From the Department of Genetics, Warsaw University
and Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland and the ¶ Section
for Molecular Biology, Swammerdam Institute for Life Sciences,
University of Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands
Received for publication, August 13, 2002, and in revised form, September 25, 2002
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ABSTRACT |
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The yeast mitochondrial degradosome (mtEXO) is an
NTP-dependent exoribonuclease involved in mitochondrial RNA
metabolism. Previous purifications suggested that it was composed of
three subunits. Our results suggest that the degradosome is composed of
only two large subunits: an RNase and a RNA helicase encoded by nuclear
genes DSS1 and SUV3, respectively, and that it
co-purifies with mitochondrial ribosomes. We have found that the
purified degradosome has RNA helicase activity that precedes and is
essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a
strong accumulation of mitochondrial mRNA and rRNA precursors not
processed at 3'- and 5'-ends. The observed accumulation of precursors
is probably the result of lack of degradation rather than direct
inhibition of processing. We suggest that the degradosome is a central
part of a mitochondrial RNA surveillance system responsible for
degradation of aberrant and unprocessed RNAs.
RNA turnover is a very important step in regulation of gene
expression. RNA degradation is mediated mostly by large multiprotein complexes like the degradosome in bacteria (1) or the exosome in the
cytoplasm of eukaryotes (2). The function and composition of these
complexes has been the subject of intensive investigations in recent
years. Much less is known about enzymes involved in RNA turnover in
mitochondria (3-5).
We use the yeast Saccharomyces cerevisiae as a model to
study mitochondrial RNA metabolism. There are only 3 known
ribonucleases that are involved in RNA turnover in yeast mitochondria:
Ynt20 (6), Nuc1 (7), and the multiprotein complex known as the mitochondrial degradosome or MtEXO (4). Because YNT20 and
NUC1 are not essential for mitochondrial gene expression,
their function is redundant (6, 8). In contrast to this, the
mitochondrial degradosome is necessary for mitochondrial biogenesis and
mutations in its subunits lead to respiratory incompetence (9, 10).
The yeast mitochondrial degradosome was initially identified as a
hydrolytic NTP-dependent 3' Inactivation of either the SUV3 or DSS1 genes
gave similar phenotypes: respiratory incompetence, very strong
inhibition of mitochondrial translation, and a variety of disturbances
of RNA processing and stability, which finally lead to the loss of
mitochondrial genomes (10, 13-15). Initially the research on
SUV3 mutations was concentrated on their effect on intron
metabolism. It has been shown that the In the present study we have purified the degradosome using tandem
affinity chromatography (17) and shown that in contrast to previously
published data it contains only 2 large subunits (Suv3p and Dss1p). The
complex co-purifies with mitochondrial ribosomes. We have shown that
the degradosome has RNA helicase activity, which precedes and is
essential for RNA hydrolysis by this complex. We present data that the
Dss1 protein is responsible for exoribonuclease activity of the
degradosome and that this activity is essential for mitochondrial
biogenesis. Finally, we have analyzed the influence of inactivation of
degradosome components on mitochondrial RNA processing and have shown
that the lack of degradosome activity causes aberrations of all
processing steps of rRNA and mRNA, but not tRNA.
Strains Used and Constructed--
For an analysis of phenotypes
of SUV3 and DSS1 disruptions we used the
previously described strains: BWG
Strains used for degradosome purifications were derived from W303.
SUVTAP and DSSTAP were constructed by in vivo recombination as described in Ref. 17 using plasmid pBS1539 as template. Strain DSSSER was constructed by in vivo recombination. Strain W303
was transformed by the PCR product obtained on the template of total DNA from DSSTAP strain and primers: SER
(TTATACAGGTCGACCTTTTAGACATGAAATGATTGGAGCTAAACAATCTTTGACAGTAAC) and CON
(AACATGAATTAACACCATCGCAGCAACGAG). Primer SER contains the DSS1 gene
coding sequence with a mutation changing tyrosine 814 to serine. The
proper integration was confirmed by Western blotting and sequencing of
PCR products. Diploid strains W303/DSSTAP and W303/DSSSER were
constructed by genetic crosses.
Isolation of Mitochondria--
Mitochondria were isolated as
described previously (18). For mitochondrial RNA analysis yeast were
grown in YPD medium (2% bactopeptone, 1% yeast extract Difco) with
2% glucose, and for degradosome purification in YPD medium containing
2% glycerol and 1% ethanol.
