The EMBO Journal Vol.18 No.15 pp.4321–4331, 1999
Characterization of Sm-like proteins in yeast and
their association with U6 snRNA
Andrew E.Mayes, Loredana Verdone,
Pierre Legrain1and Jean D.Beggs2
Institute of Cell and Molecular Biology, University of Edinburgh,
King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK and
1Laboratoire du Me ´tabolisme des ARN, Institut Pasteur, 25–28 rue du
Dr Roux, 75724, Paris Cedex 15, France
Seven Sm proteins associate with U1, U2, U4 and U5
spliceosomal snRNAs and influence snRNP biogenesis.
Here we describe a novel set of Sm-like (Lsm) proteins
in Saccharomyces cerevisiae that interact with each
other and with U6 snRNA. Seven Lsm proteins co-
immunoprecipitate with the previously characterized
Lsm4p (Uss1p) and interact with each other in two-
hybrid analyses. Free U6 and U4/U6 duplexed RNAs
co-immunoprecipitate with seven of the Lsm proteins
that are essential for the stable accumulation of U6
snRNA. Analyses of U4/U6 di-snRNPs and U4/U6·U5
tri-snRNPs in Lsm-depleted strains suggest that Lsm
proteins may play a role in facilitating conformational
rearrangements of the U6 snRNP in the association–
dissociation cycle of spliceosome complexes. Thus, Lsm
proteins form a complex that differs from the canonical
Sm complex in its RNA association(s) and function.
We discuss the possible existence and functions of
alternative Lsm complexes, including the likelihood
that they are involved in processes other than pre-
Keywords: Lsm/Sm proteins/snRNP/splicing/U6 snRNP
Pre-mRNA splicing in eukaryotes occurs in the spliceo-
some, a multimeric ribonucleoprotein (RNP) complex
composed of five snRNAs (U1, U2, U4, U5 and U6) and
a large number of proteins. The snRNAs, in the form
of RNA–protein complexes (snRNPs), assemble on the
substrate pre-mRNA in an ordered manner (Moore et al.,
1993). The U1 and U2 snRNPs bind at the 5? splice site
and branch point of the intron, respectively, and then the
U4, U5 and U6 snRNAs are added in the form of a pre-
assembled U4/U6·U5 tri-snRNP complex. The U4 and U6
snRNAs share extensive sequence complementarity and
are found base paired in a U4/U6 duplex particle, although
an independent U6 particle also exists, as U6 is more
abundant than U4. In the spliceosome, a number of
important RNA conformational changes occur, the most
dramatic of which is destabilization of the U4/U6 duplex,
which frees U6 to interact with U2 snRNA prior to
are highly dynamic structures, and proteins are believed
© European Molecular Biology Organization
to play critical regulatory roles in splicing complex
assembly and turnover (Staley and Guthrie, 1998).
The snRNP proteins are considered to fall into two
classes, the Sm (or core) proteins, which are associated
with the U1, U2, U4 and U5 snRNAs, and the snRNP-
specific proteins each of which associates with only one
snRNP species. There are also many non-snRNP proteins
which interact with the spliceosome during spliceo-
some assembly, the splicing reactions or spliceosome
disassembly (reviewed in Moore et al., 1993; Kra ¨mer,
The human Sm proteins cross-react with antisera from
patients suffering from the autoimmune disorder systemic
lupus erythematosus and were named B/B?, D1, D2, D3,
E, F and G based on their relative mobility in SDS–
polyacrylamide gels (Lerner and Steitz, 1979; van
Venrooij, 1987). These proteins play important roles in
the biogenesis of the snRNP particles, associating in the
cytosol with a structurally conserved region known as the
Sm site (Branlant et al., 1982) in the newly transcribed
and exported U1, U2, U4 and U5 snRNAs (Mattaj and
DeRobertis, 1985). This acts as a signal for the hyper-
methylation of the 5? cap of these snRNAs (Mattaj, 1986)
which, together with the Sm proteins, forms a bipartite
nuclear localization signal for the snRNP (Fischer and
Lu ¨hrmann, 1990; Hamm et al., 1990). Maturation of the
snRNP then continues in the nucleus with the addition of
the snRNP-specific proteins (Zieve and Sauterer, 1990).
Saccharomyces cerevisiae possesses a homologous set
of seven Sm proteins (Smb, Smd1, Smd2, etc.; Neubauer
et al., 1997; Gottschalk et al., 1998). In addition to sharing
sequence identities with the human proteins, the yeast Sm
proteins have similar properties to their human counter-
parts; indeed the human SmD1 and SmE proteins can
functionally complement null alleles of the respective
yeast genes (Rymond et al., 1993; Bordonne ´ and Tarassov,
1996). However, very little is known about the pathway
of snRNP biogenesis in yeast.
Sequence comparisons of the Sm proteins from a range
of species led to the identification of a conserved motif,
the Sm or snRNP core protein motif (Cooper et al., 1995;
Hermann et al., 1995; Se ´raphin, 1995). The motif is
composed of two conserved blocks of amino acids (32
and 14 residues) separated by a non-conserved spacer
region of variable length. Truncation of the Sm motif of
either human SmB? or SmD3 prevents these proteins
forming a complex, suggesting that both conserved regions
are required for intermolecular interactions (Hermann
et al., 1995; Camasses et al., 1998).
In addition to these canonical Sm proteins, other
sequences containing the Sm motif have been identified
in S.cerevisiae. Uss1p, which was identified genetically
and was characterized biochemically as a novel splicing
factor (Cooper et al., 1995), and Smx4p, which was
A.E.Mayes et al.
