Download Structure and assembly of the spliceosomal small nuclear

222
Structure and assembly of the spliceosomal small
nuclear ribonucleoprotein particles
Christian Kambach*, Stefan Walke† and Kiyoshi Nagai‡
The spliceosome is a macromolecular assembly that carries
out the excision of introns from nuclear pre-mRNAs. It consists
of four large RNA–protein complexes, called the U1, U2,
U4/U6 and U5 small nuclear ribonucleoproteins (snRNPs),
and many protein factors. Crystal structures of seven protein
components and fragments of the U1 and U2 small nuclear
RNAs have been determined in the form of RNA–protein and
protein–protein complexes. Together with electron microscopy
studies of the snRNPs, these structures have begun to provide
important insights into the architecture of the snRNPs and the
mechanisms of RNA–protein and protein–protein recognition.
Addresses
Medical Research Council Laboratory of Molecular Biology, Hills Road,
Cambridge, CB2 2QH, UK
*e-mail: [email protected]
† e-mail: [email protected]
‡ e-mail: [email protected]
Correspondence: Kiyoshi Nagai
Current Opinion in Structural Biology 1999, 9:222–230
http://biomednet.com/elecref/0959440X00900222
© Elsevier Science Ltd ISSN 0959-440X
Abbreviations
EF
elongation factor
LRR
leucine-rich repeat
2,2,7-trimethylguanosine
m3G
N7-monomethylguanosine
m7G
rmsd
root mean square deviation
RNP
ribonucleoprotein
RRM
RNA recognition motif
snRNA small nuclear RNA
snRNP small nuclear RNP particle
Introduction
Most eukaryotic genes contain noncoding intervening
sequences (introns) that have to be removed from the primary mRNA transcript prior to translation into protein. In
the nucleus, introns are excised by two successive transesterification reactions within a macromolecular assembly
called the spliceosome [1–4]. In the first step, the 5′ splice
site is attacked by the 2′ hydroxyl group of a conserved
adenosine at a position known as the branch point within
the intron, such that the 5′ exon is cleaved off and the 5′
end of the intron is ligated to the 2′ hydroxyl group of the
branch point adenosine, resulting in a circular lariat intron
intermediate. In the second step, the 3′ hydroxyl group of
the 5′ exon attacks the phosphodiester bond at the 3′
intron–exon junction, resulting in the ligation of the two
exons and liberation of the intron [1–4].
The major components of the spliceosome are four
RNA–protein complexes, the U1, U2, U4/U6 and U5
snRNPs (small nuclear ribonucleoprotein particles). The
snRNPs are named after their RNA components. For
example, the U1 snRNP contains U1 small nuclear RNA
(snRNA). The U4 and U6 snRNAs are found extensively
base paired in a single particle (U4/U6 snRNP). These
snRNPs assemble onto the pre-mRNA through an ordered
pathway [1,2]. In contrast to group II self-splicing introns,
which are excised by an analogous two-step trans-esterification reaction through the folding of the well-conserved
intron sequences [5], nuclear pre-mRNA introns contain
only short conserved sequences at the 5′ and 3′ splice sites
and at the branch point (followed by the polypyrimidine
tract in metazoan introns) and thus require trans-acting factors in order to splice [1–4]. The U1 and U2 snRNPs bind
to the 5′ splice site and the branch point of the pre-mRNA,
respectively, and a pre-assembled U4/U6•U5 tri-snRNP
then joins the complex. Genetic and biochemical experiments have revealed an intricate network of interactions
between pre-mRNA and snRNAs, and between the
snRNAs, that undergoes an extensive rearrangement during the course of the splicing reaction. In the spliceosome,
the extensive base pairing between the U4 and U6 snRNAs
is unwound and the U6 snRNA subsequently base pairs
with both U2 snRNA and the 5′ splice site [1–4,6]. A highly conserved loop in the U5 snRNA interacts with the exon
sequences at the 5′ and 3′ splice sites and these interactions
are important for the second trans-esterification step [7–9].
Thus, nuclear pre-mRNA splicing is a highly dynamic
process and protein components play important regulatory
roles in the assembly of the snRNPs and the rearrangement
of the network of RNA–RNA interactions [2,10,11].
Spliceosomes sediment at 50–60S, corresponding to an
approximate molecular weight of 4.8 MDa [12]. This
indicates a complexity that is comparable to the ribosome
and, to date, some 80–100 protein factors have been
shown to be involved in metazoan splicing [10,11,13–15].
Spliceosomal proteins can be divided into those that are
tightly associated with snRNPs and the non-snRNP
splicing factors (for reviews, see [2,10,11,16–18]. Based
on functionality and sequence similarities to known proteins, many spliceosomal proteins have been classified as
being ATPases, helicases, protein kinases, GTPases or
peptidyl-prolyl cis/trans isomerases and are often related
to members of their respective class with known structures [2,4,11]. This suggests that these proteins may be
involved in the regulation of the spliceosomal assembly.
In fact, the splicing reaction is inhibited by the addition
of phosphatase inhibitors or nonhydrolysable ATP analogues [19,20]. Proteins containing helicase motifs are
likely to be involved in the rearrangement of the
RNA–RNA interaction network [2,4,10,11]. GTPases and
peptidyl-prolyl cis/trans isomerases may take part in the
Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai
223
Figure 1
Structure and assembly of the Sm proteins. (a) Crystal structure of the D3
protein (front view), with the hydrogen-bond network involving Tyr62 and
the highly conserved residues Glu36, Asn40, Arg64 and Gly65 shown.
The structure is shown in a ribbon representation, with helix A in red, β
strands 1, 2 and 3 (blue) are made from residues within the Sm1 motif
and β strands 4 and 5 (yellow) are made from residues within the Sm2
motif. (b) The D3 protein (side view), showing the heavily bent strands β2,
β3 and β4. Color scheme as in (a). (c) Crystal structure of the D3 (gold)
and B (blue) protein dimer. (d) A ribbon model of the Sm protein
assembly in the core snRNP domain. (e) A surface representation of the
Sm protein assembly, with the electrostatic surface potential shown (blue,
positive; red, negative). Reproduced with permission from [32••].
conformational changes that occur within the spliceosome [10]. Protein sequence motifs found in the
spliceosomal proteins include the ribonucleoprotein
(RNP) motif or RNA recognition motif (RRM), the Sm
motif, the GTPase motif, zinc fingers, leucine-rich
repeats (LLRs), K homology (KH) domains, doublestranded RNA binding domains (dsRBDs), the DEAD
(DEAH) box, the RGG box and the WD repeat. For a
comprehensive listing of the motifs found in spliceosomal
proteins, see Burge et al. [2]. The discovery of these
motifs provoked interesting speculation concerning the
origin and evolution of the splicing machinery.
detail, both genetically and biochemically [1–4,7–11,16,17];
however, the gap between our current understanding at the
biochemical level and our knowledge of the underlying
structural requirements at a molecular level has yet to be
closed. The recent crystal structure determination of the catalytic core of a group I self-splicing intron illustrates the
power of structural analysis in understanding catalytic mechanisms [21,22••]. Of the many components of the nuclear
pre-mRNA splicing machinery, the crystal structures of
seven snRNP-associated proteins have been determined,
three of those as complexes with their snRNA targets. These
structures have given considerable insight into the molecular
mechanisms of RNA–protein and protein–protein recognition between the spliceosomal components. This review will
concentrate on illustrating how this knowledge will help us
to understand snRNP assembly and architecture.