Western Blotting--
Proteins were resolved on 12% SDS-PAGE
and then transferred to a Hybond-C membrane. Proteins were detected
with the following antibodies: tandem affinity purification
method (TAP)1 tag,
peroxidase-antiperoxidase (Sigma), NAM9 (19), and visualized using ECL from Amersham Biosciences.
Extract Preparation and Purification of TAP-tagged
Complexes--
Total native extracts were prepared as described
previously (4). For preparation of native mitochondrial extracts
mitochondria isolated from 2.5 liters of yeast culture were resuspended
in 5 ml of lysis buffer (20 mM Tris, pH 8.0, 150 mM KCl, 2% Triton X-100, 0.5 mM EDTA, 2×
complete protease inhibitor (Roche Molecular Biochemicals), 1 mM phenylmethylsulfonyl fluoride) incubated with rotation
for 15 min in 4 °C, centrifuged 20,000 × g at
4 °C, and the supernatant was collected. The TAP purification
procedure was performed exactly as described (17) with the exception
that 5 ml of mitochondrial extract was mixed with 5 ml of IPP150 buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.1% Nonidet
P-40) and directly loaded on a IgG affinity column without dialysis.
For SDS-PAGE, proteins were precipitated by tricholoroacetic acid. For
biochemical analysis, protein complexes were dialyzed to buffer
containing 50% glycerol 10 mM Tris, pH 8.0, 50 mM KCl, 10 mM MgCl2, and 1 mM dithiothreitol.
Protein Identification--
Proteins were identified by the Mass
Spectrometry Laboratory at the Institute of Biochemistry and
Biophysics, Polish Academy of Sciences. In-gel digestion of the protein
was performed by the protocol of Shevchenko et al. (20),
using bovine trypsin (Promega). Q-Time of flight mass spectrometer
(Micromass) was used for peptide mass fingerprinting and peptide
sequencing by tandem mass spectrometry. The Suv3 protein was identified
by peptide mass fingerprinting with peptide coverage of 18% protein.
Dss1p and ribosomal proteins were identified by peptide sequencing. Sequenced peptides were for: Dss1p (VELDHTR, QYLTVTSPLR,
LINSDFQLITK, NSNAVIFGEGFNK, DISALYPSVIQLLK, ELDNDQATETVVDR,
LYDLTNIEELQWK); Mrpl3p (FLPESELAK, STVNEIPESVASK, LQLPNELTYSTLSR,
MEPFEFTLGR, FFNNSLNSK, SIIAAIWAVTEQK, SPVFIVHVFSGEETLGEGYGSSLK);
Mrpl40p (GQPDLIIPWPKPDPIIIPWPKPDPIDVQTNLATDPVIAREQTFWVDSVVR, VFEFLEK); Mrp1p (GLFSIEGLQK, EVSYIPLLAIDASPK); and Mrpl35p
(YSPPEHIDEIFRMSYDFLEQR, DIIDYDVPVYR, LETLAAIPDTLPTLVPR, FVVWVFR).
Isolation of Ribosomes--
Mitochondrial ribosomes were
isolated by centrifugation through sucrose cushion as described
previously (19).
Analysis of Degradosome Enzymatic
Activities--
NTP-dependent exoribonuclease activity was
analyzed as described previously (4).
RNA Helicase Assays--
RNA helicase activity was assayed by
the strand displacement method. The partially double-stranded RNA
substrate was prepared as described in Ref. 21 with the exception that
substrates after annealing were purified by 15% native acrylamide gel
electrophoresis. The RNA helicase activity assay was performed in a
20-µl reaction volume containing 10 mM Tris-Cl, pH 8.0, 25 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 1 mM CTP, and 100 fmol of 32P-labeled partial
duplex RNA substrate at 30 °C. In a time course aliquots (5 µl)
were analyzed on a native 8% polyacrylamide gel containing 0.5× Tris
borate-EDTA and 0.1% SDS. Strand separation was visualized by autoradiography.
RNA Isolation--
RNA was isolated from mitochondria by the
guanidinium thiocyanate-acid phenol procedure using TriReagent (MRC,
Cincinnati, OH). RNA was normalized with respect to the amount
of COX1 mRNA assayed by Northern blot.