Table I. Saccharomyces cerevisiae Sm-like proteins and their genes
Gene nameaORF namea
Other namesSm protein
aAs available via the SGD database (http://genome-www.stanford.edu/
bAs determined by Fromont-Racine et al. (1997).
cAs calculated from the amino acid sequence.
dBoeck et al. (1998);eSe ´raphin (1995);fCooper et al. (1995);gParkes
and Johnston (1992).
recognized by its genomic sequence as containing an
Sm motif (Se ´raphin, 1995), were reported to associate
primarily with free U6 and U4/U6 particles. Another of
the Sm-like proteins, Spb8p, has been proposed to play a
role in the decapping of mRNAs (Boeck et al., 1998).
Searches of the S.cerevisiae genome database identified
other open reading frames (ORFs) encoding putative Sm-
like proteins. Four of these proteins, together with Uss1p,
Smx4p and Spb8p, were grouped into seven sub-families
with the human and yeast canonical Sm proteins on the
basis of sequence similarity (Fromont-Racine et al., 1997).
To simplify the nomenclature, it has been decided to
name (or rename in some cases) the genes encoding these
Sm-like proteins LSM (Like Sm; Table I). Another two
hypothetical proteins, Lsm8p (Yjr022p) and Smx1p
(Ycr020-Ap), contain the Sm motif but are not structurally
similar to any particular sub-family in the sequence
alignment. Nevertheless, two-hybrid screens with Lsm8p
as bait identified interactions with several of the Lsm
proteins (Fromont-Racine et al., 1997; unpublished data),
and Lsm8p was itself identified in a two-hybrid screen
with Hsh49p, the yeast homologue of the human
splicing factor SAP49 (Fromont-Racine et al., 1997). In
contrast, exhaustive two-hybrid screens with Smx1p as
baitdid not produceany
(P.Legrain and M.Fromont-Racine, unpublished data), and
a protein A-tagged Smx1p showed no interaction with the
spliceosomal snRNAs (Se ´raphin, 1995).
Thus structural similarity to the Sm proteins together
with the two-hybrid data suggest that some of the Lsm
proteins might also form a complex, and the inclusion of
Lsm3p (Smx4p) and Lsm4p (Uss1p) in this set may
indicate a U6 snRNA association. Superficially at least,
the intrinsic differences between U6 and the other
spliceosomal snRNAs would suggest that a U6-associated
complex might have a fundamentally different role from
that of the canonical Sm core complex. The sequence of
U6 snRNA is the most highly conserved of all the
spliceosomal snRNAs (Brow and Guthrie, 1988), but it
lacks a recognized Sm site, and does not associate with
the canonical Sm proteins. Unlike the other spliceosomal
snRNAs which are products of RNA polymerase II, U6
is produced by RNA polymerase III. Since U6 snRNA
has a γ-methyl triphosphate cap structure which is not
hypermethylated, and is thought to be largely retained in
the nucleus (at least in higher eukaryotes where this has
been studied; reviewed in Reddy and Busch, 1988), neither
of the two primary roles defined for the Sm core complex
(signals for hypermethylation and nuclear import) is
generally considered to be required for the biogenesis of
the U6 snRNP.
We present here an analysis of the molecular
interactions of the yeast Lsm proteins. We show that their
genes are required for normal growth, and that seven are
required for the maintenance of normal levels of U6
snRNA. We provide evidence that these seven proteins
form a complex, are associated with U6 snRNA and
influence the efficiency of pre-mRNA splicing through
effects on various U6 snRNA-containing complexes in
the spliceosome assembly–dissociation cycle.
Two-hybrid interactions between Lsm proteins
A two-hybrid direct mating approach was used to
investigate all potential pairwise interactions between the
Lsm proteins 1–8. Smx1p was not analysed, as the primary
goal was to investigate a potential role in pre-mRNA
splicing, and the evidence available suggested no involve-
ment of Smx1p with the splicing machinery (see
above). Several canonical Sm protein-encoding ORFs and
fragments of Lsm4p were included as controls. Bait and
prey fusions in haploid yeasts were combined by mating
in all pairwise combinations, with resistance to defined
levels of 3-aminotriazole (3-AT) used as a guide to the
strength of any interaction seen. As variance between the
expression levels and stability of different bait and prey
proteins may affect these measurements, they are only a
rough guide to the strength of interactions between differ-
ent pairs of proteins. However, the number of diploid
strains capable of surviving on medium containing up to
50 mM 3-AT does suggest a number of strong, and
specific two-hybrid interactions (Figure 1). Other than an
interaction between the Lsm4 and Smb fusions (only in
the absence of 3-AT), the Lsm proteins did not interact
with the canonical Sm proteins, whereas the Smb and
Smd3 fusions interacted as expected. Instead, the Lsm
proteins appeared to form many interactions with each
other. Also, no significantly strong homotypic interactions
were seen. Thus, those diploids which were able to survive
on 3-AT are believed to represent specific associations
between the two-hybrid fusion proteins and not aspecific
interactions between any two proteins containing the Sm
fold. It should be noted, however, that some of the
observed interactions may be indirect, as discussed below.
