From the Wellcome Trust Centre for Cell Biology, Swann Building, King's Buildings, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom
Received for publication, August 29, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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Depletion of any of the essential Lsm
proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led
to the accumulation of many aberrant processing intermediates,
indicating that an Lsm complex is required to maintain the normally
strict order of processing events. In addition, high levels of
degradation products derived from both precursors and mature rRNAs
accumulated in Lsm-depleted strains. Depletion of the essential Lsm
proteins reduced the apparent processivity of both 5' and 3'
exonuclease activities involved in 5.8S rRNA processing, and the
degradation intermediates that accumulated were consistent with
inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species
could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or
non-tagged control strains, suggesting their direct interaction with an
Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein
interactions and structural changes required during ribosomal subunit assembly.
The yeast 18S, 5.8S, and 25S rRNAs are transcribed by RNA
polymerase I as a single precursor, the 35S pre-rRNA, which undergoes complex post-transcriptional processing to remove the external transcribed spacers (5'-ETS and
3'-ETS)1 and internal
transcribed spacers (ITS1 and ITS2) to release mature rRNAs (see Fig.
1A). This process involves multiple endonucleolytic and
exonucleolytic steps (see Fig. 1B) and is largely carried out in the nucleolus. In Saccharomyces cerevisiae, enzymes
directly involved in these reactions include the endonucleases RNase
MRP and Rnt1p, the 5' All of the enzymes known to process the pre-rRNA also process other RNA
species. Rnt1p, the exosome, and Rex proteins generate the 3'-ends of
small nuclear and small nucleolar RNAs (snRNAs and snoRNAs), whereas
Xrn1p and Rat1p produce 5'-ends of intron-encoded and polycistronic
snoRNAs (2, 6-13). Likewise, degradation of many RNAs, including
cytoplasmic messenger RNAs (mRNAs) and nuclear pre-mRNAs,
involves pre-rRNA processing exonucleases: the exosome, Xrn1p, and
Rat1p (14-18).
Sm-like (Lsm) proteins have been identified in all kingdoms of life and
participate in numerous RNA processing and degradation pathways. The Sm
and Lsm complexes are all likely to form similar structures with
seven-membered rings (or six in the case of Escherichia coli
Hfq) with a central hole, through which the RNA may pass (19-24). An
Lsm2-8p complex associates with U6 snRNA and is important for U6
accumulation, U6 snRNP biogenesis, and pre-mRNA splicing (19,
25-29). A complex of Lsm1-7p functions in cytoplasmic mRNA degradation, promoting mRNA decapping and 5' degradation, probably via interactions with the decapping enzymes, Dcp1p and Dcp2p, and the
5' Sm-like proteins from Bacteria and Archaea have been shown to
form homomeric ring structures (21, 23, 24) indicating that their
general functions are universally conserved. The E. coli
proteins are known to facilitate RNA-RNA interactions (23, 24), whereas
an Archaeal Sm-like protein associates with the RNase P RNA, and its
gene is located in an operon with a ribosomal protein, suggesting a
role in ribosome synthesis or function (21). Here, we report that yeast
Lsm2-8p are required for maintenance of the normal order of pre-rRNA
processing steps and the stability of both the pre-rRNAs and rRNAs.
Strains--
Growth and handling of S. cerevisiae
were by standard techniques. The transformation procedure was as
described previously (36). Yeast strains used and constructed in this
study are listed in Table I. StrainYJK34
was constructed by PCR strategy as described (37); construction was
confirmed by PCR analysis, and expression of TAP-Lsm3p was tested by
Western blotting using PAP antibodies.
RNA Extraction, Northern Hybridization, and Primer
Extension--
For depletion of the essential Lsm proteins, cells were
harvested at intervals following a shift from RSG medium (1%
Bacto-yeast extract, 2% Bacto-peptone, 2% galactose, 2% sucrose, 2%
raffinose), or YPGal medium (1% Bacto-yeast extract, 2%
Bacto-peptone, 2% galactose), to YPD medium (1% Bacto-yeast
extract, 2% Bacto-peptone, 2% glucose). Otherwise strains were grown
in YPD medium. The lsm-
For RNA hybridization and primer extension, the following
oligonucleotides were used: 001 (27SA-2),
5'-CCAGTTACGAAAATTCTTG; 002 (20S-2), 5'-GCTCTTTGCTCTTGCC; 003 (27SA-3), 5'-TGTTACCTCTGGGCCC; 004 (20S), 5'-CGGTTTTAATTGTCCTA; 006 (27SB), 5'-GGCCAGCAATTTCAAGTTA; 007 (25S+40), 5'-CTCCGCTTATTGATATGC;
008 (18S+34), 5'-CATGGCTTAATCTTTGAGAC; 011 (18S+186),
5'-TCTCTTCCAAAGGGTCG; 013 (RNA2.1), 5'-AGATTAGCCGCAGTTGG; 017 (5.8S+30), 5'-GCGTTGTTCATCGATGC; 020 (ITS2-5'B),
5'-TGAGAAGGAAATGACGCT; 022 (25S/3'ETS), 5'-GAAATAAAAAACAAATCAGAC; 026 (5'ETS+911), 5'-CCAGATAACTATCTTAAAAG; 029 (18S+1785),
5'-TAATGATCCTTCCGCA; 030 (18S+668), 5'-TTGGAAATCCAGTACACG; 033 (5'ETS+278), 5'-CGCTGCTCACCAATGG; 053 (3'ETS+180),
5'-TGGTACACTCTTACACAC; 471 (SmX3),
5'-ACGCCTACACGATGGTTGACCAGGCCTTTGAGGA.
Immunoprecipitation--
Whole cells extracts were prepared as
described (37) using extract equivalent to 1.6 × 1010
cells. Affinity purification of TAP-tagged Lsm3p protein was performed
as described (37) with rabbit IgG-agarose beads (Sigma). Untagged
isogenic strain (YJV140) was utilized as control. Immunoprecipitation from lysates of the HA::Lsm1 and
isogenic wild-type (BMA64) strains was performed using rat monoclonal
anti-HA high affinity Ab (Roche Molecular Biochemicals) bound to
Protein G-agarose (Roche Molecular Biochemicals). Copurified RNAs were
recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol
precipitation. Precursors and mature RNAs were identified by Northern
hybridizations and primer extension analysis.