Northern Blotting--
For Northern blot analysis RNA was
resolved in 1% agarose gels containing 0.925% formaldehyde, and 1×
NBC buffer (0.5 M boric acid, 10 mM sodium
citrate, 50 mM NaOH), transferred to nylon membranes by
passive diffusion or resolved in 6% denaturing acrylamide gels and
transferred to nylon membranes by electrophoretic transfer in 0.5 TBE
buffer in a trans-blot cell apparatus (Bio-Rad). Blots were washed 2 times with 2× SSC. RNA was immobilized by UV cross-linking and
incubation at 80 °C in vacuum, hybridized at 55 °C for
oligonucleotide probes or at 65 °C for DNA fragment probes in: 7%
SDS, 0.5 M Na3PO4, pH 7.4, 1 mM EDTA, 1% bovine serum albumin buffer.
Oligonucleotides used for hybridization were as follows: COB
mRNA (TATCTATGTATTAATTTAATTATATATTATTTATTAACTCTACCGAT),
COB 3'-precursor (TATTATTATTATTTAATTTTTATAAATTAGAGATAT),
VAR1 mRNA
(AAATATAATAGAAAAAAGAGTATTATATATTAATATAAAATATATTAATAT), VAR1
precursor (GAAGGAGTTTGGTTAAAGAAGATAAAGAATAAAA), 15 S rRNA (TATAAGCCCACCGCAGGTTCCCCTACGGTAACTGTA), and 15 S rRNA 3'-precursor (ATATAATATTAATATTTATATTAATATTTAGATTAATATTTA).
Probes for tRNA detection were the products of PCR using radiolabeled
[32P]dATP. One of the primers was biotinylated and
strands were resolved on streptavidin paramagnetic beads (Dynal) using
NaOH as for solid phase nuclease S1 mapping. For tRNA Val and Thr,
primers were: Valth P (AAATAAATATTATTATATTAAGATAG) and Valth L + 5'-biotin (AATAATATTATAATAAATTTATAAATG) and both strands were used as
separate probes. For tRNA, Ala I Ile primers were tRANL1
(TATAATTTTATATTTTAATATAGG) and TRNAP + 5'-biotin
(GAAACTAACAGGGATTGAACC) and as a probe was used for the strand
containing biotin.
S1 Nuclease Mapping--
3'-Ends of COX1 and
COB mRNAs were mapped by the classical S1 mapping method
using single-stranded probes (22). 3'-Ends for 21 S rRNA and 15 S rRNA
were mapped by solid phase S1 nuclease mapping (23). For
COX1 mRNA, the DNA fragment excised by
EcoRI and BstYI restriction enzymes from the
pUCCOX1 plasmid containing the 3'-end of the COX1 gene
labeled at the 3'-end with [32P]dATP using the Klenow
fragment (Fermentas) was used as a probe. Single-stranded RNA was
purified by denaturing acrylamide gel electrophoresis. Plasmid pUCCOX1
was constructed by cloning the PCR product obtained using primers, MWG1
(TAAAATGGAACTAAT), MWG2 (TGTGGCTTCCCAATGCATTTCTTAGG), and mitochondrial
DNA as a template, digested with NsiI and BstYI
into pUC18 vector digested with NsiI and
BamHI.
For COB mRNA, the DNA fragment excised by
Csp6I and BamHI restriction enzymes from pUCCOB
plasmid containing the 3'-end of COB, labeled at the 3'-end
with [32P]dATP using Klenow fragment (Fermentas) was used
as a probe. Single-stranded DNA was purified by denaturing acrylamide
gel electrophoresis. Plasmid pUCCOB was constructed by cloning of the
PCR product obtained using the primers: MWG23
(CGGGATCCTAGGACAAATTGGAGGTGCC), MWG 24 (CGGAATCAAATCTCCTTGCGGGGTCC),
and the template of mitochondrial DNA and digested with
EcoRI and BstYI into pUC18 vector digested with
EcoRI and BamHI. For 15 S rRNA, the PCR product
obtained using primers 15SL (acaggcgttacattgttgtc) and 15SP
(CCCTTATTTATTTAAAGAAGATG) digested with Sau3A restricton
enzyme was used as a probe. For 21 S rRNA, the PCR product obtained
using primers 21SNL2 (AGTACGCAAGGACCATAATG) and 21SR2
(TTTCGATTACAAAACGAATGC) digested with the NcoI restriction enzyme was used as a probe.