Lsm4p is complexed with each of the other seven
To facilitate more direct analyses of Lsm protein inter-
actions, functional haemagglutinin (HA)-tagged versions
of the proteins were constructed (Materials and methods;
Table II). When anti-Lsm4p antibodies (Cooper et al.,
1995) were used to immunoprecipitate Lsm4p from
extracts containing HA-tagged proteins, each of the other
seven HA-tagged Lsm proteins was co-immunoprecipi-
tated, whereas a control HA-tagged protein (Gal4-AD)
Sm-like proteins and U6 snRNA
Table II. Saccharomyces cerevisiae strains
MATa/α trp1∆1, his3∆200, ura3-1, leu2-3,-112, ade2-1, can1-100
MATa/α trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100
MATa/α trp1∆99, his3∆200, ura3∆99, leu2∆1, ade2-101, cir°
MATα trp1∆1, his3∆200, ura3-1, leu2-3,-112, ade2-1, can1-100
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100
MATα trp1∆1, his3∆200, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm6∆::HIS3
MATα trp1∆1, his3∆200, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm7∆::HIS3
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm1∆::TRP1
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 HA:LSM1
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm5∆::TRP1 [pACTIIst-LSM5]
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 [pBM125-HA:LSM3]
MATα trp1∆1, his3∆200, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm2∆::HIS3 [pBM125-LSM2-HA]
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm8∆::TRP1 [pBM125-HA:LSM8]
MATα trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm5∆::TRP1 [pBM125-HA:LSM5]
MATa trp1∆1, his3-11,-15, ura3-1, leu2-3,-112, ade2-1, can1-100 lsm8∆::TRP1 [pACTIIst-LSM8]
MATa ade1-101, his3-∆1, trp1-289, ura3-52, LEU2-GAL1-LSM4
MATa gal4-542, gal80-538, ade2-101, his3∆200, trp1-901, leu2-3,-112, ura3-52, lys2-801 URA3::
(GAL4 17mers)3-CYC1-lacZlys2::GAL1UAS-HIS3 cyhr2
MATa ade2, gal4-542, gal80-538, trp1-901, leu2-3,-112, his3∆200, lys2-801am, URA3:: (lexAop)8-LacZ,
MATα gal4-542, gal80-538, ade2-101, his3∆200, trp1-901, leu2-3,-112, ura3-52, URA3::GAL1-lacZ
Hollenberg et al. (1995)
aDiploid strains are isogenic for the auxotrophic markers described.
cHA epitope tag strains used for immunoprecipitations.
The lsm deletion strains carrying the HIS3 or TRP1 markers are derived from BMA38 and BMA64, respectively.
Fig. 1. Two-hybrid direct matings of Lsm proteins. Haploid strains
expressing the bait or prey fusions indicated were mated on YPDA
medium and the growth of diploid cells was assayed by replica-plating
to selective medium containing the concentrations indicated of 3-AT.
The figure shows the highest 3-AT concentration at which each diploid
displayed growth after 3 days at 30°C. Lsm4∆p, amino acids 1–92 of
Lsm4p; Lsm4-A, amino acids 1–74 of Lsm4p; Lsm4-B, amino
acids 46–86 of Lsm4p.
was not (Figure 2). Thus, each Lsm protein can associate
in a complex, or complexes, with the Lsm4 protein.
The exact composition of such a complex(es) cannot be
determined in this experiment, nor whether all the Lsm
proteins can associate simultaneously with Lsm4p.
Characterization of the LSM genes
Of the genes encoding Lsm proteins in S.cerevisiae, only
LSM4 (USS1) has been characterized extensively (Cooper
et al., 1995). In order to characterize the other LSM ORFs,
targeted gene deletions were carried out (Figure 3A;
Materials and methods). Three of the genes, LSM1, LSM6
and LSM7, were found to be dispensable for cell viability;
however, the deletions caused slow growth at 23 and
30°C, and failure to grow at 37°C (Figure 3B and data
not shown). The remaining four genes, LSM2, LSM3,
LSM5 and LSM8, were essential for cell viability and, for
these genes, a GAL1-regulated copy of the ORF (PGAL1-
LSMX) permitted growth with galactose but not with
glucose as sole carbon source (Figure 3B and data not
shown). Thus, for each LSM gene, a strain displaying a
conditional growth phenotype was constructed, either
temperature-sensitive (referred to below simply as lsm
strains; see Table II) or carbon source-regulated. Repre-
sentative growth curves are shown in Figure 3B.
Analysis of pre-mRNA splicing in the conditional
In order to determine whether the in vivo depletion of the
Lsm proteins causes a defect in pre-mRNA splicing,
Northern blot analysis was performed. Figure 4 shows
A.E.Mayes et al.
Fig. 2. Co-immunoprecipitation of Lsm proteins with antiserum
against Lsm4p. Extracts from strains (AEMY28, AEMY29, AEMY31,
AEMY33, AEMY34, AEMY35 and LMA4-2A), each producing a
different HA-tagged Lsm protein or BMA64 [pACTIIst] (HA-tagged
Gal4-AD control), were subjected to immunoprecipitation with Lsm4p
antibodies. All incubations and washes contained 150 mM NaCl. The
precipitated proteins were fractionated by 15% (w/v) SDS–PAGE and
electroblotted. Immunodetection was with anti-HA antibodies and anti-
mouse HRP-conjugated secondary antibodies, and was visualized by
ECL. The positions of the tagged fusion proteins are marked by
that following a shift to the restrictive conditions, cells
depleted of Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7
or Lsm8 protein accumulated intron-containing pre-U3
RNAs, indicating a splicing defect.
Analysis of small RNAs in the conditional strains
Cooper et al. (1995) observed that when Lsm4p was
depleted in vivo there was a concomitant reduction in the
level of U6 snRNA. The effect of a shift to the restrictive
conditions on the levels of the spliceosomal snRNAs in
the lsm conditional strains was therefore examined by
in the levels of the spliceosomal snRNAs extracted from
the lsm1 strain compared with those from the parental
strain when the cells were grown at 37°C. However, for
each of the other conditional strains, a decrease in the
level of U6 snRNA was observed upon a switch to the
restrictive conditions. The level of U6 snRNA was lower
in the lsm6 and lsm7 strains even at the permissive
temperature, so the effect of shifting to the restrictive
conditions was less obvious than for the gal-regulated
strains. Indeed it may be that the defect causing cessation
of growth at 37°C in the lsm6 strain is not directly related
to U6 snRNA function, but rather to some other defect
arising from the lack of the Lsm6 protein (see Discussion).
