From the Cambridge Institute for Medical Research,
University of Cambridge, Department of Clinical Biochemistry, Hills
Road, Cambridge CB2 2XY, United Kingdom, and the
§ Department of Chemistry, Macquarie University, Sydney,
New South Wales 2109,
School of Physics and
School of Biochemistry and Molecular
Genetics, University of New South Wales, Sydney,
New South Wales 2052, and ** Centre for Immunology, St
Vincent's Hospital,
Darlinghurst, New South Wales 2010, Australia
Received for publication, November 20, 2002, and in revised form, March 4, 2003
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ABSTRACT |
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Sm and Sm-like proteins are key components of
small ribonucleoproteins involved in many RNA and DNA processing
pathways. In eukaryotes, these complexes contain seven unique Sm or
Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a
single polypeptide chain. Here we show that single Sm and Lsm proteins
from yeast also have the capacity to assemble into homo-oligomeric
rings. Formation of homo-oligomers by the spliceosomal small nuclear
ribonucleoprotein components SmE and SmF preclude hetero-interactions
vital to formation of functional small nuclear RNP complexes in
vivo. To better understand these unusual complexes, we have
determined the crystal structure of the homomeric assembly of the
spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF
complex forms a homomeric ring but in an entirely novel arrangement
whereby two heptameric rings form a co-axially stacked dimer via
interactions mediated by the variable loops of the individual SmF
protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only
to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.
Sm and Sm-like (Lsm)1
proteins are core components of ribonucleoprotein (RNP) complexes
involved in many nucleic acid processing events within the eukaryotic
cell nucleus. The most highly characterized Sm/Lsm-containing RNPs are
those involved in pre-mRNA splicing, the U1, U2, U4/U6, and U5
small nuclear RNPs (snRNPs) (1), whereas others are known to be
important for telomere replication (2), trans-splicing (3),
and mRNA degradation (4, 5).
All Sm/Lsm proteins contain two regions of conserved sequence, termed
the "Sm motifs," separated by a segment of variable length and
composition (6-8). They possess a common structure (the Sm domain)
consisting of a five-stranded anti-parallel In eukaryotes, it is a ring of seven different but specific Sm/Lsm
proteins that binds to small nuclear RNA at a poly(U) sequence (the
"Sm-binding motif") to form the core of each snRNP complex. For example, the spliceosomal snRNPs contain one copy each of the seven
proteins SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (15), assemblies of
which are seen as ring structures when examined by electron microscopy
(16, 17). The RNA binding interaction of the Sm/Lsm heptamers is
governed by stacking of bases with conserved residues lining the inner
surface on one face of the ring (12, 18, 19). The dominating features
on the opposite ring face are the variable loops (loop L4) from
individual Sm subunits.
Lsm genes have been identified in prokaryotic archaeal species,
although only 1-3 Sm-like proteins are usually encoded per genome,
compared with the 16 or more found for eukaryotic organisms (10,
20-22). In line with this observation, these single proteins form
highly stable homo-oligomers with comparable RNA-binding affinities to
the more complex mixed heptamers of the eukaryotic snRNPs (10-12, 23).
Very recently it has been determined that the bacterial protein Hfq is
a homolog of the Sm family (24, 25), mediating RNA-RNA interactions as
a homo-hexameric assembly.
In this study we have focused on the functional organization of
recombinant versions of Sm and Lsm proteins from the simple eukaryote
Saccharomyces cerevisiae. We show that the three yeast proteins SmE, SmF, and Lsm3 are able to form stable homo-oligomeric ring structures in solution, in a similar manner to their archaeal and
bacterial Lsm counterparts. SmE and SmF, thought to form a direct
pairwise interaction within spliceosomal snRNPs in vivo, do
not associate with each other when in these stable homo-oligomeric states. The ring-forming Sm/Lsm proteins are shown to bind poly(U) RNA.
Surprisingly, in contrast to Sm/Lsm complexes described previously, the
SmF complex actively binds single-stranded DNA as well as poly(U) RNA.
The structure of SmF determined by x-ray crystallography reveals an Sm
protein assembly with a novel higher order arrangement of 14 monomers.
Cloning of Yeast Sm and Lsm Genes--
Genes encoding yeast
proteins SmE, SmF, Lsm9, and Lsm3 were isolated from S. cerevisiae genomic DNA by PCR. Primers were designed to
incorporate NdeI and EcoRI restriction sites at
the 5' and 3' ends of the genes, respectively. Genes were inserted into
the expression plasmid pETMCSIII, which relies on transcription by T7
RNA polymerase and results in a hexa-His fusion at the N terminus of
expressed proteins (26). The plasmid was transformed into the
BL21(DE3)/pLysS Escherichia coli strain for expression.