The multiple RNA–RNA, RNA–protein and protein–protein interactions that are essential for the fidelity and
efficiency of the splicing reaction have been studied in great
224
Macromolecular assemblages
The core small nuclear ribonucleoprotein
domain
U snRNPs contain two classes of proteins: those specific to
a given snRNP and those that are common to the U1, U2,
U4 and U5 snRNPs [2,10]. The latter are called core or Sm
proteins and they assemble on the snRNAs into a globular
structure called the core snRNP domain. The Sm-proteinbinding site (the Sm site) is a short, conserved uridine-rich
sequence present in the U1, U2, U4 and U5 snRNAs.
Eight generic Sm proteins have been identified in snRNPs
purified from a HeLa cell nuclear extract [17]. They are
named, in order of decreasing size, B′/B, D3, D2, D1, E, F
and G. The B and B′ proteins arise from a single gene by
alternative splicing and differ only in 11 residues at their
C termini [23,24]. These Sm proteins contain a conserved
sequence motif in two segments, Sm1 and Sm2, which are
connected by a linker of variable length [25–27]. The Sm
motif is related to no known protein sequence motif and,
hence, these proteins form a distinct protein family.
Core domain assembly is marked by several distinct intermediates. In the absence of snRNA, the Sm proteins exist
as three subcomplexes, D1D2, D3B (or D3B′) and EFG.
The EFG subcomplex binds, together with the D1D2 subcomplex, to the U snRNA to form the stable subcore
domain, which is then joined by the D3B (or D3B′) heterodimer to complete core domain assembly [28]. Neither
the individual Sm proteins nor individual Sm subcomplexes bind to snRNA. Core domain formation is an essential
step in U snRNP biogenesis and occurs in the cytoplasm
after the nuclear export of newly transcribed U snRNAs
containing the N7-monomethylguanosine (m7G) cap [29].
Core assembly triggers hypermethylation of the m7G cap
to a 2,2,7-trimethylguanosine (m3G) cap structure. The
core domain and the m3G cap act as a bipartite nuclear
import signal and the pre-snRNP matures in the nucleus
by association with specific proteins. The nuclear import
of the U4 and U5 snRNPs depends less on the presence of
the m3G cap than the U1 and U2 snRNPs [30,31••].
Recently, the crystal structures of two Sm protein subcomplexes, D1D2 and D3B, have been solved [32••]. The
four Sm proteins show a common fold containing a short,
N-terminal α helix followed by a five-stranded, antiparallel β sheet (Figure 1a, b). Strands 1–3 of the β sheet are
made from residues within the Sm1 motif, the linker of
variable length between the two motifs forms a connecting loop and Sm2 motif residues constitute β strands 4 and
5. Strands 2, 3, and 4 are heavily bent. Strand 5 loops back
over the bent strands to pair with strand 1. The main
interaction interface in both complexes comprises β strand
4 of one partner (D2 or B) pairing with β strand 5 of the
other (D1 or D3, respectively), thereby continuing the β
sheet throughout the complex (Figure 1c). The D1D2 and
D3B subcomplexes reveal a high degree of structural similarity at the level of both the individual protein fold and
the dimer architecture: a superposition of the individual
Cα backbones atoms within the Sm1 and Sm2 motifs of
the two dimers as rigid bodies yields an rmsd of 0.9 Å. The
D1D2 and D3B dimer structures show that each Sm protein can have two neighbours: one pairing with its β4
strand and the other pairing with its β5 strand. A model of
a higher order structure could be built by adding a
monomer one by one using the same subunit interactions.
This leads to the conclusion that seven core proteins
could form a complete ring [32••].
Figure 2
(a)
(b)
U1 70K
protein
m3G cap
SF3b protein
complex
U1A protein
U1 snRNA
Core domain
U1 snRNP
m3G cap
(c)
(d)
SF3a protein
m3G cap
complex
U2B′′–U2A′
protein complex
Core domain
U2 snRNA
U2 snRNP
U6 snRNA 3′ end
Base paired
U4/U6 snRNAs
Loop 1
Core domain
m G cap
U4/U6 snRNP
20S U5 snRNP
U5 snRNA
3
U4/U6.U5 snRNP
Current Opinion in Structural Biology
Electron micrographs of negatively stained splicesomal snRNPs with their interpretations. (a) U1snRNP, (b) U2 snRNP, (c) U4/U6 snRNP and
(d) U4/U6•U5 tri-snRNP. The electron micrographs were kindly provided by B Kastner. Adpated with permission from [38]
Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai
Co-immunoprecipitation and yeast two-hybrid systems were
used to investigate pairwise interactions of the Sm proteins
[33•,34•]. Kambach et al. [32••] have been able to arrange all
seven Sm proteins within a seven-membered ring
(Figure 1d) in a manner that is consistent with all the known
pairwise interactions [33•,34•]. The heptameric ring is the
only core domain model that is consistent with all the structural, biochemical and genetic data currently available. The
inner surface lining the ring bears a high concentration of
positive charges (Figure 1e). The central hole (20 Å diameter) is large enough to accommodate the single-stranded
RNA of the Sm site. It is therefore probable that the snRNA
binds to or is threaded through this hole. The AUU
sequence of the Sm site [35] has been shown to cross-link
with the G Sm protein by UV light. Core protein binding
studies of U1 and U5 snRNAs and their variants showed that,
not only the Sm-site sequence, but also the distance between
the Sm site and both the flanking stems and the internal loop
present in the flanking stem of U5 snRNA can affect the
binding of the core protein [36]. This suggests that the flanking stems also interact extensively with the core proteins.
Further characterisation of the RNA-binding interface of the
core proteins has to await more detailed cross-linking studies
and the crystal structure of the assembled complex.