Although the kinetics of U6 decline were slower for the
PGAL1-regulated strains, these had the lowest level of U6
after 24 h at the restrictive conditions, resembling the
effect of depleting Lsm4p (Cooper et al., 1995). The slow
decline of U6 snRNA with these strains may be due to
the time needed to titrate out the Lsm protein after
repression of transcription, compared with the more rapid
effect of heat treatment on the temperature-sensitive
strains. From these data, it was concluded that in addition
to Lsm4p, functional Lsm2, Lsm3, Lsm5, Lsm6, Lsm7
and Lsm8 proteins are needed for the normal accumulation
of the U6 snRNA in vivo. A slight decrease in the level
of U5LsnRNA was seen in PGAL1-LSM2 and PGAL1-LSM3,
Fig. 3. Deletion of genes encoding Lsm proteins. (A) Scheme for the
production of conditional strains. (B) Examples of growth analyses of
the conditional strains. Cells were grown to mid-logarithmic phase,
harvested, washed and resuspended in the appropriate pre-warmed
medium. OD600was followed with dilutions made where necessary to
maintain the cells in logarithmic growth. The growth temperatures or
carbon sources (in the case of PGAL1-LSM3 and its control grown at
30°C) are indicated to the right of each line. x-axis, time in hours after
shift to the experimental conditions; y-axis, OD600values. These
analyses were also performed for the other conditional strains and
produced similar results (data not shown).
while the level of U5Sincreased in PGAL1-LSM2 and
PGAL1-LSM8 under restrictive conditions. The significance
of this remains unclear.
To investigate the specificity of these effects on
spliceosomal RNAs, the levels of several other small RNA
species were examined: P RNA (the RNA component of
RNase P), MRP RNA (the RNA component of RNase
MRP) and 5S rRNA. The levels of most of these RNA
species were less obviously affected in the conditional
strains; however, there was a slight and reproducible
reduction in the level of pre-P RNA in PGAL1-LSM2,
PGAL1-LSM5 and PGAL1-LSM8 cells grown in glucose
(Figure 5B). In addition, the level of pre-5S RNA declined
in all except lsm1 cells under restrictive conditions (data
not shown), and mature 5S rRNA was slightly reduced in
PGAL1-LSM2, PGAL1-LSM5 and PGAL1-LSM8 cells. Ab-
normalities in pre-tRNAs were also observed in several
lsm strains (our unpublished results). Like U6 snRNA,
the 5S rRNA, tRNAs and P RNA are products of RNA
polymerase III. Althoughwe cannot exclude the possibility
that the effects on these RNAs may be due to indirect
effects of Lsm protein depletion (e.g. as a result of
Sm-like proteins and U6 snRNA
disruption of splicing), considering the proposal of
Pannone et al. (1998) that La protein and Lsm8p
collaborate in the stabilization of U6 and other polymerase
III-transcribed small RNAs, these results suggest that all
except Lsm1p may contribute to this activity.
Effect of U6 snRNA overproduction in conditional
Given the reduction in the U6 snRNA levels seen for
most of the lsm conditional strains, the effect of over-
partially suppressed the growth defect of lsm6 and lsm7
cells at 37°C, the (previously) restrictive temperature
(Figure 6A), and of PGAL1-LSM2, PGAL1-LSM3 and PGAL1-
LSM4 cells on glucose medium (Figure 6B).
The best complementation was for the PGAL1-LSM5
and PGAL1-LSM8 cells, for which overproduction of U6
permitted full growth on glucose medium (Figure 6B).
In contrast, overproducing U6 snRNA (confirmed by
Northern blot analysis) had no discernible effect on the
Fig. 4. Effect of in vivo inactivation or depletion of Lsm proteins on
splicing. (A) RNA was extracted from the gal-regulated strains and
from the wild-type parent grown continuously in galactose or shifted
to glucose medium for 12 h. RNA was separated in a 6% (w/v)
denaturing polyacrylamide gel, electroblotted and hybridized with a
radiolabelled oligonucleotide complementary to the U3 snoRNAs.
(B) RNA was extracted and analysed as above for lsm or wild-type
cells grown at 30°C. *represents a stable breakdown product of
pre-U3 RNA (Hughes and Ares, 1991)
Fig. 5. Effect of in vivo inactivation or depletion of Lsm proteins on the levels of small RNAs. (A) The conditional strains and wild-type parents
were grown under the restrictive conditions for the times indicated. RNAs were analysed as described for Figure 4 and probed with radiolabelled
oligonucleotides complementary to the spliceosomal snRNAs. (B) The same membrane was stripped and reprobed for P (or pre-P) RNA, MRP RNA
and 5S rRNA.
growth of the lsm1 strain at 30 or 37°C. (Figure 6A).
Presumably this reflects the fact that the level of U6
Thus, the overproduction of U6 snRNA can, to some
extent, compensate for the loss of the Lsm2, Lsm3, Lsm4,
Lsm5, Lsm6, Lsm7 and Lsm8 proteins, suggesting that at
least part of the physiological function of these proteins
is directly related to the stable accumulation of U6 snRNA.
Co-immunoprecipitation of snRNAs with tagged
Northern analysis of spliceosomal snRNAs co-immuno-
precipitating with each HA-tagged Lsm protein showed
that Lsm2, Lsm5, Lsm6, Lsm7 and Lsm8 proteins all co-
precipitated U6 snRNA and U4 snRNA (Figure 7A), as
was previously reported for Lsm3p (Se ´raphin, 1995)
and Lsm4p (Cooper et al., 1995). Some U5 snRNA also
co-precipitated with Lsm2p, and Lsm5p, presumably due
to precipitation of the U4/U6·U5 tri-snRNP (and supported
by co-precipitation of several of the Lsm proteins by
antibodies against Prp8p which is present in tri-snRNPs;
V.Vidal and J.D.Beggs, unpublished results). No immuno-
precipitation of U1 or U2 snRNA was detected for any
of the Lsm proteins. When the membranes in Figure 7A
were reprobed for P RNA, MRP RNA and 5S rRNA,
none of these RNA species was detected in any of the
proteins do not possess a general RNA-binding capacity.