The single cysteine mutants (C16S)SmE and (C75S)SmF were made from
pETMCSIII-derived plasmids using the Stratagene
QuikChangeTM Site-directed Mutagenesis kit according
to the manufacturer's instructions. The (C75S)SmF gene was also
inserted into the pGEX-4T-2 plasmid (Amersham Biosciences) for
alternative expression as a glutathione S-transferase (GST)
fusion protein. The gene was cloned from the pETMCSIII-derived plasmid
by PCR using a 5' primer that allowed insertion of the gene into
pGEX-4T-2 using the BamHI and EcoRI restriction
enzymes. This plasmid was transformed into the BL21 E. coli
strain for protein expression.
Protein Expression and Purification--
Cells were grown at
37 °C in LB broth (containing appropriate antibiotics), and protein
expression was induced by addition of 0.1 mM
isopropyl- Molecular Weight Estimation--
Proteins were analyzed by
SDS-PAGE using the Tricine buffering system (27) or in solution by
analytical gel filtration with Superose® 12 and Superdex 75 (Amersham
Biosciences). Gel-filtration columns were run in 10 mM Tris
(pH 8.0), 200 mM NaCl at a flow rate of 60 µl/min.
Elution and void volumes were calibrated using standard globular
proteins and blue dextran (28).
Electron Microscopy--
Protein samples of (C75S)SmF,
(C16S)SmE, Lsm3, and MtLsm Protein Affinity Studies--
Cultures of E. coli
strains expressing bait GST-(C75S)SmF were mixed with cultures
expressing prey His-tagged (C16S)SmE. The mixed cells were lysed with a
French press in lysis buffer (see above). A 500-µl aliquot of the
clarified lysate was incubated with 50 µl of glutathione-agarose and
bound protein pelleted. The pellet was washed several times with
buffer, and bound and unbound fractions were examined by SDS-PAGE.
Biosensor interaction studies were performed with a BIAcore2000 surface
plasmon resonance instrument. His-tagged (C16S)SmE (75 µl, 200 nM) introduced to a Ni-NTA chip at a flow rate of 5 µl/min resulted in an increase of 3,300 response units above base
line. Binding of (C75S)SmF (derived from the GST fusion protein by
thrombin proteolysis) to His-tagged (C16S)SmE was tested by injecting
15 µl of 15 nM protein (i.e. 150 nM) at a flow rate of 5 µl/min. Similarly, binding of
GST-(C75S)SmF fusion protein was tested by injecting 15 µl of protein
at a concentration of 590 nM.
Crystallization of SmF--
Following gel filtration, His-tagged
versions of SmF and (C75S)SmF in Tris/NaCl (plus 10 mM
dithiothreitol for SmF) were concentrated to 20 mg/ml. Crystals of both
proteins were grown by sitting drop vapor diffusion in a reservoir
containing 24% polyethylene glycol 3350, 0.1 M Tris (pH
8.5), and 0.1 M sodium acetate. Diffraction quality
crystals measuring 2 × 0.5 × 0.5 mm formed within several weeks. From these conditions, two tetragonal crystal forms have been
identified, P4122 and P43212.
Data Collection and Structure Determination--
Data from the
P4122 crystal form of SmF was collected at beamline 14-4 at
the European Synchrotron Radiation Facility. Data to 2.8 Å resolution
was collected at 100 K after cryoprotecting the crystal in mother
liquor plus 10% glycerol. Images were integrated using MOSFLM (29) and
scaled using CCP4 programs (30). As a starting point for molecular
replacement, the MtLsm
Data from cryo-cooled crystals of the (C75S)SmF protein in space group
P43212 was collected on beamline BL9-2 at the
Stanford Synchrotron Radiation Laboratory using an ADSC Quantum4 CCD
detector with x-rays at 1.0 Å wavelength. As calculations indicated
two heptameric rings per asymmetric unit in this crystal form (35), a
molecular replacement model was constructed by pairing the refined SmF
heptameric structure (above) with its strongly interacting symmetry
equivalent. Molecular replacement with AMORE showed that this dimeric
arrangement of the heptameric rings constituted the asymmetric unit of
the P43212 crystal form. The structure of 14 subunits was rebuilt using the O program (33) and refined with REFMAC5
to yield final Rcryst and Rfree
values of 29.2 and 29.7%, respectively (Table I).