The U1, U2, U4/U6 and U5 snRNPs each show a globular
domain in negatively stained electron micrographs [37],
similar to the appearance of an in vitro reconstituted core
domain (Figure 2). This domain remains intact even when
the specific proteins are depleted from the snRNPs
[37,38]. The overall dimensions of the core domains (about
80 Å in diameter) are in good agreement with the seven
subunit model of the proposed core domain [32••].
In addition to the canonical Sm proteins, closely related Smlike proteins have been found in Saccharomyces cerevisiae and
man. Two of these proteins (Uss1p and SmX3) have been
shown to be associated with U6 snRNA in yeast [25,27]. It
is now believed that the U6 snRNP contains a full set of Smlike proteins that form a core-domain-like assembly in the
U6 snRNP, both in yeast and in man (J Beggs, B Séraphin,
T Achsel, R Lührmann, personal communication). Proteins
bearing strong similarity to the Sm proteins have also been
found in the archeon Archaeoglobus fulgidus [39]. This shows
that ancestral Sm proteins appear early in evolution.
Proteins of the Sm family are more widespread than originally thought and may have diverse functions.
U1 small nuclear ribonucleoprotein particle
Figure 3a shows a schematic representation of the human
U1 snRNP [1,3]. The U1 snRNA contains 163 nucleotides
with a m3G cap and forms four stem–loops. The ACUUACCU sequence present at the 5´ end is complementary
to the conserved sequence (AGGURAGU) at the 5′ splice
site of pre-mRNA and plays an important role in binding
the U1 snRNP to the 5′ splice site. The Sm site (AUUUGUGG) present in the single-stranded region is the
binding site for the core Sm proteins. In addition, human
225
U1 snRNP contains three specific proteins, named U1
70K, U1A and U1C. U1 70K and U1A bind to stem–loops
I and II within the U1 snRNA, respectively (Figure 3a).
The U1A protein contains two RNP domains (or RRMs) that
are linked by a protease-sensitive peptide. The N-terminal
RNP domain of U1A is necessary and sufficient for binding
to U1 snRNA [40,41]. The crystal structure of the N-terminal fragment of the U1A protein in complex with its U1
snRNA hairpin II binding site provides important insights
into RNA recognition by RNP domains [42] (Figure 4a). The
RNP domain contains a four-stranded antiparallel β sheet
flanked on one side by two α helices [43]. The 10 nucleotide
loop of the U1 snRNA hairpin II (stem–loop II) binds on the
surface of the β sheet as an open structure. The first seven
nucleotides of stem–loop II, AUUGCAC, and the loop-closing C⋅G base pair form an intricate hydrogen-bond network
with the sidechains and the mainchain of the U1A protein.
These nucleotides show stacking interactions with either an
adjacent RNA base or a protein sidechain, or both, that stabilise the hydrogen-bond network with the protein by
restricting the orientation of the RNA bases [42].
The U1 70K protein contains a single copy of the RNP
motif (or RRM) around residues 100–180, followed by a
highly charged C-terminal tail with alternating
Arg-(Glu/Asp) and Arg-Ser repeats [44]. A fragment of the
U1 70K protein containing the RNP motif is alone capable
of binding stem–loop I of U1 snRNA. The N-terminal fragment containing residues 1–97 (with no known sequence
motifs) can, however, be incorporated into the core U1
snRNP consisting of the U1 snRNA and the core Sm proteins [45]. The N-terminal fragment of the U1 70K protein
preceding the RNP motif is unable to bind U1 snRNA on
its own, but it interacts with the core domain through protein–protein interactions. The U1 70K protein can be
chemically cross-linked to the B and D2 proteins [45].
The U1C protein binds to the U1 snRNP only in the presence of both the core domain and the U1 70K protein, and
does not bind the U1 snRNA on its own. This indicates that
the U1C protein probably binds both to the U1 70K protein
and to the Sm proteins. The latter contact is corroborated
by the observation of a cross-link between U1C and the Sm
B protein [46]. U1 snRNPs depleted of the U1C protein fail
to bind to pre-mRNA. The U1C protein was proposed to
form a noncanonical Cys2-His2-type zinc finger domain
near its N terminus. This segment is sufficient to restore
the binding of the U1 snRNP to the 5′ splice site [47]. The
U1C protein apparently alters the conformation of the 5′
end of U1 snRNA so that it can pair with the 5′ splice site.
Electron micrographs of the U1 snRNP show two protuberances emerging from the central globular core snRNP
domain [48] (Figure 2a). Antibodies specific to the U1A and
U1 70K proteins were used to identify each of the proteins
in a single protuberance. The protuberance corresponding
to the U1 70K protein was found to be closer to the
226
Macromolecular assemblages
Figure 3
U1A
protein
(a)
G
U
U
A
C A
C
C
U
A
60- U
U
C
G
G
A
U1 70K
G
protein
C
G
G
50- G
A
40
A AG
G
G U GG U U U U C CC
C I
A
A C C A A G A GG
C U AGU
U
G
20
G
30
A
C
G
G
U1C protein
10- U
C
C
A
U
U
C
m32, 2, 7 G p p p A U A
II
C
U
C
C
G
G
A
U
Core RNP
G
domain
U -80
G
C
U
G
150
A
C
IV U C
C
U
G
C
G C
C
C G
U -90
G C
100
G
U
U
CAA
CG A U U U C C C
C
U
A
III
A U
G C U A A A G GG U G U
C
G C
A
110
C
G C 160
U 120
C
140 G
130
G
G C
C A U A A U U U G U G G U A G U G U G OH
Sm site
5′ss
(b)
III
UU
C G
G C
A U
G C
I
G C
AA
A
UU
UA G
U U
U U
U
A
II
120 A U
C G
a U 60
U
G C
U6-I
C G-20
G C
G C
G C
A U
U A 140
G U
AG
50 C G
C
10-C
A U6-II
A U
U
A
U A
U2B′′ protein
bp
G
C G
A U
2, 2, 7
90
100
110G C
160
C
GpppAU
CGCUU
A
AAGUGUAGUAUC
U
GUUCUU
AC
UC
m3
A
AAUAUAUUAAA
GGAUUUUUG
GAGCAG
C
U
A U UG
G
150
30
40
C
A
U
U A
GCAUCG CCUGG
Sm site
C
70 C G
IV A
C G
CGUGGC GGACCU
G
U A 80
A
Core RNP
C C AU
C G
C 180AA
COH
domain
U
C
170
A C
U
IIb
SF3b
SF3a
Schematic representation of the U1 and U2
snRNPs. (a) The interaction between the U1
snRNA and protein components within the U1
snRNP. The Sm proteins (B/B′ D3, D2, D1, E,
F and G) assemble around the Sm site,
forming the core RNP domain. The U1 70K
and U1A proteins bind to stem–loops I and II,
respectively. The U1C protein does not bind
to U1 snRNA on its own and requires the
presence of the U1 70K protein. The
nucleotides near the 5′ end of U1 snRNA
(5′ss) are known to pair with the conserved
sequence at the 5′ splice site of the premRNA. For the crystal structure of U1A bound
to hairpin II, see Figure 4a [42]. A model of
the core RNP domain is shown in Figure 1e
[32••]. (b) The domain structure of the U2
snRNP inferred from biochemical data and
electron microscopy. The Sm proteins
assemble around the Sm site to form the core
RNP domain, as in U1 snRNP (a). The
U2B′′–U2A′(LRR) protein complex binds
stem–loop IV of U2 snRNA. The crystal
structure of this complex is shown in
Figure 4b [49•]. The SF3a complex joins the
large domain consisting of the core RNP
domain and the U2B′′–U2A′(LRR) complex.