With Lsm1p, no snRNAs were immunoprecipitated
under these conditions (150 mM NaCl; Figure 7A)
despite efficient precipitation of the protein (data not
shown). A very low level of U6 snRNA could be co-
immunoprecipitated with Lsm1p at 50 mM but not at
higher salt concentrations (data not shown).
with each of the Lsm proteins was in the free or the U4
A.E.Mayes et al.
Fig. 6. Effect of overproduction of U6 snRNA on growth of
conditional strains. (A) Ten-fold serial dilutions of each conditional
strain transformed with either vector (YEp24) or U6 encoded on a
high copy number plasmid (pYX117; Hu et al., 1994) were spotted on
selective medium and incubated at 30 or 37°C for 3 days. (B) Ten-fold
serial dilutions of gal-regulated strains transformed with either vector
(YEp13 or YEp24) or U6 encoded on a high copy number plasmid
(pYX172 or pYX117; Hu et al., 1994) were spotted on selective
galactose or glucose medium and incubated at 30°C for 3 days.
base-paired form, the co-precipitated RNA was analysed
under non-denaturing conditions that preserve the U4/U6
base pairing (Brow and Guthrie, 1988). The HA-tagged
Lsm2, Lsm3, Lsm5, Lsm6, Lsm7 and Lsm8 proteins co-
precipitated both free U6 and base-paired U4/U6
(Figure 7B and data not shown). In conclusion, all
except Lsm1p associate stably with U6 snRNA, remaining
associated with this RNA as it interacts with U4 snRNA,
and at least transiently as the U4/U6·U5 tri-snRNPs form.
Analyses of snRNP complexes
The state of the U4 and U6 snRNAs was examined in
extracts from lsm6 and lsm7 cells (deletion strains which
contain no Lsm6p or Lsm7p, respectively). Compared
with extracts from control strains, the lsm extracts con-
tained greatly reduced levels of free U6 snRNP even at
30°C, and accumulated free U4 snRNP (Figure 8A). The
majority of the residual U6 was complexed with U4. As
lsm7 extract contains a high level of U4/U6 duplex, it
was of interest to investigate the U4/U6·U5 tri-snRNP
content of this extract. Prp8p antibodies were used to
immunoprecipitate tri-snRNPs (Cooper et al., 1995), and
the levels of the U4, U5 and U6 snRNAs that co-
precipitated were compared between lsm7 and wild-type
extracts (Figure 8B). The levels of U5 were similar (Prp8p
is a U5 snRNP protein), whereas the levels of U4 and U6
in the lsm7 precipitate were reproducibly only one-third
of wild-type levels. Therefore, although the two extracts
contained similar levels of U4/U6 duplexed RNAs, in the
absence of Lsm7p less of this was in the form of
Searches of the complete S.cerevisiae genome database
identified 16 putative ORFs encoding proteins with Sm
motifs (Fromont-Racine et al., 1997). Three of the encoded
proteins had already been identified and characterized as
homologues of canonical Sm proteins: Smd1p (Rymond,
1993; Rymond et al., 1993), Smd3p (Roy et al., 1995)
and Smep (Bordonne ´ and Tarassov, 1996), while four
others had been shown to be associated with U1 snRNA
but not characterized further (Neubauer et al., 1997;
Gottschalk et al., 1998). Based on the two-hybrid inter-
here, eight Sm-like or Lsm proteins appear capable of
associating with one another to form a novel complex or
complexes. Seven of these proteins (Lsm2, Lsm3, Lsm4,
Lsm5, Lsm6, Lsm7 and Lsm8) associate with U6 snRNA
and are required for maintenance of normal U6 snRNA
levels and for pre-mRNA splicing.
The two-hybrid data for the Lsm proteins suggest
greater promiscuity in their mutual protein interactions
for particular Sm protein pairings (Fury et al., 1997;
Camasses et al., 1998). However, no strong homotypic
interactions were seen and, with the exception of Smbp
and Lsm4p, several canonical Sm proteins that were
tested did not interact with the Lsm proteins. The failure
of Lsm4-Ap and Lsm4-Bp to interact indicates the require-
ment for both Sm motifs. An analogous situation has been
reported for the canonical Sm proteins, where deletion of
either of the Sm motifs leads to loss of their interaction
(Hermann et al., 1995; Camasses et al., 1998). The two-
hybrid approach may also detect indirect interactions,
mediated by a bridging protein or RNA. The apparent
promiscuity of the Lsm proteins may reflect indirect
interactions in some cases. In exhaustive two-hybrid
screens of a genomic library using many bait proteins
including dozens of splicing factors, Lsm proteins were
found as prey almost exclusively by Lsm proteins as baits
unpublished results). Thus the Lsm proteins have strong,
highly specific interactions with each other.
It is not known whether either the two-hybrid inter-
actions or the co-immunoprecipitation of pairs of Lsm
proteins is facilitated by U6 snRNA. However, it is
interesting to note the strong co-immunoprecipitation of
HA-Lsm1p with Lsm4p (in 150 mM NaCl), given that
U6 snRNA did not co-precipitate with Lsm1p in the same
conditions. Thus Lsm1p and Lsm4p may interact in the
absence of U6 snRNA.