The coordinates and structure factors for both the P4122
and the P43212 structures of yeast SmF have
been deposited with the Protein Data Bank (accession codes 1N9R and
1N9S).
RNA/DNA Binding Assays--
RNA/DNA oligonucleotides
were 32P-labeled at their 5' ends, purified on a 10%
polyacrylamide/7 M urea gel, and recovered by n-butanol precipitation (36). Labeled oligonucleotides were heat-denatured (2 min, 95 °C) and immediately cooled on ice before addition of reaction mixture. RNA binding mixture (15 µl) containing 20 fmol of RNA/DNA oligonucleotides, 5 µg of protein, and 1.5 µl of
binding buffer (20 mM Tris/HCl (pH 8.0), 70 mM
KCl, 5 mM MgCl2, 0.5 mM
CaCl2, 0.1 mM EDTA, 7% (w/v) glycerol, 4 mM dithiothreitol, and 20 units of ribonuclease inhibitor)
were incubated at 22 °C for 30 min. Oligonucleotide species were
fractionated on a 5% native polyacrylamide gel in Tris borate/EDTA
buffer (with 3 mM Recombinant SmE, SmF, and Lsm3 Form Homo-oligomeric Rings--
In
order to characterize the yeast Sm and Lsm proteins, we have produced
several as recombinant molecules in E. coli. SmE, SmF, and Lsm3 when expressed in bacteria were found to be soluble and
easily purified in high yields by affinity chromatography. As there was
some evidence of a proportion of covalently linked dimers within
samples of His-tagged SmE and SmF on SDS-PAGE, non-native disulfide
bond formation was avoided by mutating the single non-conserved cysteine residues of each sequence to serine, resulting in His-tagged (C16S)SmE and (C75S)SmF. These site-specific mutants yielded single bands by electrophoresis, yet otherwise appeared to possess an identical fold to proteins of native sequence as judged by
gel-filtration, electron microscopy, and crystallography (see below).
The molecular integrity of the recombinant products was verified by
electro-spray mass spectrometry.
Gel-filtration chromatograms were used to ascertain the oligomeric
composition of the various recombinant proteins in solution. By
including in each run samples of archaeal MtLsm
Transmission electron micrographs of the complexes of (C16S)SmE,
(C75S)SmF, and Lsm3 stained with uranyl acetate were examined to
determine the organization of the oligomeric assemblies. In all cases,
ring structures are revealed (Fig.
2A). Each protein complex is
the same dimension and is similar in size to the structure of the
MtLsm Complexes of SmE and SmF Do Not Interact in Vitro--
SmE and SmF
are known to interact with each other, both within RNA-free hexameric
complexes with SmG and in intact splicing snRNPs (38, 39). Two-hybrid
studies and immunoprecipitations have also detected a direct pairwise
interaction between these two core Sm molecules (39-41). The specific
pairing of these proteins is a key event in the formation of a
functional spliceosomal Sm complex (9, 39). We therefore examined
whether the recombinant molecules are capable of a similar pairing
in vitro. With (C16S)SmE attached to the sensor chip surface
via its N-terminal hexa-His affinity tag, no binding event could be
detected upon introduction of either (C75S)SmF or the heavier variant
GST-(C75S)SmF (Fig. 2B). Similarly, no mass change was
detected when the biosensor experiment was configured for binding of
GST-(C16S)SmE to His-tagged (C75S)SmF on the Ni-NTA chip.
We were also unable to detect any co-complex of GST-(C75S)SmF and
(C16S)SmE in solution phase following the mixing of expression host
cell lysate and pull-down with glutathione-agarose (not shown). The
lack of observable interaction between the recombinant forms of SmE and
SmF does not exclude very weak interactions between the proteins but
certainly rules out the possibility in vitro of the 1:1
complex formation thought to occur in native snRNPs. It appears that
the formation of such a mixed complex is inhibited by the highly
organized and stable oligomeric states formed separately by each
protein component.
Crystal Structure of SmF--
We have determined the
crystallographic structures of the homo-oligomeric complexes of both
SmF and the mutant form (C75S)SmF. Both crystallize under identical
conditions, although we have observed that (C75S)SmF crystallizes in
two different forms (P43212 and
P4122) while so far observing only the P4122
form for native SmF. We report here the structure of SmF in the
P4122 crystal form and (C75S)SmF in the
P43212 form. The statistics of data collection
and structure refinement are given in Table
I. The two structures were determined
sequentially by molecular replacement using the previously solved
MtLsm
SmF from S. cerevisiae forms a homo-heptameric ring (Fig.