Pulse-chase Labeling--
Metabolic labeling of RNA was
performed as described previously (41). The
GAL::lsm3 strain was pre-grown in
galactose minimal medium lacking uracil and transferred to glucose
minimal medium for 8.5 h. The isogenic WT strain (YJK53) was grown
directly in glucose minimal medium. Cells at 0.3 A600 nm were labeled with
[3H]uracil for 1 min followed by a chase with excess
unlabeled uracil for 1, 2.5, 5, 10, 20, and 60 min.
Depletion of Any Essential Lsm Protein Leads to the Appearance of
Aberrant Pre-rRNAs and Degradation Intermediates--
Pre-rRNA
processing (Fig. 1) was assessed by
pulse-chase labeling with [H3]uracil in the
GAL-lsm3 strain in which expression of the essential Lsm3p
was under GAL control. Cells were labeled with
[3H]uracil for 1 min followed by a chase with excess
unlabeled uracil for the times indicated (Fig.
2). Overall incorporation into pre-rRNA and rRNA was strongly reduced in the Lsm3p-depleted strain. Labeling was performed following growth on glucose medium for 8.5 h, prior to the appearance of any clear growth defect (27, 35, and data not
shown), so the reduced incorporation is unlikely to be a simple consequence of growth inhibition.
Early steps in pre-rRNA processing were strongly retarded in the
Lsm3p-depleted strain, leading to substantial accumulation of the 35S
pre-rRNA (Fig. 2A, lanes 7-12). This was
accompanied by a delay in the synthesis of the 27S and 20S pre-rRNAs.
Little 27SA2 pre-RNA was synthesized in the mutant, so the
27SB pre-rRNA was presumably generated largely by cleavage at sites
A3 and B1L. Mature 18S and 25S rRNAs were
synthesized with considerable retardation, but their relative ratio was
not clearly altered. Synthesis of the mature 5.8S rRNA was also
strongly retarded in the GAL::lsm3 strain (Fig. 2B). The aberrant 23S RNA was detected together
with other aberrant RNA intermediates (marked with asterisks
in Fig. 2, A and B; see Fig. 1C for
the identities of aberrant pre-rRNA species seen in Lsm-depleted
strains). We conclude that the depletion of Lsm3p inhibits pre-rRNA
processing. The 35S pre-rRNA was present even after 60-min chase (Fig.
2A, lane 12) and in other experiments was shown
to persist at 2-, 4-, 6-, and 12-h chase time points (data not shown).
This is probably a consequence of degradation of the labeled pre-rRNA
and rRNAs in the Lsm3p-depleted strain, with subsequent
re-incorporation of the labeled nucleotides, which cannot be chased by
exogenous uracil. As previously reported (35), synthesis of
tRNAs was also retarded in the strain depleted of Lsm3p.
Pre-rRNA processing was analyzed in more detail by Northern
hybridization (Figs.
3-5).
The essential Lsm proteins, Lsm2-5p and Lsm8p, were placed under
GAL control (strains
GAL::lsm2,
GAL::lsm3, GAL::lsm4,
GAL::lsm5, and
GAL::lsm8) (27) and depleted by
transferring the strains from permissive RSG medium (0-h samples) to
repressive glucose medium. The genes encoding non-essential Lsm
proteins, Lsm1p and Lsm6-7p, were deleted, giving rise to
temperature-sensitive strains (strains lsm1-
Clear pre-rRNA processing defects were seen in the
GAL::lsm2 to lsm5 and
GAL::lsm8 strains at early times of
depletion (shown for GAL::lsm3 in Fig.
3, the complete set of strains are shown for low molecular weight
pre-rRNAs in Fig. 5). Even on galactose medium some elevation was seen
in the level of the 35S pre-rRNA in the
GAL::lsm3 strain, but other processing
intermediates were present at wild-type levels. Processing was clearly
defective 6 h after transfer to glucose medium, before the
appearance of any detectable growth defect (27, 35, and data not
shown). The 35S primary transcript was further elevated, accompanied by
the appearance of the aberrant 23S RNA and depletion of the
27SA2 and 20S pre-rRNAs (Fig. 3, A-C). This
phenotype is characteristic of the inhibition of pre-rRNA processing at
sites A0 to A2. The level of the 27SB pre-rRNA
was less strongly reduced at 6 h but was clearly reduced at later
time points (Fig. 3D), as were the mature 25S and 18S rRNAs
(Fig. 3E).
In addition to the loss of the normal pre-rRNA processing
intermediates, there was a dramatic accumulation of aberrant pre-rRNAs in the Lsm3p and other Lsm-depleted strains. These included the 23S and
21S RNAs, which have been seen in several other pre-rRNA processing
mutants, as well as many unusual intermediates, some of which are
indicated in Fig. 1C. These included fragments extending from the 5'-end of the transcript to site D (5'ETS-D), from
A1 to B1, and from site D to the 3'-end of the
25S rRNA (D-B2) (Fig. 3; see also Fig.
1C). None of these species have been observed in the
wild-type or reported for previously characterized pre-rRNA processing
mutants. The appearance of these RNAs indicates that processing events
in the 5'-ETS, ITS1, and ITS2 do not occur in the normal order
following depletion of the Lsm complex.
Additionally, a set of truncated and heterogeneous pre-rRNA-derived
species, which are presumed to be degradation intermediates, strongly
accumulated upon depletion of each of the essential Lsm proteins
(bracketed and indicated with an asterisk in Fig.
3). Degradation intermediates were readily observed with probes
directed against each of the pre-rRNA spacer regions, and
the total signal for these heterogeneous species was equal to or
greater than the pre-rRNAs in the wild-type strain, indicating
substantial degradation of all regions of the pre-rRNA (Fig. 3 and data
not shown). In addition, probes against the mature 18S and 25S rRNAs
detected fragments apparently derived from their breakdown (Figs.