From the analysis of snRNA levels in the conditional
strains, it is evident that the level (and presumably the
of U6 snRNA is dependent on the presence of seven
Sm-like proteins and U6 snRNA
Fig. 7. Co-immunoprecipitation of snRNAs with Lsm proteins. (A) Extracts from strains each producing an HA-tagged Lsm protein or HA-tagged
control protein (all as in Figure 2) were incubated with anti-HA antibodies. Total lanes contain RNA from one-fifth the amount of extract used in
the immunoprecipitation reactions. RNAs were analysed as described in Figure 4. Note that HA-tagged Lsm2p is precipitated relatively inefficiently
by anti-HA antibodies (data not shown), presumably due to poor availability of the epitope tag. (B) Non-denaturing analysis of U6 snRNA
co-immunoprecipitated with Lsm proteins. The RNAs were extracted from the precipitates and analysed by non-denaturing electrophoresis, Northern
blotting and probing for U6 snRNA. Total as in (A).
Fig. 8. Analysis of U4/U6 and U4/U6·U5 complexes in lsm deletion
strains. (A) Non-denaturing analysis of U4 and U6 snRNAs in the
lsm6 and lsm7 deletion strains. Extracts are from the lsm6 (AEMY19)
and lsm7 (AEMY22) deletion strains, and LSM6 (AEMY34) and
LSM7 (AEMY35) controls grown at 30°C. Total RNA from each
extract was analysed as in Figure 7B. As a control, a sample of RNA
was denatured by boiling prior to loading on the gel. (B) Analysis of
tri-snRNPs in the lsm7 extract. Extracts from the lsm7 deletion and
LSM7 control strains were incubated with anti-Prp8p antibodies and
the immunoprecipitated RNAs were extracted and analysed by
denaturing gel electrophoresis as for Figure 4, probing for U4, U5 and
U6 snRNAs. The levels of these RNAs were compared by
functional Lsm proteins. This is similar to the effect on
the U1, U2, U4 and U5 snRNAs of depleting Sm proteins
(e.g. Rymond, 1993).
Pannone et al. (1998) reported similar effects of La
protein in the biogenesis of the U6 snRNP, suggesting
that the La protein (which associates with nascent U6 and
other RNA polymerase III transcripts) acts as a chaperone,
stabilizing the U6 snRNA structure in a conformation
suitable for the formation of the U6 snRNP. These authors
described a synthetic lethal interaction between the genes
encoding the La and Lsm8 proteins, and proposed that
Lsm8p may be the first U6-specific protein that binds to
U6 snRNA, although no evidence was presented for a
direct interaction between these two proteins. Since the
La protein is non-essential, it may be the Lsm complex
which is really the chaperone. The La protein may function
by ‘handing off’ (as proposed by Herschlag, 1995) the
U6 snRNA from the transcription machinery to the Lsm
proteins, which in turn facilitate di- and tri-snRNP forma-
tion. Subtle but reproducible effects of Lsm protein
depletion on levels of pre-P, pre-5S and pre-tRNAs also
support the proposal of Pannone et al. (1998). Although
none of these other RNAs was observed to co-precipitate
with any of the HA-tagged Lsm proteins in extracts, Lsm
proteins appear to bind to some of these RNAs in
vitro (L.Verdone and J.D.Beggs, unpublished results). The
specificity of RNA binding by these proteins is currently
being investigated more fully. U6 snRNA is different from
continued association with the Lsm proteins for its stable
accumulation as an RNP. The mature forms of the other
polymerase III products associate with different sets of
Analysis of the U4 and U6 snRNAs under non-
denaturing conditions showed that in the absence of either
Lsm6p or Lsm7p, free U6 snRNPs were severely depleted,
and free U4 particles accumulated, presumably as a
consequence of the reduced level of U6 in these cells.
An effect on U4/U6·U5 tri-snRNP formation was also
observed; with extract lacking Lsm7p (or Lsm4p;
Cooper et al., 1995), the amount of tri-snRNPs co-
immunoprecipitating with Prp8p was reduced, although
the level of U4/U6 duplex was normal. Thus Lsm4p and
Lsm7p, at least, may play a role in tri-snRNP formation
and/or stabilization. The co-precipitation of U5 snRNA
with some of the HA-tagged Lsm proteins (Figure 7)
A.E.Mayes et al.
Fig. 9. Sequence alignment of Sm-like proteins. (A) Putative homologues of Lsm2p were identified by BLAST searches (http://www.ncbi.
nlm.nih.gov/cgi-bin/BLAST/nph-blast?Jform?0) and aligned using the PILEUP program in GCG10. Identities and similarities are highlighted using
BOXSHADE 3.21 (http://www.isrec.isb-sib.ch/software/BOX_form.html). The positions of the Sm motifs 1 and 2 are indicated. White on black
represents amino acid identity in at least five of the nine sequences; black on grey represents conservation of the nature of the amino acid at that
site. Accepted groupings were M?I?V?L, K?R?H, F?Y?W, S?T, E?D, A?G, Q?N. The accession numbers of the proteins are as follows:
Homo sapiens, AA315292; Mus musculus, U85207; Branchiostoma floridae, Z83273; Drosophila melanogaster, AA821196; Brugia malayi,
AA228204; Caenorhabditis elegans, Z81118; Arabidopsis thaliana, AC005278; Saccharomyces cerevisiae, P38203; Schizosaccharomyces pombe,
AL034491. The sequences shown for the C.elegans and A.thaliana proteins differ from those in the database due to the predicted use of other splice
sites. The conceptual translations used here produce proteins of the size expected for Lsm2p homologues, whilst those in the database give larger
products with a disrupted Sm architecture. (B) Sm-like proteins from ancient organisms. Polypeptide sequences containing the Sm motifs were
identified from BLAST searches of protein databases. Sequences were aligned with identities and similarities highlighted as in (A). MTH649,
Methanobacterium thermoautotrophicum protein (accession No. AE000845); MTH1440, M.thermoautotrophicum protein (accession No. AE000905);
AF362, Archaeoglobus fulgidus protein (accession No. AE001079); AF875, A.fulgidus protein (accession No. AE001044); P. minut., Pedinomonas
minutissima protein (accession No. U58510).
and with untagged Lsm4p indicates at least a transient
association of Lsm proteins with tri-snRNPs.