The SF3b complex is thought form a large
domain at the 5′ end of the U2 snRNA. The
U6-I and U6-II sequences highlighted are
known to pair with U6 snRNA within the
spliceosome, after the pairing of U4 and U6
snRNAs is unwound. The branch point (bp)
sequence highlighted pairs with the
conserved sequence at the branch point
within the intron. Adapted with permission
from [18].
U2A′ protein
Current Opinion in Structural Biology
antibody bound to the m3G cap of U1 RNA than that corresponding to the U1A protein [48]. The sizes of the
protuberances are consistent with those predicted from the
molecular weights of the U1A and U1 70K proteins.
U2 small nuclear ribonucleoprotein particle
Figure 3b shows a schematic representation of the human
U2 snRNP [1,3,17]. The U2 snRNA, containing 187
nucleotides, forms four stem–loop structures. The Sm site
and the regions that base pair with the branch point and the
U6 snRNA are highlighted. The 12S U2 snRNP purified
under high salt conditions contains two U2-specific proteins, the U2B′′ and U2A′ proteins, in addition to the core
Sm proteins, whereas the 17S U2 snRNP purified under
low salt conditions contains nine additional proteins,
including the heteromeric splicing factors SF3a and SF3b
[2,10,11]. The role of the U2A′ and U2B′′ proteins in splicing has long remained elusive, but recent evidence from
S. cerevisiae indicates that these two proteins are required
for the integration of the U2 snRNP into the pre-spliceosome [49••]. The 5′ half of the U2 snRNA is extensively
modified (2′-O-methyl groups and pseudouridines). The
requirements of these modifications for both the conversion of the 12S U2 snRNP into the (spliceosome assembly
competent) 17S U2 snRNP particle and the subsequent
splicing activities were addressed by Yu et al. [50••]. The
authors found a correlation between the extent of
nucleotide modification and U2 snRNP function in splicing
and concluded that these modifications within the 27
nucleotides at the 5′ end of the U2 snRNA are essential for
the 12S→17S U2 RNP conversion.
The crystal structure of the U2B′′–U2A′ protein complex
bound to a fragment of U2 snRNA has been determined
at 2.4 Å resolution [51••] (Figure 4b). The U2B′′ protein,
containing two RNP domains, is closely related to the
Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai
227
Figure 4
Crystal structure of the U1A protein and the
U2B′′–U2A′(LRR) protein complex bound to
their cognate RNA hairpins. (a) A surface
representation of a complex between the U1A
protein (white) and U1 snRNA stem–loop II
[42]. Amino acid residues specific to the U1A
protein are highlighted in red. (b) A surface
representation of the ternary complex
between the U2B′′–U2A′(LRR) proteins and a
fragment of U2 snRNA stem–loop IV. The
U2B′′ protein is in white, with amino acid
residues specific to U2B′′ highlighted in
yellow. The U2A′(LRR) protein is shown in
blue. Reproduced with permission from [51••].
(a)
(b)
U1A
U2B"
A11
C12
A11 G12
C10
C10
U2A’
U13
U13
C14
G9
C15
G9
U8
U7
U8
C16
C15
A14
U7
A6
C5•G16 base pair
U1A protein [41]. The U1A protein binds to stem–loop II
of U1 snRNA on its own, whereas the U2B′′ protein binds
to stem–loop IV of U2 snRNA only in complex with the
U2A′ protein [41], which is a member of the LRR protein
family [52]. A complex between a fragment of the U2B′′
protein containing the N-terminal 96 residues and the
U2A′(LRR) protein shows high affinity and specificity for
stem–loop IV of U2 snRNA [41]. This U2B′′ fragment differs from the U1A protein in only 25 positions. Stem–loop
II of U1 snRNA contains a 10 nucleotide loop, with the
AUUGCACUCC sequence closed by a C⋅G base pair,
whereas stem–loop IV of U2 snRNA contains an 11
nucleotide loop, with the AUUGCAGUACC sequence
closed by a U⋅U base pair [41,51••]. The LRRs in the
U2A′(LRR) protein form a solenoid that is similar to, but
more irregular than, those found in the porcine ribonuclease inhibitor [52]. Helix A of the U2B′′ protein fits into
the concave surface of the parallel β sheet of the LRRs,
and the N-terminal and C-terminal arms flanking the
LRRs embrace the N-terminal RNP domain of the U2B′′
protein. Scherly et al. [53] showed that two amino acid
replacements, Asp24→Glu and Lys28→Arg, in the U1A
protein allow it to form a stable complex with the
U2A′(LRR) protein. The guanidinium group of Arg28
from the U2B′′ protein protrudes to form hydrogen bonds
with Thr69 and Glu92 of the U2A′(LRR) protein. In the
crystal structure of the U1A protein in complex with
snRNA stem–loop II, the first seven loop nucleotides,
AUUGCAC, and the loop-closing C⋅G base pair are recognised by the U1A protein, but the last three loop
nucleotide bases are not [42]. In the U2B′′–U2A′(LRR)
complex with a fragment of U2 snRNA stem–loop IV, all
the loop nucleotides are involved in extensive interactions
with the U2B′′ protein. The double-stranded stem of U2
snRNA hairpin IV interacts with the U2A′(LRR) protein
and, hence, extends in a different direction from the RNP
domain than that of U1 snRNA hairpin II [51••]. The differences between the two RNP proteins (U2B′′ and U1A)
and the presence of the U2A′(LRR) protein allow the two
A6
U1 RNA
U5•U17 base pair
cognate complexes to form a distinct network of interactions, even for the first six loop nucleotides that are
conserved between the two RNA hairpins. These interactions cannot be formed by the noncognate complexes,
resulting in a highly specific complex.