3E and 4).
Following transfer of the GAL::prp45
(data not shown) or GAL::syf3 strain
(Fig. 3) to glucose medium, the time course and degree of pre-mRNA
splicing inhibition was comparable to the Lsm3p-depleted strain (see
Fig. 5I below) and pre-rRNA processing was inhibited, with
accumulation of 35S, 23S, and 21S species and reduced levels of
27SA2 20S pre-rRNAs. However, the pre-rRNA processing
defect was distinctly different from that seen on Lsm3p depletion. In particular, no accumulation of aberrant intermediates or breakdown products was observed in the GAL-syf3 strain, and levels of
the 20S pre-rRNA and mature rRNAs were not strongly reduced, probably because growth is inhibited for other reasons allowing a reduced rate
of ribosome synthesis to maintain normal rRNA levels at the reduced
growth rate. Depletion of Lsm1-7p (but not Lsm8p) also reduces 5'
degradation of cytoplasmic mRNA (32). Strains carrying the
xrn1-
Strains lacking the non-essential proteins Lsm1p, 6p, or 7p showed only
mild defects in the processing of high molecular weight pre-rRNAs (data
not shown), although accumulation of 35S and 23S was seen, indicative
of a delay in cleavage at sites A0-A2. In the
lsm1-
Fusion constructs between the DNA binding domain of Gal4p and Lsm2p and
Lsm5p, which were constructed for use in two-hybrid analyses (50),
confer partial temperature sensitivity for growth and cytoplasmic
mRNA degradation in lsm2-
We conclude that depletion of any essential Lsm protein causes some
inhibition of pre-rRNA processing accompanied by disorganization of the
processing pathway leading to the production of several unusual
pre-rRNAs. Degradation of these species appears to be inefficient
leading to the accumulation of abundant heterogeneous intermediates.
Depletion of the Essential Lsm Proteins Provokes Degradation of the
Mature rRNAs--
The apparent degradation of mature rRNAs seen
following depletion of any essential Lsm protein (shown for Lsm3p in
Fig. 4) was further investigated by Northern hybridization. The mature 25S (Fig. 4A) and 18S (Fig. 4C) rRNAs were
strongly depleted, with the appearance of abundant truncated products
(Fig. 4, B and D). In contrast, much less
depletion of the 5.8S rRNA was seen (Fig. 4E). Because 25S
and 5.8S are cosynthesized and base-paired, this shows that the loss of
mature 25S is due largely to post-synthesis degradation, rather than
impaired synthesis.
The identities of the 18S rRNA breakdown products were examined in more
detail using probes against different regions of the 18S rRNA (Fig.
4D, lanes 11-14). The most 5' probe (008)
detects products that are 3'-truncated but not 5'-truncated. Probes
located at positions 186 and 668 from the 5'-end of 18S (011 and 030) detect both 5'- and 3'-shortened species. The 3' probe (029) detects 5'-truncated but not 3'-truncated RNAs. This analysis shows that the
majority of the 18S rRNA is degraded 3'
The exosome complex functions in the degradation of pre-rRNA spacers
and aberrant pre-rRNA intermediates (51, 52) and is a likely candidate
to also degrade the rRNAs. Processing of the 3'-end of the 5.8S rRNA
involves the exosome (53), and the mature 5.8S rRNA may therefore be a
poor substrate for degradation. We conclude that depletion of any
essential Lsm protein provokes the degradation of both pre-rRNAs and
mature rRNAs. Degradation of mature tRNAs and tRNA precursors was also
observed in the strains lacking Lsm proteins, particularly the
essential Lsm2-5p or Lsm8p (35).
Depletion of the Essential Lsm Proteins Inhibits 5' and 3'
Processing of 5.8S rRNA and Exosome-mediated Spacer
Degradation--
Consistent with the high molecular weight RNA
analysis, dramatic accumulation of aberrant precursors to the 5.8S rRNA
was seen in strains depleted for any of the essential Lsm proteins (Fig. 5). The 27SB pre-rRNA is 5'-processed at two closely located sites, B1L and B1S, prior to cleavage at site
C2 to generate the 7SL and 7SS
pre-rRNA, the major 3'-extended precursor to 5.8S rRNA (see Fig.
1B). The 7S pre-rRNA is 3'-processed by the exosome complex
(53-55). This reaction normally proceeds with high processivity but is
multistep generating, successively, the 5.8S+30 pre-RNAs and 6S
pre-rRNAs. Processing of 5.8S+30 specifically requires the Rrp6p
component of the exosome.
Northern hybridization with a probe specific for the 3'-extended 5.8S
species (probe 020; Fig. 5) showed that depletion of any essential Lsm
protein (Fig. 5A, lanes 3-17) resulted in some decrease in the level of the 7S pre-rRNA and mature 5.8S (Fig. 5F), and the loss of the 5.8S+30 and 6S pre-rRNAs. Aberrant
species intermediates in size between 7S and 5.8S+30 were also
accumulated. The most abundant of these comigrated with RNAs observed
in exosome mutants (data not shown) (11), consistent with inhibition of the exosome complex in the Lsm-depleted cells. Less striking effects were seen in strains lacking the non-essential proteins, Lsm1p, 6p, or
7p, but the 7S pre-rRNA was clearly reduced in lsm6-
In addition to processing the 3'-end of the 5.8S rRNA, the exosome
normally degrades the 5'-ETS region of the pre-rRNA from site
A0 to the 5'-end of the transcript (51). In strains
depleted of the essential Lsm proteins, the 5'-ETS-A0
fragment accumulated together with shorter fragments (Fig.
5A, lanes 3-17). Weaker accumulation of
intermediates was seen in strains lacking Lsm6p or Lsm7p (Fig.