The yeast Prp24 protein has been identified as a factor
involved in the formation and disassembly of U4/U6
duplexes (Ghetti et al., 1995; Jandrositz and Guthrie,
1995). Interestingly, prp24 mutants resemble lsm mutants
in having reduced levels of U6 snRNA and, in extracts
containing destabilized U4/U6, Prp24p co-precipitated
small amounts of free U4 as well as free U6 and base-
paired U4/U6 (Ragunathan and Guthrie, 1998a). Prp24p
has been demonstrated to promote the annealing of U4
and U6 snRNPs in yeast extracts; however, the annealing
activity of Prp24p with deproteinized U4 and U6 snRNAs
was markedly lower, prompting the suggestion that other
proteins contribute to the rate of annealing (Ragunathan
and Guthrie, 1998a). Thus, Lsm proteins may cooperate
with Prp24p in promoting the association of the U4
and U6 snRNAs. In support of such a model, genetic
interactions between LSM4 and PRP24 have been
observed (A.E.Mayes, M.Cooper and J.D.Beggs, un-
published results), and Prp24p has been identified in
exhaustive two-hybrid screens with several Lsm proteins
RNA–protein interactions are expected to respond to
changing circumstances faster than RNA–RNA inter-
actions (Herschlag, 1995); thus the Lsm proteins may
have a chaperone-like function to facilitate U4/U6 dimer
formation and possibly also in tri-snRNP assembly,
minimizing the energy required to drive conformational
rearrangements. This would be additional to the roles of
Prp24p as a ‘matchmaker’ (Herschlag, 1995) in the
association of U4 with U6, and of the RNA-unwinding
protein Brr2p in their dissociation (Ragunathan and
In summary, we propose that the Lsm proteins form a
complex on free U6 RNA that is either newly synthesized
(and may be La-associated; Pannone et al., 1998) or
released from dissociating spliceosomes. This complex
protects the RNA against degradation and may facilitate
subsequent conformational rearrangements of the RNA
and/or of other proteins involved in the association of U6
with U4 and then with U5 snRNPs as these particles form
tri-snRNPs. The weak co-precipitation of U5 snRNA with
the Lsm proteins suggests that either the epitopes become
masked in the tri-snRNPs or the Lsm proteins dissociate
from the tri-snRNPs soon after their formation and/or the
destabilization of the U4/U6 interaction in tri-snRNPs.
Sm-like proteins and U6 snRNA
Following completion of the splicing reaction, the U6
snRNA must reassociate with the U4 snRNA, and in cells
depleted of Lsm proteins this U6 snRNA is degraded,
resulting in the accumulation of free U4 snRNPs.
An interesting question that remains is the subcellular
localization of the Lsm proteins and of U6 snRNA. Most
current evidence suggests a nuclear localization for U6
snRNA; however, U6 snRNA free from U4 snRNA has
been reported to be present and matured in the cytosol of
mouse fibroblasts prior to nuclear import and association
with U4 snRNA (Fury and Zieve, 1996). Obviously, an
additional role for the Lsm protein complex may be to
act as (part of) a nuclear localization signal analogous to
the canonical Sm proteins, facilitating the nuclear import
of U6 snRNA that might be present transiently in the
cytosol, for example immediately after mitosis.
The stoichiometry of the proteins present in the
canonical Sm complex has been studied (Raker et al.,
1996; Plessel et al., 1997), and a structural model has
been proposed in which a single copy of each protein is
present in a seven-membered complex (Kambach et al.,
1999). Data presented here suggest that seven interacting
Lsm proteins associate with U6 snRNA and, although it
is not demonstrated that all interact simultaneously in the
by analogy with the canonical Sm complex.
In striking contrast to the others, depletion of Lsm1
had no effect on the level of U6 (or other polymerase III
transcripts) or on the efficiency of pre-mRNA splicing,
and HA-tagged Lsm1p did not associate stably with U6
snRNA. Therefore, although Lsm1 can interact with the
other Lsm proteins, it is not a component of the
U6-associated complex. It is therefore conceivable that
there is more than one form of Lsm complex with
alternative protein compositions, which might have
distinct functions and/or substrate specificities. This is
also suggested by the fact that, in most cases, the growth
defect caused by the depletion of the Lsm proteins is not
fully suppressed by the overproduction of U6 snRNA
(Figure 6). This would be expected if Lsm proteins have
another essential function that is independent of U6.
Clearly, the proposed role of Lsm1p in decapping mRNA
(Boeck et al., 1998) suggests a possible function for
another Lsm complex. Indeed, data from two-hybrid
screens indicate that several Lsm proteins interact with
factorsinvolved in mRNA
M.Fromont-Racine, J.D.Beggs and P.Legrain, unpublished
results), and there is direct evidence that multiple Lsm
proteins influence mRNA decapping (R.Parker, personal
Database searches reveal that some Lsm proteins appear
to have been conserved through evolution. A sequence
alignment (Figure 9A) of Lsm2p structural homologues
from a number of higher organisms shows that the
sequence identities extend beyond the Sm motifs. Lsm2p
has 63% identity (75% similarity) to the human sequence,
whilst the other S.cerevisiae Lsm proteins all have ?32%
identity (41% similarity). Also, the identification of an
Sm-like protein and a U6-like RNA in the miniaturized
genome of Pedinomonas minutissima suggests that these
are ancient macromolecules (Gilson and McFadden,
1996).The presenceof Smmotif sequencesinthe genomes
of Methanobacterium thermoautotrophicum and Archaeo-
globus fulgidus (Klenk et al., 1997; Smith et al., 1997)
reinforces this theory (see Figure 9B). Since neither
of these archaebacteria contains recognizable splicing
machinery, these Sm-like proteins may affect processes
more fundamental than RNA splicing. Eukaryotes may
have enlarged the Lsm protein family, and recruited Lsm
proteins to function in pre-mRNA splicing in addition to
their roles in other cellular processes.