Electron micrographs of negatively stained 12S U2
snRNPs show a small domain attached to the core domain
that could be identified as being the U2B′′–U2A′ protein
complex bound to U2 snRNA hairpin IV [38,54]. In contrast, the larger 17S U2 snRNP particle contains nine
additional U2-specific proteins, including the SF3a and
SF3b complexes, and, thus, shows a second large domain
consisting of SF3b connected to the core domain, with a
single-stranded region of U2 snRNA appearing like a filament (Figure 2b) [54]. SF3a associates with the core
proteins and the U2B′′–U2A′ protein complex.
U4/U6, U5 and tri-(U4/U6•U5) small
nuclear ribonucleoproteins
The U4/U6 snRNP isolated from a HeLa cell nuclear
extract contains U4 and U6 snRNAs, which are extensively base paired, and the core Sm proteins bound to the Sm
site of the U4 snRNA. Electron micrographs of the U4/U6
snRNPs show a globular core domain and the Y-shaped filamentous structure of the base paired U4 and U6 snRNAs
protruding from the core domain [55] (Figure 2c).
Human U5 snRNA contains a long stem–loop structure, with
two internal loops followed by a single-stranded region and a
short stem–loop structure. The core Sm proteins bind to the
Sm site within the single-stranded region. The highly conserved loop I of U5 snRNA plays an important role in
aligning the 5′ and 3′ exons for ligation during the second
trans-esterification reaction [7,8]. The human 20S U5
snRNP is far more complex than the U1 and U2 snRNPs
(Figure 2d). It contains nine U5-specific proteins (220, 200,
116, 110, 102, 100, 52, 40 and 15 kDa), in addition to the core
Sm proteins (Figure 2d). The 200 kDa protein contains two
228
Macromolecular assemblages
RNA helicase motifs (DEIH and DDAH), whereas the
100 kDa protein contains a single RNA helicase motif
(DEAD) [2,4,10]. These proteins have been implicated in
the rearrangement of RNA–RNA interactions during the
splicing reaction. The 116 kDa protein is required for the
second step of splicing and bears high homology to the GTPbinding ribosomal translocase elongation factor (EF)-2 [10].
The 116 kDa protein can be cross-linked to a stable hairpin
inserted between the branch point and 3′ splice site and it
has been proposed that the 116 kDa protein may be involved
in 3′ splice site selection by a scanning mechanism [56••].
Homologues of the 220 kDa (Prp8p), 200 kDa (Snu246p)
and 116 kDa (Snu114p) proteins have been identified in
yeast. The yeast Prp8 protein and its human counterpart (the
220 kDa protein) can be cross-linked to nucleotides around
the 5′ splice site, the branch point and 3′ splice site and are
thought to collaborate with the conserved loop I of U5
snRNA in aligning the 5′ exon and the 3′ splice site during
the second trans-esterification reaction [57–59]. Dix et al.
[60••] cross-linked proteins to U5 snRNA either uniformly or
site-specifically labelled with 4-thiouridine, within the
reconstituted U5 snRNP and located the RNA contact sites
for five yeast proteins. Prp8p is in contact with a wide region
of U5 snRNA, including the stem of conserved loop I,
whereas Snu114p was cross-linked to a 4-thiouridine that was
introduced to the 5′ strand of internal loop I.
The U4/U6 and U5 snRNPs associate, together with more
than half a dozen tri-snRNP-specific proteins, to form a trisnRNP complex (Figure 2d) [2,4,10]. This complex joins
the U1 and U2 snRNPs assembled on the pre-mRNA.
Conclusions
As the discovery of introns was made only 20 years ago, our
understanding of the molecular mechanism of nuclear premRNA splicing has advanced remarkably within the
relatively short history of its research. The methods developed to study the ribosome, such as RNA footprinting,
cross-linking and immunoelectron microscopy, were
applied to and have facilitated the investigation of the
splicing machinery. The number of protein components
involved in pre-mRNA splicing is far greater than in the
ribosome and the splicing process is highly dynamic,
involving the assembly, rearrangement and disassembly of
many components. Thus, the splicing machinery is less
accessible to crystallographic analysis.
Neubauer et al. [61••] purified spliceosomes that were fully
assembled around a biotinylated, 32P-labelled pre-mRNA
substrate and rapidly characterised their protein components using mass spectrometry and an EST (expressed
sequence tag) database search after they had been isolated
from two-dimensional electrophoresis gels. This new
method, together with the complete yeast genome
sequence now available, will greatly facilitate the further
isolation and characterisation of the components involved
in splicing. Fromont-Racine et al. [62•] characterised a network of protein–protein interactions within yeast
spliceosomal snRNPs using exhaustive two-hybrid screening. This will complement the biochemical investigation
of protein–protein interactions within the splicing machinery. A detailed structural knowledge of the components of
the splicing machinery, as well as their interactions, are
essential to understanding the architecture of the snRNPs
and their assembly.
Crystallisation and X-ray analyses of either the snRNPs
or their large domains may be possible. The recent
progress in ribosome crystallography, providing the first
electron density map of a 50S ribosomal subunit at a resolution higher than 10 Å, is extremely encouraging
([63••]; see the review by Agrawal and Frank, this issue,
pp 215–221). The vast amount of biochemical data and
the 15 high-resolution structures of ribosomal proteins
[64] will greatly facilitate the initial interpretation of the
electron density map. Crystallographic data at high resolution will eventually provide details of its architecture at
or near atomic resolution. The interactions between the
ribosome and many essential factors, such as tRNA,
mRNA, EF-Tu and EF-G, are being studied in low-resolution maps obtained using (cryo) electron microscopy
([64]; see the review by Agrawal and Frank, this issue,
pp 215–221). This hybrid approach has proved to be
extremely powerful, creating working models of the ribosome at increasingly higher resolution.
Understanding the splicing process requires the structural
knowledge of many intermediate states. Such intermediate
states may be isolated using mutant substrate pre-mRNAs or
mutant protein factors that allow the accumulation of intermediate species. Biochemical heterogeneity or the inherent
flexibility of such complexes might preclude single-particle
cryoelectron microscopy at high resolution, but even low-resolution structures, together with the crystal structures of their
constituents, will provide valuable information.
Acknowledgements
The authors thank Andy Newman, Chris Oubridge, Richard Bayliss and
Phil Evans for critical reading of the manuscript, Berthold Kastner for
allowing us to include his electron micrographs and present and past
members of the group for their contributions to the project. The work was
supported by the Medical Research Council and Human Frontier Science
Program. CK was supported by NATO and EU fellowships, and SW by
Boehringer Ingelheim and EU Marie Curie studentships.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Moore MJ, Query C, Sharp PA: Splicing of precursors to
messenger RNAs by the spliceosome. In The RNA World. Edited
by Gesteland RF, Atkins JF. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press; 1993:303-357.