Primer Extension--
5 µg of mitochondrial RNA were
hybridized with 2 pM [ Mapping the 3'-End of COB mRNA by Ribonuclease T1
Digestion--
The procedure was very similar to that described
previously (24). 5 µg of mitochondrial RNA were hybridized to 1 pM oligonucleotide CobT1
(TATCTATGTATTAATTTAATTATATATTATTTATTAACTCTACCGAT) in 20 µl by heating at 65 °C for 5 min, and slow renaturation to
37 °C (90-120 min). Heteroduplexes were ethanol precipitated,
resuspended in 5 µl of digestion buffer (20 mM
sodium citrate, pH 5.0, 10 mM EDTA, 5 units of RNase T1
(Invitrogen)), and incubated at 37 °C for 30 min. 5 µl of
formamide were added to the samples. The samples were heated at
75 °C for 3 min and resolved in 8% acrylamide, 7 M urea
gel, transferred to nylon membrane by electrophoresis transfer in 0.5 TBE buffer in a trans-blot cell (Bio-Rad) and hybridized using
32P-end labeled CobT1 oligonucleotide. The accompanying
DNA sequencing reactions were used as molecular weight markers.
The Mitochondrial Degradosome Contains Only Two Large Subunits,
Dss1p and Suv3p--
To determine the structure and in
vitro activity of the degradosome we decided to purify this
protein complex by the TAP method (17). In our previous work (4) we
constructed a yeast strain with C-terminal Suv3 TAP tag fusion (named
SUVTAP). In addition, for the purpose of this study we constructed a
C-terminal Dss1p TAP tag fusion strain (DSSTAP). Western blot
analysis of total native yeast extracts using anti-TAP antibodies
revealed that in such conditions Dss1p always partially degrades (Fig.
1). Therefore we developed a method of
degradosome isolation from purified mitochondria that minimizes protein
degradation. In such conditions, using either Suv3-TAP fusion or
Dss1-TAP fusion the purified degradosomes were active as a
NTP-dependent exoribonuclease and contained only 2 large
proteins migrating as 75 and 105 kDa that we assumed to be the Suv3p
and Dss1p proteins (Fig. 2B).
The two proteins were excised from the gel and subjected to mass
spectrometric analysis. As expected they represented SUV3
and DSS1 gene products. This result correlates with the
predicted molecular masses of the Suv3p (81 kDa) and Dss1p (108 kDa).
The differences in gel migration of Suv3p and Dss1p in purifications
from strains DSSTAP and SUVTAP were because of the rest of the TAP tag
in Dss1p and Suv3p proteins, respectively. There was no 90-kDa protein,
which was previously believed to correspond to Suv3p (12). SDS-PAGE
analysis of protein samples from mock TAP purification shows no
proteins, confirming specificity of purification (Fig. 2B).
On the basis of these results we suggest that the degradosome contains
only two large subunits: Suv3p and Dss1p.
The Mitochondrial Degradosome Complexes Co-purify with
Mitochondrial Ribosomes--
In addition to Suv3p and Dss1p, the
TAP-purified degradosome preparations contained smaller proteins. Their
concentrations changed slightly from purification to purification (data
not shown). On the basis of mass spectrometry analysis we identified
three proteins from the large ribosomal subunit (Mrpl3p, Mrpl40p, and Mrpl35p) and one protein from the small subunit (Mrp1p). This suggested
the association of the degradosome complexes with ribosomes. Therefore,
we purified mitochondrial ribosomes by centrifugation through a sucrose
cushion and analyzed the presence of the degradosome subunit Dss1p and
ribosomal proteins by immunoblotting analysis using antibodies specific
for TAP tag and the ribosomal protein Nam9p (Fig.
3). Our analyses indicated that the Dss1p
protein was present exclusively in the ribosomal fraction, suggesting that in vivo all degradosome particles are associated with
ribosomes.
The Mitochondrial Degradosome Has RNA Helicase Activity Followed by
Exoribonuclease Activity--
RNA-dependent NTPase
activity of the degradosome and homology of Suv3 protein to known RNA
helicases suggested that the degradosome has RNA helicase activity, but
so far no biochemical data had been reported. Our attempts to isolate
Suv3p and Dss1p in a variety of heterologous expression systems failed,
therefore we performed an RNA helicase assay by a strand displacement
method using the degradosome purified from the DSSTAP strain (Fig.
4). As can be seen in Fig. 4 the
degradosome has the ability to unwind double-stranded RNA regions; this
results in formation of single-stranded RNA that was subsequently
degraded exoribonucleolytically. There was no partial degradation of
the substrate before unwinding, so RNA helicase activity precedes
exoribonuclease activity of this complex. This result is in agreement
with the previously reported absolute requirement for nucleotide
triphosphates for RNA degradation by MtEXO (11).