3), where each subunit binds to its
neighbor via
With stacking of
Examination of the dimeric interface reveals the unique interactions
responsible for the dimerization of SmF heptamers into an oligomeric
complex of 14 subunits, which results in the burial of 1,030 Å2 of accessible surface per heptamer. The stacking of two
rings is a result of hydrophobic interactions between Val60
and Val63 residues on one L4 loop and His65 on
the opposite L4 face, as well as a specific hydrogen bond between the
two histidine residues (His65) close to opposing L4-loops
(Fig. 3D). It is conjectured that this interaction would be
destabilized at low pH due to the protonation of each imidazole group.
RNA and DNA Binding by Oligomeric Sm/Lsm Protein
Complexes--
To investigate the binding properties of the prepared
Sm/Lsm protein complexes, we carried out electrophoretic mobility shift assays with 32P-labeled synthetic oligonucleotides
containing the specific U-rich sequences to which these proteins bind
in vivo. The human U snRNP core assembly has been shown to
have the same high affinity for AAUUUUU as it does for the complete Sm
site sequence AAUUUUUGA, and results in a stable complex (16). For our
study, the RNA sequences U5 and
A2U5 were screened, as well as the
corresponding DNA oligonucleotide, A2T5.
Fig. 4 shows that a strong band shift
occurs for A2U5 in the presence of either
(C75S)SmF or MtLsm Several previous reports show that homotypic interactions between
eukaryotic Sm/Lsm proteins are able to occur, at least in vitro. Using two-hybrid methods, others have identified weak
interactions between yeast SmE and itself (40, 41). GST interaction
studies have shown that the trypanosomal SmB protein is able to
interact with itself (42). However, to the best of our knowledge, this is the first report of Sm/Lsm proteins from a eukaryotic organism forming stable homo-oligomers with a ring-like morphology. This overall
structure is similar to that of the hetero-heptamers at the heart of
known Sm/Lsm-containing snRNPs. Furthermore, these complexes not only
bind specifically to identical poly(U) RNA sequences as native Sm/Lsm
assemblies, they also bind to single-stranded poly(dT) DNA. This
binding has not been observed previously.
The heptamer unit of the SmF 14-mer is very similar to previously
observed archaeal Lsm heptamers (Table
III), generally within an R.M.S.D. of
1.4-1.6 Å for all structures (M. thermoautotrophicum 1I81 (10) and 1JRI; A. fulgidus
1I5L and 1I4K (12)). The exception is the Lsm from Pyrobaculum
aerophilum (11), with an R.M.S.D. of 2.0-2.4 Å from all other
heptamers. This difference appears to be due to the packing of two
aromatic residues (Phe/Tyr) on the interface of the archaeal Lsm
proteins and SmF (Tyr38 and Tyr80 in SmF). In
P. aerophilum Tyr38 is replaced by an
isoleucine residue, which alters the monomer packing geometry.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-sheet preceded by a
short
-helix at the N terminus (9-12). The variable region of
sequence comprises the flexible loop L4 connecting strands
3 and
4. The highly bent
-sheet forms a curved shape that encompasses a
hydrophobic core extending to both edges of the molecule. This structure dictates that Sm/Lsm proteins have a preference for forming
closed ring oligomers of seven subunits, whereby each subunit interacts
with its neighbors through a combination of
-strand pairing and
extensive hydrophobic contacts. In the cases of the bacterial Sm
homolog Hfq and Sm2 from Archaeoglobus fulgidus, homomeric
hexamer assemblies of the fold have been observed (13, 14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside to the culture medium at A595 of 0.5-0.6. Harvested cells were
lysed by a French press in lysis buffer (20 mM Tris (pH
8.5), 50 mM NaH2PO4, 100 mM NaCl) containing 0.5% (w/v) Tween 20® (Calbiochem), 10 µg/ml RNase A, and protease inhibitor mixture (Sigma). The lysate was
clarified by centrifugation (30 min at 15,000 × g,
4 °C), and expressed proteins were purified on Ni-NTA or
glutathione-Sepharose affinity media as appropriate. The yeast protein
Lsm9 was refolded from inclusion bodies by solubilizing in urea (8 M), glycerol (10%), imidazole (10 mM), binding
to Ni-NTA-agarose and elution with gradual dilutions of urea. All
proteins were further purified by gel-filtration chromatography using
10 mM Tris (pH 8.0), 200 mM NaCl as running
buffer. Recombinant production of the Methanobacterium thermoautotrophicum Lsm protein (MtLsm
) has been described
previously (10). For RNA binding assays, the site-specific mutants
MtLsm
(R72L) and MtLsm
(R72L/N48A) were also prepared.