In this work, we have investigated the association of
the Lsm proteins with U6 snRNA and their consequent
role in pre-mRNA splicing. However, there are strong
indications that these proteins may have more general
functions. Hopefully, the functional characterization of a
number of interacting factors that were identified in a
systematic programme of exhaustive two-hybrid screens
with Lsm proteins as baits will give new clues as to the
roles, locations and other associations of these novel
Materials and methods
The genotypes of S.cerevisiae strains used in this work are listed in
Table II. Yeast cells were propagated and sporulated as described by
Cooper et al. (1995). Yeast transformations were performed as in Geitz
et al. (1992).
Two-hybrid direct matings
The two-hybrid bait vectors were pAS2∆∆ (Gal4 DNA-binding domain;
Fromont-Racine et al., 1997) and pBTM116 (LexA DNA-binding
domain; Vojtek et al., 1993). Two-hybrid prey were constructed using
pACTIIst (Fromont-Racine et al., 1997), except those for Smb, Smd1
and Smd3, which were as reported by Fromont-Racine et al. (1997).
Yeast strains CG1945 (for Gal4 bait fusions), L40 (for LexA bait fusions)
or Y187 (for prey fusions) were grown on selective media. Bait and
prey strains were mated by replica-plating onto rich medium, and
diploids were grown on medium selecting for both the bait and prey
plasmids, then tested on histidine-free medium for a successful two-
hybrid interaction. The stringency of the interaction was tested by growth
of the diploids on selective medium containing up to 50 mM 3-AT.
Gene deletions and complementations
All primers for PCR were based on the coding sequences in the
Saccharomyces Genome Database (http://genome-www.stanford.edu/
Saccharomyces/). Gene deletions were made by replacing the entire
coding sequence with either a HIS3 or TRP1 cassette (Baudin et al.,
1993). Each deletion was made in two genetic backgrounds, BMA38 or
BMA64 (Table II) and JDY6 (data not shown); the same results were
obtained in each strain. The diploids were sporulated at 23°C and viable
progeny were scored for the appropriate auxotrophic marker, and for
growth at 23, 30 and 37°C. For essential genes, the diploid strain was
transformed with a PGAL1-regulated version of the gene, sporulated and
haploid progeny were tested for complementation of the deletion. LSM5
and LSM8 extensively overlap uncharacterized ORFs on the other strand
(previously unannotated and YJR023c, respectively) which were also
disrupted by the knockout deletions. However, prey fusion constructs
encoding Lsm5p or Lsm8p, but not the putative products of the other
strand, complement the growth defect caused by the deletions, thus
confirming that LSM5 and LSM8 are essential.
HA-tagging of the Lsm proteins
The Lsm proteins were tagged with a single HA epitope (nine amino
acids) at their N-termini, with the exception of Lsm2p which was
C-terminally tagged. The HA-tagged LSM2, LSM3, LSM5 and LSM8
genes were cloned in pBM125. LSM6 and LSM7 were cloned (with the
intron of LSM7 removed) in YCpIF16 to generate PGAL1-regulated,
HA-tagged versions. The two-hybrid prey fusions of LSM5 and LSM8
have an HA tag and were used for the immunoprecipitation studies. The
chromosomal LSM1 gene was tagged by replacing lsm1∆::TRP1 in
AEMY24, with HA-tagged LSM1 sequence. The functional ability of
each tagged protein was confirmed by rescue of the growth defect of
the gene-deleted strains.
A.E.Mayes et al.
RNA extraction and analysis
RNA was extracted from yeast cells by the method of Schmitt et al.
(1990). Denaturing Northern analysis and probes for detecting the small
RNAs were as described by Cooper et al. (1995). Oligonucleotides were
provided by D.Tollervey for the detection of: P RNA, ATTTCTG-
ATAACAACGGTCGG; MRP RNA, AATAGAGGTACCAGGTCAA-
GAAGC; 5S rRNA, CTACTCGGTCAGGCTC; and U3 (2?-O-methyl-
RNA), UUAUGGGACUUGUU. Non-denaturing conditions for the
extraction of complexed U4/U6 snRNAs from yeast cell extracts were
as in Brow and Guthrie (1988).
Yeast cell extracts were prepared as described by Lin et al. (1985).
Immunoprecipitation of RNAs was as described by Cooper et al. (1995).
Co-precipitated proteins were analysed by SDS–PAGE and Western
analysis, and visualized by enhanced chemiluminescence (ECL,
Amersham). Antibodies for immunodetection and immunoprecipitation
were: anti-HA antibodies (Boehringer Mannheim), rabbit polyclonal
anti-Lsm4p (anti-Uss1p) or anti-Prp8p antibodies (Cooper et al., 1995),
and anti-mouse horseradish peroxidase (HRP)-conjugated second
We are very grateful to M.Fromont-Racine for the LSM8 deletion strain,
to M.Fromont-Racine and J.-C.Rain for providing the LSM8 two-hybrid
constructs prior to publication, to M.Cooper for lsm4∆, lsm4-A and
lsm4-B constructs,and to Jeremy Brown,Roy Parker andDavid Tollervey
for helpful comments on this manuscript. A.E.M. was the recipient of
a Wellcome Trust Prize Studentship. J.D.B. holds a Royal Society
Cephalosporin Fund Senior Research Fellowship. This work was partly
funded by Wellcome Trust Grant 044374 and EU Biotech Grant 95007
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genome sequence of
Received February 1, 1999; revised June 11, 1999;
accepted June 15, 1999