2.
Burge CB, Tuschl TH, Sharp PA: Splicing of precursors to mRNAs
by the spliceosome. In The RNA World, edn 2. Edited by
Gesteland RF, Cech T, Atkins JF. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press; 1999:525-560.
3.
Baserga SJ, Steitz JA: The diverse world of small
ribonucleoproteins. In The RNA World, edn 2. Edited by
Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai
Gesteland RF, Cech T, Atkins JF. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press; 1993:359-381.
4.
Staley JP, Guthrie C: Mechanical devices of the spliceosome:
motors, clocks, springs, and things. Cell 1998, 92:315-326.
5.
Qin PZ, Pyle AM: The architectural organization and mechanistic
function of group II intron structural elements. Curr Opin Struct
Biol 1998, 8:301-308.
6.
Madhani HD, Guthrie C: Dynamic RNA-RNA interactions in the
spliceosome. Annu Rev Genet 1994, 28:1-26.
7.
Newman AJ, Norman C: U5 snRNA interacts with exon sequences
at 5¢ and 3¢ splice sites. Cell 1992, 68:743-754.
8.
Sontheimer EJ, Steitz JA: The U5 and U6 small nuclear RNAs as
active site components of the spliceosome. Science 1993,
262:1989-1996.
9.
O’Keefe RT, Norman C, Newman AJ: The invariant U5 snRNA loop 1
sequence is dispensable for the first catalytic step of splicing in
yeast. Cell 1996, 86:679-689.
10. Will CL, Lührmann R: Protein functions in pre-mRNA splicing. Curr
Opin Cell Biol 1997, 9:320-328.
11. Krämer A: The structure and function of proteins involved in
mammalian pre-mRNA splicing. Annu Rev Biochem 1996,
65:367-409.
12. Müller S, Wolpensinger B, Angenitzki M, Engel A, Sperling J, Sperling R:
A supraspliceosome model for large nuclear ribonucleoprotein
particles based on mass determinations by scanning transmission
electron microscopy. J Mol Biol 1998, 283:383-394.
13. Brody E, Abelson J: The ‘spliceosome’: yeast pre-messenger RNA
associates with a 40S complex in a splicing-dependent reaction.
Science 1985, 228:963-967.
14. Frendewey D, Keller W: Stepwise assembly of a pre-mRNA
splicing complex requires U-snRNPs and specific intron
sequences. Cell 1985, 42:355-367.
15. Grabowski PJ, Seiler SR, Sharp PA: A multicomponent complex is
involved in the splicing of messenger RNA precursors. Cell 1985,
42:345-353.
16. Beggs JD: Yeast splicing factors and genetics strategies for their
analysis. In Pre-mRNA Processing. Edited by Lamond AI. Austin, TX:
RG Landes Co; 1995:79-95.
17.
Lührmann R, Kastner B, Bach M: Structure of spliceosomal snRNPs
and their role in pre-mRNA splicing. Biochim Biophys Acta 1990,
1087:265-292.
18. Nagai K, Mattaj IW: RNA-protein interactions in the splicing
snRNPs. In RNA-Protein Interactions. Edited by Nagai K, Mattaj IW.
Oxford: Oxford University Press; 1994:150-177.
19. Mermoud JE, Cohen PTW, Lamond AI: Regulation of mammalian
spliceosome assembly by a protein phosphorylation
mechanisms. EMBO J 1994, 13:5679-5688.
20. Tazi J, Kornstädt U, Rossi F, Jeanteur P, Cathala G, Brunel C,
Lührmann R: Thiophosphorylation of U1-70K protein inhibits premRNA splicing. Nature 1993, 363:283-286.
21. Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE,
Cech TR, Doudna JA: Crystal structure of a group I ribozyme
domain: principles of RNA packing. Science 1996, 273:1678-1685.
22. Golden BL, Gooding AR, Podell ER, Cech TR: A preorganized
•• active site in the crystal structure of the Tetrahymena ribozyme.
Science 1998, 282:259-264.
This work presents the largest RNA crystal structure determined so far. The
247-nucleotide group I intron RNA folds into two separate domains that
closely pack together. The remaining cleft can bind a helical region of the 5′
splice site. The authors also identify the binding site for the catalytically
important guanosine cofactor. This study provides the first example of an
RNA structure in which the active site is preorganised and ready for catalysis. Such a structural organisation has so far only been known for protein
enzymes and should help to close the gap between the RNA and protein
world even further.
23. Chu J-L, Elkon KB: The small nuclear ribonucleoproteins, SmB and
B¢, are products of a single gene. Gene 1991, 97:311-312.
24. van Dam A, Winkel I, Zijlstra Baalbergen J, Smeenk R, Cuypers HT:
Cloned human snRNP proteins B and B¢ differ only in their
carboxy-terminal part. EMBO J 1989, 8:3853-3860.
229
25. Séraphin B: Sm and Sm-like proteins belong to a large family:
identification of proteins of the U6 as well as the U1, U2, U4 and
U5 snRNPs. EMBO J 1995, 14:2089-2098.
26. Hermann H, Fabrizio P, Raker VA, Foulaki K, Hornig H, Brahms H,
Lührmann R: snRNP Sm proteins share two evolutionarily
conserved sequence motifs which are involved in Sm proteinprotein interactions. EMBO J 1995, 14:2076-2088.
27.
Cooper M, Johnston LH, Beggs JD: Identification and
characterization of Uss1p (Sdb23p): a novel U6 snRNAassociated protein with significant similarity to core proteins of
small nuclear ribonucleoproteins. EMBO J 1995, 14:2066-2075.
28. Raker VA, Plessel G, Lührmann R: The snRNP core assembly pathway:
identification of stable core protein heteromeric complexes and an
snRNP subcore particle in vitro. EMBO J 1996, 15:2256-2269.
29. Mattaj IW, De Robertis EM: Nuclear segregation of U2 snRNA
requires binding of specific snRNP proteins. Cell 1985, 40:111-118.
30. Fischer U, Darzynkiewicz E, Tahara SM, Dathan NA, Lührman R,
Mattaj IW: Diversity of signals required for nuclear accumulation
of U snRNPs and variety in the pathways of nuclear transport.
J Cell Biol 1991, 113:705-714.
31. Palacios I, Hetzer M, Adam SA, Mattaj IW: Nuclear import of U
•• snRNPs requires importin beta. EMBO J 1997, 16:6783-6792.