were diluted to 10-100 µg/ml in 10 mM Tris (pH 8.0), 100 mM NaCl. Copper grids
(400 µm mesh) were coated with an ultra-thin layer of carbon (~10
nm). Preparations were stained for 10 s with 5.0% uranyl acetate,
2.0% acetic acid after binding the protein for 5 s. Grids were
examined using a Philips CM10 transmission electron microscope, and
electron micrographs were recorded at a magnification of
×73,000-145,000.
heptameric structure (Protein Data Bank code
1I81) was converted to polyserine and truncated by removal of the
N-terminal helix of each protein chain. Molecular replacement was
performed with AMORE (31) using data from 15 to 4 Å. Very clear
rotationally related solutions constituted the top six results, with a
seventh rotationally related solution appearing at the eighth position.
The top solution (correlation coefficient 37.0, R factor
48.7) was subjected to rigid body and simulated annealing refinement in
CNS (32), resulting in a model with Rcryst = 40% and Rfree = 41% and a clearly
interpretable electron density map. The model was rebuilt in the O
program (33) and subjected to TLS refinement with REFMAC5 (34) to final
Rcryst and Rfree values
of 25.4 and 26.7%, respectively (Table I). There is one heptameric
ring in the asymmetric unit, but visual inspection revealed a striking
co-axially stacked interaction with a symmetry-related heptamer. This
involves contacts between all seven of the variable loops on one face
of the structure with all seven of the loops from the symmetry-related
molecule (see text for discussion).
-mercaptoethanol) and detected by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
characterized by us
previously (10) as an oligomeric complex of seven subunits, we were
able to compare the relative sizes of the homo-oligomers formed by
(C16S)SmE, (C75S)SmF, and Lsm3 (Fig. 1).
Of the group of proteins described here, only Lsm9 and MtLsm
samples
elute with an apparent molecular weight corresponding to a monomer (9 and 8 kDa, respectively). The majority of the (C16S)SmE sample elutes
as a molecule with an apparent molecular mass of 55-60 kDa and
at the same volume as the MtLsm
heptamer. The chromatograms of both
(C16S)SmE and SmE often contained an additional shoulder corresponding
to a size of ~110 kDa, indicating some dimerization of the major SmE
species (Fig. 1A). The proteins (C75S)SmF and Lsm3, however,
each elute as single species with apparent molecular masses of 100-110
and 95-105 kDa, respectively, i.e. they appear to be twice
the molecular size of the (C16S)SmE and MtLsm
oligomers. The large
(C75S)SmF and Lsm3 complexes are particularly stable, as shown by their
electrophoretic properties (Fig. 1B); the large (C75S)SmF
oligomer withstands 2% SDS and boiling for 5 min, and this treatment
merely halves the size of the Lsm3 complex. In contrast, (C16S)SmE is
more readily converted to a monomeric species. These results suggest
that Lsm3 and (C75S)SmF associate in a similar dimeric arrangement of
two ~55-kDa complexes in solution, with the Lsm3 dimerization being
somewhat less stable. Note that the (C75S)SmF oligomer is significantly
thermostable, resisting unfolding after heating to 65 °C, a point at
which the (C16S)SmE complex is dissociated (Fig. 1C).
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Fig. 1.
Gel filtration and electrophoresis of
recombinant Sm/Lsm proteins. A, gel filtration of
Sm/Lsm proteins on a Superdex 75 column in 10 mM Tris (pH
8.0), 200 mM NaCl. (C75S)SmF and Lsm3 appear to form
complexes twice the size of (C16S)SmE and the archaeal protein MtLsm
which forms a heptameric ring (10). Dimerization of the (C16S)SmE
complex is sometimes observed in solution (*). B,
silver-stained SDS-PAGE shows samples boiled for 5 min: lane
M, 10-kDa marker; lane 1, MtLsm
;
lane 2, MtLsm
; lane 3,
(C16S)SmE; lane 4, (C75S)SmF; lane
5, Lsm3; and lane 6, Lsm9.
