of Cyclin/cdk2 Activity. Mol. Cell. Biol.
22: 7459-7472
[Abstract]
[Full Text]
-
Sanchez, R., Marzluff, W. F.
(2002). The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro. Mol. Cell. Biol.
22: 7093-7104
[Abstract]
[Full Text]
-
Dominski, Z., Yang, X.-c., Raska, C. S., Santiago, C., Borchers, C. H., Duronio, R. J., Marzluff, W. F.
(2002). 3' End Processing of Drosophilamelanogaster Histone Pre-mRNAs: Requirement for Phosphorylated Drosophila Stem-Loop Binding Protein and Coevolution of the Histone Pre-mRNA Processing System. Mol. Cell. Biol.
22: 6648-6660
[Abstract]
[Full Text]
-
Lanzotti, D. J., Kaygun, H., Yang, X., Duronio, R. J., Marzluff, W. F.
(2002). Developmental Control of Histone mRNA and dSLBP Synthesis during Drosophila Embryogenesis and the Role of dSLBP in Histone mRNA 3' End Processing In Vivo. Mol. Cell. Biol.
22: 2267-2282
[Abstract]
[Full Text]
-
Cho, H. D., Tomita, K., Suzuki, T., Weiner, A. M.
(2002). U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase). J. Biol. Chem.
277: 3447-3455
[Abstract]
[Full Text]
-
Allard, P., Champigny, M. J., Skoggard, S., Erkmann, J. A., Whitfield, M. L., Marzluff, W. F., Clarke, H. J.
(2002). Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo. J. Cell Sci.
115: 4577-4586
[Abstract]
[Full Text]
-
Dominski, Z., Erkmann, J. A., Yang, X., Sanchez, R., Marzluff, W. F.
(2002). A novel zinc finger protein is associated with U7 snRNP and interacts with the stem-loop binding protein in the histone pre-mRNP to stimulate 3'-end processing. Genes Dev.
16: 58-71
[Abstract]
[Full Text]
Top
Abstract
Introduction