5B, lanes 20-25). The degradation remains
predominately 3'
In addition to the species below 7S, the Lsm-depleted strains also
accumulated larger 5.8S-related RNA species. The largest species
detected with probe 020 (Fig. 5A) has the gel mobility and
hybridization pattern expected for a species that extends from site
A2 in ITS1 to site C2, the 3'-end of the 7S
pre-rRNA (see Fig. 1C for schematics of aberrant
processing intermediates detected in lsm mutant strains).
Northern analyses with probes hybridizing 5' to 5.8S (003 and 001; Fig.
5, D and E), identified RNAs that extend from
sites A2 and A3, within ITS1, to site E (the
3'-end of the 5.8S rRNA). Accumulation of the
A2-C2, A2-E, and
A3-E fragments was also observed in the strains deleted
for the non-essential proteins Lsm6 and 7p (Fig. 5, D and
E, lanes 20-25) even under the permissive
conditions at 23 °C. Notably, the A3-E species did not
visibly accumulate in the GAL-lsm8 strain (Fig.
5E, lanes 15-17) but was clearly accumulated in
the lsm1-
The appearance of the A2-C2,
A2-E, and A3-E species shows that processing
in ITS2 had occurred prior to the completion of processing in ITS1. We
conclude that the normally strict order of processing is disorganized
in the Lsm-depleted strains. Further evidence for disruption of the
normal order of processing came from the detection of intermediates
that appear to extend from site D (the 3'-end of the 18S rRNA) to site
B1, encompassing the entire ITS1 region (Fig. 5,
D-F). Fragments extending from site D to sites
A2 and A3 within ITS1 also accumulated in the
lsm2-8 mutants.
The processing of 5.8S precursors was also examined in the
GAL::prp45 (data not shown) and
GAL::syf3 strains (Fig. 5, G
and H). Depletion of Syf3p also affected the processing of
5.8S rRNA, with reduced levels of the 7S and 6S pre-rRNAs, but no
accumulation of aberrant precursors was observed in Syf3p-depleted
cells. The defects in splicing activity, as assessed by the level of
pre-U3, were comparable in the
GAL::lsm3 and
GAL::syf3 strains (Fig.
5I).
Depletion of Lsm Proteins Inhibits 5' Processing of 5.8S
rRNA--
The 5'-end of the major, short form of the 5.8S rRNA
(5.8SS) is generated by 5'
The pre-rRNA primary transcript is cleaved in the 3'-ETS by the
endonuclease Rnt1p at sites 14 and 49nts 3' to the mature 25S (site
B0) generating the 35S pre-rRNA (1, 57). These cleavages
can be detected by primer extension on the cleaved 3' fragment and were
unaffected in the Lsm3p and Lsm8p-depleted strains (Fig. 6G,
lanes 2 and 3; and data not shown). The cleaved fragment is
stabilized by mutation of Rat1p (Fig. 6G, lane 4) allowing the identity
of the stops to be confirmed.
We conclude that the Lsm proteins are required for the normal, highly
processive activities of both 5' Lsm3p Is Associated with Pre-rRNAs--
To test for a physical
association of the Lsm complex with pre-rRNAs, immunoprecipitation was
performed using a strain expressing Lsm3p with a C-terminal, tandem
affinity purification (TAP) tag, under the control of the endogenous
promoter (31, 37). As a control, precipitation was also performed using
a previously described strain expressing HA-tagged Lsm1p (27).
Low molecular weight RNAs were separated on a poly-acrylamide gel and
analyzed by Northern hybridization (Fig.
7, A-G). The 7S pre-rRNA was
clearly recovered in the immunoprecipitate from the TAP-Lsm3 strain
(Fig. 7A, lane 4) but not from an otherwise isogenic wild-type strain (Fig. 7A, lane 3) or
from the HA-Lsm1 strain (Fig. 7A, lane 7). In
contrast, the smaller 6S and 5.8S+30 pre-rRNAs (Fig. 7B,
lane 4), the pre-5S rRNA (Fig. 7D) and mature MRP
RNA (Fig. 7F) were recovered at the same levels as in the non-tagged strain. The mature 5.8S and 5S rRNAs were coprecipitated with both TAP-Lsm3 and HA-Lsm1 (Fig. 7, C and E),
presumably reflecting the reported association of Lsm1-7p with
polysomes (58). The Lsm1-7p complex is reported to associate with
deadenylated mRNAs to promote decapping and 5'
High molecular weight RNAs were analyzed by primer extension (Fig. 7,
G-P). Several pre-rRNAs tested (35S, 33S,
27SA2, 27SA3, 27SBL,
27SBs, and 26S) were detectably coprecipitated with
TAP-Lsm3. Most of these species were not coprecipitated with HA-Lsm1
above the background in the wild-type control strains, although some coprecipitation was seen for the 35S pre-rRNA. The significance of this
is unclear. The 20S pre-rRNA is reported to undergo late dimethylation
at A1779 and A1780 after export to the
cytoplasm (59), which can be detected by primer extension analysis
(Fig. 7P). The dimethylated pre-rRNA was
coprecipitated with TAP-Lsm3, but weaker coprecipitation was also seen
for HA-Lsm1. Accumulation of the 20S pre-rRNA was also seen in the
lsm1-
The yield of coprecipitated pre-RNA was low but comparable to results
reported for other proteins associated with pre-ribosomal particles
(60, 61) and to the efficiency of precipitation of deadenylated
mRNAs (Ref. 32 and Fig. 7G). These results indicate that
Lsm3p transiently associates with pre-ribosomal particles, presumably
as a component of an Lsm complex.
Ribosome synthesis is a highly complex process that requires
80 r proteins and 5 kB of rRNA, as well as ~140
non-ribosomal protein factors and ~100 snoRNPs. Moreover, the very
compact structures of the mature ribosomal subunits revealed by recent
structural studies (62-64) appear to be incompatible with ribosomal
protein assembly and, particularly, with binding of the many snoRNPs to the mature rRNA regions. Efficient assembly and disassembly of the
ribosome synthesis machinery is therefore certain to involve extensive
structural reorganization and to require many cofactors. Numerous yeast
proteins, identified via defects in pre-rRNA processing and/or defects
in export of ribosomal subunits to the cytoplasm, do not appear to
function directly in pre-rRNA cleavage. In addition to the Lsm proteins
discussed here, these include at least 17 putative
ATP-dependent RNA helicases, four GTPases, and an
AAA-ATPase (3, 4, 60, 65, 66). All of these are predicted to function
in the organization of the RNP structure of the ribosomal subunits.