C, thermostability of (C16S)SmE and (C75S)SmF homo-oligomers
measured from peak areas in gel-filtration traces following 15 min of
incubation in a water bath. The SmF complex is resistant to
denaturation to 65 °C.
heptameric ring, showing a diameter of ~8 nm and a central
area of stain ~2 nm wide. As the rings of (C75S)SmF and Lsm3 appear
to be equal in dimension to those of MtLsm
and (C16S)SmE, despite
having twice their effective molecular size in solution, this suggests
that the former proteins are organized as co-axially stacked double
rings. The ultra-structures of these yeast homo-oligomeric assemblies
are highly similar to the hetero-heptameric Sm/Lsm protein complexes
seen at the core of snRNPs from HeLa cells (16, 17, 20, 37).
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Fig. 2.
A, electron micrographs of Sm/Lsm
homo-oligomers. Purified proteins were bound to carbon-coated grids and
negatively stained with uranyl acetate. The upper panels
show selected fields 100 nm in width for samples of MtLsm ,
(C16S)SmE, (C75S)SmF, and Lsm3. White arrows indicate a
representative ring-shaped particle. The lower panels (10 nm
in width) show galleries of four typical particles. B,
biosensor response for Ni-NTA chip with bound His-tagged (C16S)SmE
(upper sensorgram) probed with 15 µl of 15 nM (C75S)SmF
(1), 150 nM (C75S)SmF (2), and 590 nM GST-(C75S)SmF (3). Lower sensorgram shows
reference channel response with no chelated protein; inset
shows the net difference.
heptamer as a starting model to first solve the structure of
the SmF P4122 crystal form.
Statistics of data collection and structure refinement
-strand pairing and hydrophobic interactions. Each SmF
polypeptide chain essentially adopts an identical fold to that defined
for its human and archaeal Sm and Lsm counterparts (9-12), a curved
-sheet composed of five anti-parallel strands. These strands
comprise residues His25-Leu31 (
1),
Thr36-Asp46 (
2),
Asn50-Val60 (
3),
Val63-Arg74 (
4), and
Tyr80-Leu84 (
5) of the SmF sequence,
respectively. In our data for SmF we do not observe any significant
density for the N-terminal
-helix common to the other Sm/Lsm
structures. Most subunits in the two SmF crystals structures show a
single-turn distorted helix near the N terminus, which in some subunits
is preceded by an extended chain, particularly in the
P43212 structure.
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Fig. 3.
Ribbon structures of the SmF
homo-oligomeric complex. A, structure of the SmF
assembly in the P4122 crystal form. A single SmF subunit is
shown at the top. The heptameric ring forms extensive
contacts with a symmetry-related heptamer in an identical arrangement
to that seen in the P43212 crystal form.
B, two heptameric rings are shown in magenta and
green, and residues in the L4 loop that form the interface
between the two rings are shown as ball and stick
models. C, predicted interactions of Sm/Lsm complexes
with nucleic acids and proteins. RNA may pass across one face of the
ring (heavy line) or through the central hole (dashed
line). We propose that one face of a single ring docks with
conserved nucleic acid Sm-binding sequences, whereas the variable loops
on the opposite loop L4 face are ideally located for interacting with
associated proteins. D, stereo-diagram showing a close-up of
the interface between two SmF heptamers (shown in tan and
green). The interaction is governed by well ordered contacts
between the variable L4 loops of individual SmF chains.
-strands of seven neighboring subunits into a ring,
two circular faces are formed, which we term the loop L4 face and the
RNA-binding face according to their most dominant structural features
(10). In the P4122 crystal form of SmF, there is one
heptameric ring per asymmetric unit (see "Experimental Procedures"), but this heptamer makes extensive contacts with a
symmetry-related complex along one face, where the two heptamers are
stacked co-axially. This interaction is governed by binding between
residues from the loop L4 faces of each heptamer, and we believe that
this dimeric arrangement corresponds to the complex that we observe in
solution and in electron micrographs. In strong support of this, our
crystal structure of (C75S)SmF in the P43212 form shows two heptamers per asymmetric unit and reveals an identical arrangement to the SmF symmetry-related dimer (Fig. 3B). The
observation of an identical 14 subunit complex in a different crystal
form strongly implies that the dimeric interaction is not simply the result of crystal packing.