The nuclear import of U small nuclear ribonucleoproteins (snRNPs) depends on
the trimethylguanosine cap and the fully assembled core domain. The addition of
the importin-β-binding domain of importin α inhibited nuclear import of U snRNP,
whereas the depletion of importin α stimulated U snRNP import. The authors
concluded that the nuclear import of U snRNP is mediated by importin β.
32. Kambach C, Walke S, Young RA, Avis JM, De la Fortelle E, Raker VA,
•• Lührmann R, Li J, Nagai K: Crystal structures of two Sm protein
complexes and their implications for the assembly of the
spliceosomal snRNPs. Cell 1999, 96:375-387.
The seven proteins (B′/B, D3, D2, D1, E, F and G) that form the core domain
in the U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs) contain
a conserved sequence called the Sm motif. The crystal structures of two Sm
protein complexes were determined. The two complex structures highlight the
conserved fold of the Sm domain, consisting of an N-terminal α helix followed
by a heavily bent, five-stranded β sheet. In addition to the very similar subunit
structures, the subunit interfaces in the D3B and the D1D2 complexes are also
conserved. The authors propose that all the Sm proteins interact in the manner observed in the two dimer structures. They proposed a core domain
model in which all seven core proteins form a closed ring-like structure. The
high density of positive charges around the inner ring suggests a potential
binding site for the RNA. The model shows a similar size and appearance to
those of the core snRNPs observed by electron microscopy.
33. Camasses A, Bragado-Nilsson E, Martin R, Séraphin B, Bordonné R:
•
Interaction with the yeast Sm core complex: from proteins to
amino acids. Mol Cell Biol 1997, 18:1956-1966.
The Sm proteins (B′/B, D3, D2, D1, E, F and G) assemble around the Sm site
in U1, U2, U4 and U5 small nuclear RNAs and form a globular core domain.
A yeast two-hybrid screen was used to find pairwise interactions between
the yeast Sm proteins. Furthermore, site-directed mutagenesis was used to
identify residues within the E protein that are important for its interaction with
the F and G proteins.
34. Fury MG, Zhang W, Christodoulopoulos I, Zieve GW: Multiple protein:
•
protein interactions between the snRNP common core proteins.
Exp Cell Res 1997, 237:63-69.
A yeast two-hybrid screen was used to find pairwise interactions between
the human Sm proteins. The results are in good agreement with the experiment on the yeast Sm proteins carried out by Camasses et al. [33•]
35. Heinrichs V, Hackl W, Lührmann R: Direct binding of small nuclear
ribonucleoprotein G to the Sm site of small nuclear RNA.
Ultraviolet light cross-linking of protein G to the AAU stretch
within the Sm site (AAUUUGUGG) of U1 small nuclear
ribonucleoprotein reconstituted in vitro. J Mol Biol 1992,
227:15-28.
36. Jarmolowski A, Mattaj IW: The determinants for Sm protein binding
to Xenopus U1 and U5 snRNAs are complex and non-identical.
EMBO J 1993, 12:223-232.
37.
Kastner B, Bach M, Lührmann R: Electron microscopy of small
nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence
for a common structure-determining principle in the major U
snRNP family. Proc Natl Acad Sci USA 1990, 87:1710-1714.
38. Kastner B: Purification and electron microscopy of spliceosomal
snRNPs. In RNP Particles, Splicing and Autoimmune Disease.
Edited by Schenkel J. Berlin: Springer Verlag; 1998:95-140.
230
Macromolecular assemblages
39. Klenk HP, Clayton RA, Tomb J-F, White O, Nelson KE, Ketchum KA,
Dodson RJ, Gwinn M, Hickey EK, Peterson JD et al.: The complete
genome sequence of the hyperthermophilic sulphate-reducing
archaeon Archaeoglobus fulgidus. Nature 1997, 390:364-370,
40. Scherly D, Boelens W, van Venrooij WJ, Dathan NA, Hamm J, Mattaj
IW: Identification of the RNA binding segment of human U1A
protein and definition of its binding site on U1 snRNA. EMBO J
1989, 8:4163-4170.
41. Scherly D, Boelens W, Dathan NA, van Venrooij WJ, Mattaj IW: Major
determinants of the specificity of interaction between small
nuclear ribonucleoprotein-U2B¢¢ and their cognate RNAs. Nature
1990, 345:502-506.
42. Oubridge C, Ito N, Evans PR, Teo HC, Nagai K: Crystal structure at
1.92 Å resolution of the RNA-binding domain of the U1A
spliceosomal protein complexed with an RNA hairpin. Nature
1994, 372:432-438.
43. Nagai K, Oubridge C, Jessen TH, Li J, Evans PR: Crystal structure of
the RNA-binding domain of the U1 small nuclear
ribonucleoprotein A. Nature 1990, 348:515-520.
44. Query CC, Bentley RC, Keene JD: A common RNA recognition
motif identified within a defined U1 RNA binding domain of the
70K U1 snRNP protein. Cell 1989, 57:89-101.
45. Nelissen RL, Will CL, van Venrooij WJ, Lührmann R: The association of
the U1-specific 70K and C proteins with U1 snRNPs is mediated in
part by common U snRNP proteins. EMBO J 1994, 13:4113-4125.
46. Nelissen RLH, Heinrichs V, Habets WJ, Simons F, Lührmann R, van
Venrooij WJ: Zinc finger-like structure in U1-specific protein C is
essential for specific binding to U1 snRNP. Nucleic Acids Res
1991, 19:449-454.
47.
Heinrichs V, Bach M, Winkelmann G, Lührmann R: U1-specific
protein C needed for efficient complex formation of U1 snRNP
with a 5¢ splice site. Science 1990, 247:69-72.
48. Kastner B, Kornstädt U, Bach M, Lührmann R: Structure of the small
nuclear RNP particle U1: identification of the two structural
protuberances with RNP-antigens A and 70K. J Cell Biol 1992,
116:839-849.
49. Caspary F, Séraphin B: The yeast U2A¢/U2B¢¢ complex is required
•• for pre-spliceosome formation. EMBO J 1998, 17:6348-6358.
An open reading frame (LEA1) encoding a protein with striking similarities to
the human U2A′ protein was found in the yeast genome sequence database.
This protein associates with U2 small nuclear RNA only in the presence of
Yib9p, the yeast counterpart of the human U2B′′ protein. In the absence of
LEA1, spliceosome assembly is blocked prior to the addition of the U2 small
nuclear ribonucleoprotein. This demonstrates that U2B′′ (Yib9p) and U2A′
(LEA1) are required for the formation of the pre-spliceosome.