Here we report that normal maturation of rRNAs also requires the
activity of Lsm proteins. Depletion of the essential proteins, Lsm2-5p
or Lsm8p, resulted in severe defects in pre-rRNA processing and
stability. Milder effects were seen in strains lacking the non-essential proteins Lsm6p or Lsm7p, and it may be that these can be
partially replaced by Lsm1p or other Sm-like proteins. The accumulation
of some aberrant pre-rRNAs was clearer in the absence of Lsm1p than
following depletion of Lsm8p, suggesting their export as components of
pre-ribosomes and cytoplasmic degradation.
An obvious question is the degree to which the observed effects
are direct? Because the Lsm proteins are involved in both mRNA
degradation and pre-mRNA splicing, indirect effects are certainly a
possibility. However, strains carrying the lsm1- Several pre-rRNA species were coprecipitated with tagged Lsm3p but not
with tagged Lsm1p or in non-tagged control strains. These included the
35S, 33S, 27SA2, 27SA3, 27SB, and 7S pre-rRNAs but not the 5.8S+30 or 6S pre-rRNAs. These observations indicate the
direct interaction of a nuclear Lsm2-8p complex with pre-ribosomes. Tagged Lsm1p coprecipitated the dimethylated, cytoplasmic form of the
20S pre-rRNA and the lsm1- During synthesis of the 3'-end of 5.8S rRNA, the 5.8S+30 and 6S
pre-rRNAs are generated by rapid, processive 3' Processing of the 5'-end of the 5.8S rRNA by 5' In wild-type cells pre-rRNA processing sites are used in strict order,
generating the predominant pathway shown in Fig. 1B. In
Lsm-depleted cells a series of intermediates were detected that are
absent or extremely rare in wild-type strains, some of which are shown
in Fig. 1C. We interpret this as evidence that the Lsm
complex helps maintain the normal order of use of the pre-rRNA
processing sites, perhaps as a consequence of helping to establish the
correct RNP structure of the pre-ribosomal particles. We cannot,
however, exclude the possibility that at least some of these
"aberrant" pre-rRNAs are normal products of the processing pathway,
which have escaped detection in wild-type cells due to very rapid
degradation. Indeed, the A2-E and A3-E
species are detectable at very low levels in wild-type strains. These
species also showed some elevation in the strain lacking Lsm1p,
suggesting that they can be exported as components of a pre-60S
particle and degraded in the cytoplasm. For none of the stable
RNA species do we have accurate information on the discard rate during
in vivo processing. However, the overexpression of several
small stable RNAs seen in exosome mutants (11) suggests that this may
be substantial in some cases.
Rapid degradation of the pre-rRNAs is seen in many other strains with
defects in pre-rRNA processing or ribosomal subunit assembly (reviewed
in Ref. 4). However, in most cases this takes the form of the
disappearance of the pre-rRNAs without accumulation of degradation
intermediates. The pre-rRNAs are large (several kilobases in length),
highly structured, and associated with many ribosomal proteins and
processing factors. The RNA degradation system, therefore, has a
remarkable capacity to open RNA structures and displace stably bound
proteins. Comparison of different probes showed strong accumulation of
degradation intermediates derived from pre-rRNAs and, more
surprisingly, the mature rRNAs in Lsm protein-depleted strains.
Intermediates with appropriate gel mobility were also clearly detected
by metabolic labeling. Accumulation of pre-rRNA degradation
intermediates was previously observed in strains lacking exosome
components (52), but the intermediates that accumulated following Lsm
protein depletion were more abundant, and their pattern was distinct
from the exosome mutants. This probably relates to the observation that
depletion of the essential Lsm proteins also inhibited 5' The basis of the degradation of the mature rRNAs is not clear. The
ribosomes may sustain damage in folding or RNP structure during normal
use, which requires a chaperone function for its repair. Alternatively,
the ribosomal subunits synthesized in the Lsm protein-depleted strains
may be partially misassembled such that they are labile to subsequent
degradation. The strain lacking the nuclear Lsm8p protein showed
substantial rRNA degradation, whereas mild effects were seen in the
strain lacking the largely cytoplasmic Lsm1p. We therefore favor the
latter model. In either case, we assume that the strong accumulation of
the degradation intermediates is enhanced by reduced degradation
activity in the lsm mutant strains.
Characterized functions of eukaryotic Lsm proteins in U6 snRNA
stability and mRNA decapping are not conserved to Archaea and are
most unlikely to represent their ancestral functions. An Archaeal Sm-like protein is encoded by a gene located in a putative operon with
ribosomal protein L37e (21) and is associated with RNase P (22), which
itself participates in pre-rRNA processing. The original Sm-like
proteins are likely to have arisen as factors involved in the synthesis
of rRNAs, tRNAs, and other small stable RNA species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' exonuclease Rat1p, and
3'
5' exonucleases, including the exosome complex, Rex1p, and
Rex2p (1, 2; reviewed in Refs. 3 and 4). In addition to the RNA
processing enzymes, around 110 other factors are known to be required
for normal pre-rRNA processing in yeast. These include several small
nucleolar ribonucleoprotein (snoRNP) particles, putative RNA helicases,
GTPases, and many other assembly factors (5). It is very likely that
these act to promote correct folding of the pre-rRNA, assembly of the
~80 ribosomal proteins, and assembly/disassembly of the processing complexes, with processing inhibition arising as a secondary
consequence of defects in the structure of the pre-ribosomal particles
(see Refs. 3 and 4).