. The position of the gel band indicates that the
RNA-protein binding interaction involves an intact Sm oligomer in both
cases. Gel shifts of similar size are obtained for the binding
assay in the presence of U5. The specificity of the
interaction is seen when key residues of the RNA-binding site in
MtLsm
, Arg72 and Asn48 (10), are altered and
result in complete abolition of binding of
A2U5. Oligomeric complexes of (C16S)SmE and
Lsm3 both showed some binding to U5, but to a lesser degree
than heptameric MtLsm
and (C75S)SmF (gels not shown). The suggestion
by Toro et al. (14) that an aromatic residue at the midpoint
of the L3 loop (e.g. Tyr48 in yeast SmF,
Phe46 in SmE, and His46 in MtLsm
) promotes
strong RNA binding is not well supported by these results, as we
observed a range of RNA interactions for Sm proteins containing this
sequence feature. Rather, our data (summarized in Table
II) indicate that it is the ability to
preform ring assemblies that promotes specific interactions of Sm
proteins with RNA. Lsm9 and MtLsm
are not able to form assemblies in
solution, and these recombinant versions give no response to RNA. DNA
gel shifts were performed with our complete set of Sm proteins and yielded the surprising result that (C75S)SmF is capable of interacting with DNA.
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Fig. 4.
Autoradiograph demonstrating in
vitro interaction of 32P-labeled oligo(U) RNA
and oligo(T) DNA with Sm/Lsm complexes. A, specific
binding of radiolabeled A2U5 RNA assayed by gel
shift and visualized on a native PAGE gel. RNA (20 fmol) was incubated
alone (lane 1) or with 5 µg of purified protein as
follows: MtLsm (lane 2), (R72L)MtLsm
mutant
(lane 3), (R72L/N48A)MtLsm
mutant (lane 4),
and (C75S)SmF (lane 5). A shift in RNA mobility, indicating
RNA-protein complex formation, is noted for MtLsm
and SmF. Mutants
of MtLsm
altered the strictly conserved RNA binding pocket.
B, gel shift assays of radiolabeled
A2T5 DNA (lane 1) and following
incubation with proteins (lanes 2-5) as described for
A. A2T5 produces a band shift for
(C75S)SmF at the same intensity as that seen for the
A2U5/(C75S)SmF interaction.
Purification and characterization of Sm/Lsm proteins
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Least squares alignment of Sm/Lsm heptamer structures
atoms used in the
superposition. Mth, M. thermoautotrophicum;
Af, A. fulgidus; Pa, P. aerophilum.
Distortion of the N-terminal helix in SmF is also seen in human SmB
(9), although the latter structure lacks an Sm-Sm interface as it is
not part of a heptameric complex in the crystal. Two factors appear to
contribute to the disruption of the helix in SmF: the presence of a
proline at residue 17, and a steric clash between Phe18 and
a residue on strand 4, Phe72. The Pro17
residue is unique to yeast (and human) SmF and SmG and is preceded by
Pro15, which is conserved in numerous Sm protein sequences.
The steric clash is likely to be absent in the heterogeneous
spliceosomal core complex, since SmF would directly interface with SmE
which instead contains a leucine residue at the position corresponding to Phe72. We note that the human SmD1-SmD2 interface (9)
also has the packing problem seen for SmF between residues 18 and 72, with SmD1 Phe6 (equivalent to Phe18) packing
against SmD2 Phe100 (equivalent to Phe72), yet
the structure manages to maintain the full N-terminal helix.
Our structure of the SmF protein complex reveals an unexpected higher
order arrangement of Sm/Lsm proteins. Although the homo-heptameric ring
formation is identical to that observed for archaeal Lsm proteins, the
dimerization of two rings is novel. Our data also suggest that Lsm3,
and to some extent SmE, are both able to organize similarly. The
dimerization of the rings is mediated by the variable L4 loop segments
that comprise one face of each heptameric complex, opposite to the ring
face containing the determinants for RNA binding (10, 12). Sequence
alignments of the Sm/Lsm protein family show loop L4 to be a region of
variable length and amino acid composition between more structurally
conserved protein segments (6-8). Thus this conformationally flexible
portion of the Sm fold appears to be a key determinant for the
protein-protein interactions we observe here. The Sm-like bacterial
homolog Hfq, which is also found organized as a co-axial dimeric ring
assembly in the crystalline state (each of six subunits), lacks this
loop L4 sequence feature. The Hfq dimer interface instead utilizes
contacts from a different portion of the Sm fold to that seen for SmF,
instead involving the edge of strand 2 via Phe60
(13).