50. Yu YT, Shu MD, Steitz JA: Modifications of U2 snRNA are required for
•• snRNP assembly and pre-mRNA splicing. EMBO J 1998,
17:5783-5795.
The U2 small nuclear (sn)RNA undergoes extensive post-transcriptional
modifications and contains 2′-O-methylated residues and pseudouridines
towards the 5′ end, as well as the 5′ trimethylguanosine cap. Various fragments of natural U2 snRNA were produced by oligonucleotide-directed
RNase H cleavage and were then joined with unmodified in vitro transcribed
RNA in order to produce chimaeric full-length U2 snRNAs. The ability of
modified RNAs to assemble into a fully functional U2 small nuclear ribonucleoprotein (snRNP) was assessed. It was found that modifications of the
first 27 residues were important for full assembly into functional U2 snRNPs.
53. Scherly D, Dathan NA, Boelens W, van Venrooij WJ, Mattaj IW: The
U2B¢¢ RNP motif as a site of protein-protein interaction. EMBO J
1990, 9:3675-3681.
54. Kastner B, Bach M, Lührmann R: Electron microscopy of snRNPs
U2, U4/6 and U5: evidence for a common structure-determining
principle in the major U snRNP family. Mol Biol Rep 1990, 14:171.
55. Kastner B, Bach M, Lührmann R. Electron microscopy of U4/U6
snRNP reveals a Y-shaped U4 and U6 RNA containing domain
protruding from the U4 core RNP. J Cell Biol 1991,
112:1065-1072.
56. Liu ZR, Laggerbauer B, Lührmann R, Smith CWJ: Crosslinking of the
•• U5 snRNP-specific 116 kDa protein to RNA hairpins that block
step 2 of splicing. RNA 1997, 3:1207-1219.
The first AG dinucleotide downstream of the branch point is usually used as
the 3′ splice site, suggesting that a 5′→3′ scanning process occurs from the
branch point to locate the 3′ splice site. The authors found that the insertion
of a stable hairpin between the branch point and 3′ splice site blocks the
second step of splicing. Using methylene blue, which induces doublestranded RNA specific UV cross-linking, the authors found that the U5-specific 116 kDa protein can be cross-linked to pre-mRNA containing a stable
hairpin between the branch point and the 3′ splice site. This experiment supports the scanning mechanism and suggests a possible role for the U5
116 kDa protein in pre-mRNA splicing.
57.
Chiara MD, Gozani O, Bennett M, Champion-Arnaulk P, Palandjian L,
Reed R: Identification of proteins that interact with exon
sequences, splice sites, and the branchpoint during each stage of
spliceosome assembly. Mol Cell Biol 1996, 16:3317-3326.
58. Reyes JL, Kois P, Konforti BB, Konarska MM: The canonical GU
dinucleotide at the 5¢ splice site is recognised by p220 of the U5
snRNP within the spliceosome. RNA 1996, 2:213-225.
59. Teigelkamp S, Newman AJ, Beggs G: Extensive interactions of
PRP8 protein with the 5¢ and 3¢ splice sites during splicing
suggest a role in stabilization of exon alignment by U5 snRNA.
EMBO J 1995, 14:2602-2612.
60. Dix I, Russell CS, O’Keefe RT, Newman AJ, Beggs JD: Protein-RNA
•• interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA
1998, 4:1675-1686.
The yeast U5 small nuclear ribonucleoprotein (snRNP) was reconstituted
using U5 small nuclear RNA either uniformly or site-specifically labelled with
photoactivatable 4-thiouridine. After UV irradiation, cross-linked proteins
were identified using antibodies or tagged proteins, providing important
information on the architecture of the U5 snRNP.
61. Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P,
•• Sleeman J, Lamond A, Mann M: Mass spectrometry and ESTdatabase searching allows characterization of the multi-protein
spliceosome complex. Nat Genet 1998, 20:46-50.
A method that allows the rapid characterisation of the protein components
of a large protein assembly such as the spliceosome is described. A biotinylated pre-mRNA substrate was used to purify the spliceosome using affinity
chromatography. Individual proteins were isolated from a two-dimensional
gel and were rapidly characterised by mass spectrometry and an expressed
sequence tag (EST) database search.
62. Fromont-Racine M, Tain JC, Legrain P: Towards a functional analysis
•
of the yeast genome through exhaustive two-hybrid screens.
Nat Genet 1997, 16:277-282.
A yeast two-hybrid screen was used to characterise an extensive network of
protein–protein interactions within the spliceosomal small nuclear ribonucleoprotein (snRNP). The authors have revealed the protein components of the
yeast U1 and U2 snRNPs and their interactions. This method has proved
useful in studying the architecture of the snRNPs.
51. Price SR, Evans PR, Nagai K: Crystal structure of the spliceosomal
•• U2B¢¢-U2A¢ protein complex bound to a fragment of U2 small
nuclear RNA. Nature 1998, 394:645-650.
This crystal structure determination provides further insight into RNA recognition. It gives an example of two highly homologous proteins, evolved from
a common ancestor, that highly discriminate between similar target RNAs.
The authors compare the crystal structures of the U2B′′–U2A′ complex with
the U1A complex, each bound to their cognate RNA hairpin loop. Only a few
amino acid substitutions between U2B′′ and U1A result in very specific
RNA-binding properties. A detailed analysis reveals how complex formation
with U2A′ modulates the RNA-binding specificity of U2B′′. This interaction
allows key amino acid residues to specifically recognise U2 small nuclear
(sn)RNA elements that are different from U1 snRNA and explains the
observed specificity.
63. Ban N, Freeborn B, Nissen P, Penczek P, Grassucci RA, Sweet R,
•• Frank J, Moore PB, Steitz TA: A 9 Å resolution X-ray
crystallographic map of the large ribosomal subunit. Cell 1998,
93:1105-1115.
The authors present the results of their crystallographic studies on the large
50S ribosomal subunit of Haloarcula marismortui. A combination of 20 Å
resolution electron microscopy image maps and a large, 18 atom tungsten
cluster for heavy-atom phasing yields an electron density map at 9 Å resolution. This map shows more details than anything previously reported. Long,
continuous features of the map are consistent with stretches of doublestranded helical RNA. Finally, the molecular architecture of the ribosome
begins to reveal its secrets.
52. Kobe B, Deisenhofer J: Structural basis of the interactions between
leucine-rich repeats and protein ligands. Nature 1995,
374:183-186.
64. Ramakrishnan V, White SW: Ribosomal protein structures: insights
into the architecture, machinery and evolution of the ribosome.
Trends Biochem Sci 1998, 23:208-212.