3' exonuclease Xrn1p (30-33). The Lsm1-7p complex also
protects mRNA 3'-ends from premature degradation by the exosome complex of 3'
5' exonucleases (34). Yeast Lsm proteins
additionally associate with precursors to RNase P RNA, suggesting a
direct role in its processing (28), and with tRNA precursors (35) but
not with the mature RNase P RNA or the related MRP RNA (28). Depletion
of the Lsm proteins does not reduce the accumulation of the mature MRP
or P RNAs, or the accumulation of any small nucleolar RNA (snoRNA)
tested (27).2
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this work
strains were pre-grown at
23 °C and transferred to 37 °C. RNA was extracted as described
previously (38). Northern hybridization and primer extension were as
described previously (39, 40). Standard 1.2% agarose/formaldehyde and
6% acrylamide/urea gels were used to analyze the high and low
molecular weight RNA species, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of the pre-rRNA and processing
pathway in S. cerevisiae. A, structure
of the 35S pre-rRNA and locations of oligonucleotide probes. Positions
of cleavage sites are shown in uppercase letters.
Oligonucleotides are numbered. B, major pre-rRNA
processing pathway. The 27SA2 pre-RNA is processed by two
alternative pathways giving rise to two forms of 5.8S rRNA, the major
5.8SS form and the minor 5.8SL form. Only the
major pathway leading to the synthesis of 5.8SS is shown.
An alternative pathway leads to cleavage at site B1L, the
5'-end of the 27SBL pre-rRNA, and mature 5.8SL
rRNA. The subsequent processing of both 27SB species is identical.
C, aberrant pre-rRNA processing intermediates detected in
Lsm-depleted strains.
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Fig. 2.
Pulse-chase analysis of pre-rRNA
processing. RNAs from the BMA64 wild-type (lanes 1-5)
and GAL-lsm3 (lanes 6-11) strains. Strains were
pre-grown in permissive RSG medium and transferred to glucose medium at
30 °C for 8.5 h. Cells at 0.3 A600 were
labeled with [3H]uracil for 1 min and then chased with
excess unlabeled uracil for the times indicated. Total RNA was
extracted and separated on 1.2% agarose/formaldehyde gel (panel
A) to analyze high molecular weight RNAs or a 6%
polyacrylamide/urea gel (panel B) to analyze low molecular
weight RNAs. The positions of mature rRNAs, tRNAs, and major pre-rRNAs
and pre-tRNAs are shown on the right. The
GAL-lsm3 panels were exposed ~10 times (panel
A) and 16 times (panel B) longer that those for the
wild-type.
,
lsm6-
, and lsm7-
) (27), which were grown in
glucose medium at 23 °C (0-h samples) and transferred to the
non-permissive temperature of 37 °C. Depletion of the essential Lsm
proteins leads to depletion of the U6 snRNA and the inhibition of
pre-mRNA splicing (19, 25-29). Inhibition of rRNA processing in
lsm strains can potentially arise as a result of the
splicing defect. Many r protein genes contain introns, and reduced r
protein synthesis leads to the inhibition of ribosome synthesis (42, 43). We therefore compared the Lsm-depleted strain to strains depleted
of known pre-mRNA splicing factors Prp45p (data not shown) (45) and
Syf3p (Figs. 3 and 5) (44).
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Fig. 3.
Normal synthesis of 18S and 25S rRNA requires
Lsm proteins. A-E, Northern analysis of high molecular
weight pre-rRNA processing in GAL-lsm3 and
GAL-syf3 mutants. Wild-type (WT, lanes
1 and 2), GAL-lsm3 (lanes 3-7),
and GAL-syf3 (lanes 8-12) strains were grown in
permissive, RSG medium (0 h) and transferred to repressive, glucose
medium at 30 °C for the times indicated. RNA was separated on a
1.2% agarose gel and hybridized with oligonucleotide probes. Probe
names are indicated in parentheses below each panel. RNA
species are shown on the right, and degradation products are
marked with an asterisk. The positions of migration of 18S
rRNA (1795 nucleotides), U2 snRNA (1175 nucleotides), and U1 snRNA (668 nucleotides) determined by hybridization of the same filter are
indicated on the left as size markers. A,
hybridization with oligonucleotide 026 complementary to the 5'-ETS/18S
boundary; B, hybridization with oligonucleotide 002 complementary to ITS1 upstream of site A2; C,
hybridization with oligonucleotide 001 complementary to ITS1 downstream
of site A3; D, hybridization with
oligonucleotide 006 complementary to ITS2; E, hybridization
with oligonucleotides 007 and 008 complementary to the mature 25S and
18S, respectively. Comigrating bands are indicated by dashes
between lanes.
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Fig. 4.
Deletion of Lsm3p leads to degradation of the
mature rRNAs. The wild-type strain (WT, lanes
1-3) and GAL-lsm3 strain (lanes 4-11) were
grown in permissive RSG medium (0 h) and transferred to repressive,
glucose medium at 30 °C for the times indicated. Probe names are
shown in parentheses. RNA species are indicated.
A and B, hybridization with the 25S 5' probe
(oligonucleotide 007: position +40). C and D,
hybridization with the 18S rRNA 5' probe (oligonucleotide 008; position
+34). Oligonucleotides 011, 030, and 029 are complementary to the 18S
rRNA at positions further 3' (+178, +668, and +1785, respectively).
These are shown in panel D as lanes 12-14 for
the GAL::lsm3 strain 30 h after
transfer to glucose medium. E, hybridization with 5.8S rRNA
probe (oligonucleotide 017). Comigrating bands are indicated by
dashes between lanes.
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Fig. 5.
Normal 5' and 3' processing of
5.8S rRNA requires Lsm proteins. A-F, Northern analysis of
5.8S rRNA processing in lsm mutant strains. RNA was
separated on a 6% polyacrylamide gel and hybridized with
oligonucleotide probes. Probe names are indicated in
parentheses on the left. RNA species are shown
between two columns. Strains carrying
GAL-regulated constructs
(GAL::lsm, lanes 3-17) and
the BMA64 wild-type strain (WT, lanes 1-2) were
grown in permissive RSG medium (0 h) and transferred to repressive,
glucose medium at 30 °C for the times indicated. Strains deleted for
Lsm1p (lanes 26-28), Lsm6p (lanes 20-22), and
Lsm7p (lanes 23-25) and the BMA64 wild-type strain
(WT, lanes 18 and 19) were pre-grown
at 23 °C (0 h) and transferred to 37 °C for the times indicated.