In vivo there are two major types of interaction governed by Sm/Lsm protein complexes; a core RNA-binding interaction that is common to all of the multitude of Sm/Lsm complexes so far identified, and interactions with many different protein factors associated with the diverse snRNP complexes. Coupled with previous structures of Sm/Lsm assemblies (10-12, 14, 18), the structure of SmF demonstrates an inherent dichotomy between the two faces of the heptameric ring; one side governs nucleic acid binding, and the other side is capable of protein-protein interactions. This suggests to us that the various Sm/Lsm complexes may have a polarity in their functional interactions within the cell, whereby RNA binds in conserved grooves on one side of the ring and proteins bind to the variable loops on the other (Fig. 3C).
In this study we have identified at least three yeast Sm and Lsm proteins that can form homo-oligomeric rings. The fact that Sm and Lsm proteins with different functions are able to self-associate in a common manner shows that this is a quite general phenomenon. Of course, one particularly important question arises, i.e. what is the role of these eukaryotic homo-assemblies in vivo? It has been rigorously shown (15) that yeast spliceosomal snRNPs contain only one copy each of SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG. Thus should stable SmE and SmF oligomeric complexes exist in vivo, they are definitely not components of mature snRNP particles. Future functional studies will need to pay particular regard to discriminating between heterotypic and homotypic protein interactions.
It is known that spliceosomal snRNP assembly, at least in higher eukaryotes, requires interactions with the SMN protein, which appears to play a role in guiding the correct associations of different Sm hetero-complexes (43-45). The results of this study clearly show that Sm/Lsm protein-protein interactions are not limited to highly specific hetero-associations. Furthermore the homo-assemblies we observe here (specifically SmE and SmF) preclude the formation of hetero-complexes required for functional snRNP formation. This demonstrates why there is a need for proteins such as SMN or the complex pIC1n (46) in assisting correct formation of eukaryotic snRNPs containing several different Sm/Lsm proteins. Without such chaperones, the necessary specificity of Sm-Sm interactions may be severely compromised.
One of the most intriguing results presented here is the affinity of
the SmF homo-oligomeric complex for both single-stranded poly(U) RNA
and poly(dT) DNA. Further studies are needed to assess the functional
significance of this affinity for single-stranded DNA. It has been
proposed that the diverse Sm/Lsm species found in eukaryotic organisms
have evolved from one single Sm protein, presumably with the ability to
form ring structures with RNA-binding affinity (9). Studies of the
bacterial Sm-like Hfq protein have given rise to the postulate that
Sm/Lsm proteins have evolved specifically to modulate RNA-RNA
interactions (24, 25). A recent review (47) has highlighted the
potential role of Sm/Lsm proteins (along with many other
RNA-interacting domains) as a simple scaffold for mediating RNA
interactions in an ancestral organism relying primarily on RNA-based
metabolism. Our work suggests an even broader role in nucleic acid
stabilization that includes single-stranded DNA as well as RNA. The
ability of archaeal and bacterial Sm/Lsm proteins to form RNA-binding
homo-oligomers has led to the general consensus that these may
represent the ancestors of the Sm/Lsm protein family. However, the
identification of eukaryotic proteins with the same self-associative
and nucleic acid-binding properties shows that these potentially
ancestral traits are common to proteins from all domains of life.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Nick Dixon (Australian National University) for providing us with the pETMCSIII plasmid, Debra Birch (Macquarie University) for technical assistance with electron microscopy, and Dr. Matthew Seaman for the gift of yeast genomic DNA. We also thank Dr. David Owen (University of Cambridge) for the contribution of some materials and equipment and Dr. Jan Löwe and Suzanne Cordell for their assistance with crystallographic data collection.
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FOOTNOTES |
---|
* This work was supported by grants from the Australian Research Council and Macquarie University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N9R and 1N9S) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Present address: Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, KY16 9ST, UK.
§§ To whom correspondence should be addressed. Tel.: 61-29850- 8282; Fax: 61-29850-8313; E-mail: bridget.mabbutt@mq.edu.au.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M211826200
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ABBREVIATIONS |
---|
The abbreviations used are:
Lsm, Sm-like;
GST, glutathione S-transferase;
MtLsm or
, M.
thermoautotrophicum Lsm
or
protein;
r.m.s.d., root mean
square deviation;
RNP, ribonucleoprotein;
snRNP, small nuclear
ribonucleoprotein;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
Ni-NTA, nickel-nitrilotriacetic acid.
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