Probes are: A, oligonucleotide 033, complementary to 5'-ETS
around position +278; B, oligonucleotide 002, complementary
to ITS1 upstream of site A2; C, oligonucleotide
003, complementary to ITS1 upstream of site A3;
D, oligonucleotide 001, complementary to ITS1 downstream of
site A3; E, oligonucleotide 020 complementary to
the 5.8S-ITS2 boundary; F, oligonucleotide 017, complementary to the mature 5.8S. G-I, comparison of low
molecular weight pre-rRNA processing and accumulation of unspliced
pre-U3 RNA (U3-int) and mature U3 in GAL-lsm3 (lanes
1-5) and GAL-syf3 strains (lanes 6-10).
Strains were grown and RNA was prepared as described for Fig. 4.
or dcp1-
mutations, which also
inhibit mRNA 5' degradation (16), have been extensively studied and
do not result in pre-rRNA processing defects that resemble those seen
in the Lsm-depleted strains (46, 47, and data not shown). A
prp2-1/xrn1-
strain (18), which is
simultaneously inhibited for splicing and mRNA turnover, also
failed to show such defects (data not shown). We conclude that the
inhibition of pre-mRNA splicing and mRNA turnover does not
generally lead to the pre-rRNA turnover defects seen upon Lsm protein
depletion. It remains formally possible that specific defects in these
activities, particularly in pre-mRNA splicing, do contribute to the
lsm pre-rRNA processing phenotype.
strain, but not the lsm6-
or
lsm7-
strains, the 20S pre-rRNA was depleted. It is
notable that processing of the 20S pre-rRNA to the mature 18S rRNA
takes place in the cytoplasm (48, 49), and Lsm1p also localizes mainly
to the cytoplasm (32).
and lsm5-
backgrounds (32, 34). However, these alleles did not clearly affect
pre-rRNA processing or stability (data not shown).
5' with minor degradation occurring in the 5'
3' direction.
strain (Fig. 5A, lanes 20-28). Growth inhibition
in the lsm6 and lsm7 strains at 37 °C is much
more rapid than in the GAL::lsm
strains, and their growth had ceased by 10 h.
5', as indicated by the cut-off in the signal at
the fragment size corresponding to the position of the probe with
respect to the 5'-end of the RNA. A fragment extending from the 5'-end
of the transcript to site A1 also accumulated in the
Lsm-depleted strain. We conclude that the activity of the exosome on
both the 7S pre-rRNA and the excised 5'-ETS region is inhibited in
Lsm-depleted strains.
strain (Fig. 5E, lanes
26-28), indicating differences in Lsm protein requirements. It is
therefore possible that some spacer fragments, including
A3-E, are exported to the cytoplasm and degraded by a
cytoplasmic pathway.
3' digestion from site
A3. This is carried out by two exonucleases, Xrn1p and
Rat1p, with the major activity probably provided by Rat1p (46). Primer
extension was performed on the
GAL::lsm3 and
GAL::lsm8 strains (shown for
GAL::lsm3 in Fig.
6; identical results were seen for
GAL::lsm8). Primer extension from
within the 5.8S rRNA showed the existence of 5'-extended forms (Fig.
6E). The 3'-region of the ITS1 is stably base-paired to the
5'-region of the 5.8S rRNA (see Fig. 6H) (56), and the major
stop observed in the Lsm3p-depleted strain lies 3 nucleotides away from
the base of this stem (arrows in Fig. 6, E and
H), consistent with a role for the Lsm complex in
facilitating progression of the 5' exonucleases into the base-paired
region. A somewhat different pattern of stops was seen in the
xrn1-
/rat1-1 strain (Fig. 6E, lane 6), but this pattern is partially allele-specific
because the combination of another RAT1 allele,
tap1-1, with xrn1-
did not yield an identical
pattern (46). As in the exonuclease mutants, the ladder extended to the
A3 cleavage site in the Lsm-depleted strains. This site was
accurate at the nucleotide level, as were other cleavage sites tested;
A0, A1, A2, B1L, and
B1S (Fig. 6, A-F).
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Fig. 6.
Depletion of Lsm proteins inhibits 5'
exonuclease activity but does not prevent accurate pre-rRNA cleavage.
A and B, primer extension analysis through 5'-ETS
in GAL-lsm3 strain using oligonucleotide 008, which
hybridizes within mature 18S. A, primer extension stop at
site A0. B, primer extension stop at site
A1. C-F, primer extension analysis through ITS1
in GAL-lsm3 strain using oligonucleotide 017 (C-E), which hybridizes within mature 5.8S and
oligonucleotide 013 (F), which hybridizes within ITS2.
C, primer extension stop at site A2.
D, primer extension stop at site A3.
E, primer extension through ITS1 until stop at site
A3. F, primer extension stops at sites
B1L and B1S. Panel D represents a
longer exposure of the same gel as panel E. G,
primer extension analysis through 3'-ETS in GAL-lsm3 strain
using oligonucleotide 053, which hybridizes downstream of position
+180. Rnt1p cleavage sites at positions +49 and +14 are ndicated.
H, predicted secondary structure of the stem-loop
structure formed in the 3' region of ITS1. The major primer extension stop
detected in the GAL-lsm3 strain is located three nucleotides
away from the base of the stem and is indicated with an
arrow (E and H). RNA from wild-type
(WT) and GAL-lsm3 strains was prepared as
described for Fig. 4. The rat1-1 and
xrn1- /rat1-1 strains were pre-grown at
23 °C and transferred to 37 °C for 2 and 3 h,
respectively.
3' and 3'
5' exonucleases in 5.8S rRNA processing, but not for the specificity of endonucleases.
3' degradation
(32, 33, 58). Consistent with this, both TAP-Lsm3 and HA-Lsm1
coprecipitated the SMX3 mRNA (Fig. 7G) with
enrichment for the shorter, deadenylated form.
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Fig. 7.
Lsm3p is associated with pre-rRNAs.
Immunoprecipitation of RNAs from strain expressing TAP-tagged Lsm3p and
HA-tagged Lsm1p. Lysates from TAP-Lsm3 strain and the
isogenic WT strain (YJV140) were immunoprecipitated with rabbit
IgG-agarose beads (Sigma). Lysates from
HA::Lsm1 and isogenic wild-type (BMA64)
strains were immunoprecipitated with rat monoclonal anti-HA Ab bound to
Protein G-agarose. RNA was recovered from the lysate (T) and
the immunoprecipitate (P) and analyzed by Northern
hybridization (A-G) or primer extension (H-P).
Probe names are indicated in parentheses. RNA species and
primer extension stops are shown on the right. Approximately
30-fold more cell-equivalents of RNA was loaded for the pellet
fractions compared with the total fraction. Probes are: A
and B, oligonucleotide 020 complementary to the 5.8S-ITS2
boundary; C, oli gonucleotide 017 complementary to the
mature 5.8S; D, oligonucleotide 266 complementary to the pre-5S; E, oligonucleotide
041 complementary to the mature 5S; F, oligonucleotide 031 complementary to the mature MRP RNA; G, oligonucleotide 471 complementary to SMX3 mRNA; H-J,
oligonucleotide 008 complementary to the mature 18S; K and
L, oligonucleotide 007 complementary to the mature 25S;
M and O, oligonucleotide 006 complementary to
ITS2; P, oligonucleotide 002 complementary to ITS1 upstream
of site A2. The primer extension stops in panel
P correspond to the 18S rRNA nucleotides
m
strain (data not shown). Taken together, these results suggest the association of Lsm1p with cytoplasmic
pre-ribosomes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutation
are impaired in mRNA decapping and degradation, whereas the Lsm6p and Lsm7p are required for the normal stability of U6, but these mutations do not strongly inhibit pre-rRNA processing, suggesting that
the Lsm complex involved in pre-rRNA processing is distinct from these
activities. Cells defective in pre-mRNA splicing are expected to
show defects in pre-rRNA processing due to reduced synthesis of
ribosomal proteins. Indeed, the original splicing mutants were
identified on the basis of their reduced total RNA synthesis (67). It
is very likely that reduced ribosomal protein synthesis contributes to
the reduction in rRNA synthesis in the GAL::lsm strains. However, the
accumulation of aberrant intermediates and degradation products appears
specific for Lsm-depleted cells. Similar phenotypes were not seen on
depletion of the splicing factors Syf3p and Prp45p or in strains
carrying the temperature sensitive prp2-1
mutation.2 Cells mutant for cytoplasmic mRNA
degradation factors, the decapping enzyme Dcp1p, the 5'
3'
exonuclease Xrn1p, and the exosome complex also
failed to show similar defects in pre-rRNA processing. Moreover, pre-rRNA processing defects were observed in the
GAL::lsm3 strain 6 h after
transfer to glucose medium, well before growth was inhibited (27,
35).
stain showed some 20S
accumulation, suggesting that it may associate with a late cytoplasmic
pre-40S particle. Direct roles for Lsm proteins in ribosome synthesis are supported by several two-hybrid interactions (50, 68). Lsm1p,
Lsm2p, and Lsm8p each interacted with the exosome component Mtr3p.
Lsm2-5 and 8p (but not Lsm6 or 7p) were each reported to interact with
ribosomal proteins. In a recent proteomic analysis Lsm8p was found also
to associate with another component of the exosome, Rrp42p (69).
5' exonuclease digestion by the exosome complex from site C2, the 3'-end
of the 7S pre-rRNA. In strains depleted of any essential Lsm protein the 5.8S+30 and 6S pre-rRNAs were lost and a ladder of 3'-extended species appeared, with accumulation of the same species seen in exosome
mutants. The ends of these RNAs correspond to sites that are required
for ITS2 processing, which may therefore represent protein binding
sites (70, 71). This suggests that the Lsm complex aids the
displacement of bound proteins during 7S processing. Degradation of the
excised 5'-ETS region by the exosome was also slowed by depletion of
the essential Lsm proteins.
3' exonucleases
Rat1p and Xrn1p is normally highly processive, and intermediates in
processing are not observed in wild-type strains. In contrast, processing intermediates were readily detectable by primer extension in
strains depleted of Lsm3 or Lsm8p, showing that the processivity of 5'
processing was reduced. Northern hybridization indicated that this was
also the case in strains depleted of the other essential Lsm proteins.
The major nuclease pause site was positioned 3 nucleotides from the
base of a stable stem-structure (56), suggesting that the exonucleases
are less able to penetrate into the base-paired region in the absence
of the Lsm complex.
3'
processing of the 5.8S rRNA. Indeed, the rRNA degradation intermediates
observed included both 5'- and 3'-truncated species. The Lsm1-7p
complex antagonizes the 3' degradation of mRNAs by the exosome
(34), so it is also possible that the degradation of otherwise correct
pre-rRNAs is permitted by Lsm protein depletion.
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ACKNOWLEDGEMENT |
---|
We thank Phil Mitchell for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institut de Biologie Moleculaire et
Cellulaire, UPR 9002 du CNRS, 15, rue R. Descartes, 67084 Strasbourg Cedex, France.
§ To whom correspondence should be addressed. Tel.: 44-131-650-7092; Fax: 44-131-650-7040; E-mail: d.tollervey@ed.ac.uk.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M208856200
2 J. Kufel, C. Allmang, E. Petfalski, J. Beggs, and D. Tollervey, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ETS, external transcribed spacers; ITS, internal transcribed spacer; snoRNP, small nucleolar ribonucleoprotein; snRNP, small nuclear ribonucleoprotein; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; Lsm, Sm-like protein; Ab, antibody; HA, hemagglutinin; WT, wild-type; MRP, mitochondrial RNA processing; TAP, tandem affinity purification; PAP, peroxidase-anti-peroxidase; KB, kilobase.
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