Dss1p Is Responsible for RNase Activity of the Degradosome,
Which Is Essential for Respiratory Competence of
Mitochondria--
RNase activity of the degradosome seems to be
associated with the Dss1 protein that has regions of homology to
bacterial RNase II. To understand the role of the Dss1p in degradosome
functions we constructed the yeast strain DSSSER containing a point
mutation within the DSS1 coding sequence and a TAP tag at
the C terminus of the protein. The tyrosine 814 residue was changed to
serine. This tyrosine is conserved in the exoribonuclease family to
which Dss1p belongs and it may be involved in the catalytic mechanism (25). The constructed strain appeared to be respiratory incompetent with very unstable mitochondrial genomes (data not shown). This indicates that this amino acid is essential for Dss1p function and
mitochondrial biogenesis.
We have tried to purify the mitochondrial degradosome from the mutant
strain and from a nonrespiring strain DSSTAP rho0 as a
control, but Western blot analysis revealed that Dss1p is unstable in
mitochondrial extracts from respiratory incompetent strains (data not
shown). To avoid this problem we constructed respiring diploid strains
W303/DSSSER and W303/DSSTAP (wild type Dss1p) as a control.
TAP-purified degradosomes were assayed for NTP-dependent
exoribonuclease and RNA helicase activity. Results presented in Fig.
5 show that the mutation Y814S of
the Dss1p causes complete loss of the exoribonuclease activity of the
degradosome but does not abolish RNA helicase activity. These results
show that Dss1p is responsible for exoribonuclease activity of the degradosome and that it is essential for proper mitochondrial biogenesis.
Lack of Degradosome Components Causes Accumulation of RNA
Precursors Not Processed at the 3'- and 5'-Ends--
Our previous
results have shown that a disruption of SUV3 or
DSS1 genes results in alterations in RNA stability and
processing in mitochondria in a system where intronless mitochondrial
genomes were introduced into mutant strains (13). 15 S rRNA and
COB mRNAs were unstable, in addition in the mutants we
detected several precursors of 21 S rRNA and VAR1, which
were not visible on Northern blots prepared from the wild-type strains.
We decided to look more carefully at mitochondrial RNA processing in
We analyzed the processing of 3'-ends of 21 S rRNA, 15 S rRNA, and
mRNA for COB and COX1 by S1 nuclease mapping
(Fig. 6A). Our results show
that the mature form of RNA is produced for all RNA classes tested.
Processing of 21 S rRNA is strongly inhibited and the precursor
accumulates. In the case of 15 S rRNA, and mRNA for COX1
and COB precursor molecules were not seen because they were
longer than the probes used for S1 nuclease mapping. Therefore we
analyzed 3'-end processing of 15 S rRNA, COB mRNA, and
VAR1 mRNA by Northern blot hybridizations using two
oligonucleotide probes: one complementary to the 3'-end of mature RNA
and the other complementary to the region adjacent to the precursor
(Fig. 6B). For all RNAs tested the accumulation of
precursors not processed at the 3'-end was observed. On autoradiograms
of Northern blots with precursor-specific probes it was impossible to
detect any new RNA classes not seen on Northern blots with
mRNA-specific probes. This suggests that all RNA detected with
precursor-specific probes contains mature RNA and there is no
accumulation of noncoding RNA classes arising after 3'-end
processing.
The mRNAs for Var1 and Cob are located at the end of polycistronic
transcripts and accumulation of very high molecular weight precursors
may suggest problems with transcription termination. This would,
however, be very difficult to prove, as the sites of transcription
termination in yeast mitochondria are not known and noncoding RNA
classes arising after 3'-end processing are very unstable.
To analyze the role of the degradosome in 5'-end RNA processing we used
the primer extension method (Fig. 7). We
analyzed all transcripts known to be specifically processed at the
5'-end: 15 S rRNA and COB, VAR1, and
ATP6/8 mRNA. As a control we used 21 S rRNA,
COX1 mRNA not requiring the 5'-end processing, and COX3 mRNA with the mature 5'-end generated by tRNA
excision. The analysis was also done for Val tRNA. For all specifically
processed RNA there was strong accumulation of precursors that are not
processed at the 5'-end. Processing of COX3 mRNA and
tRNAVal was not impaired.
The Mitochondrial Degradosome Is Not Involved in tRNA
Processing--
The mature mitochondrial tRNA is generated in three
steps: 5'-endonucleolytic cleavage by RNase P, 3'-end processing by an unidentified endonuclease, and CCA synthesis by tRNA nucleotide transferase (26). In mammalian mitochondria exoribonucleases are
involved in tRNA repair processing after incorrect CCA addition (27).
To check if the degradosome is involved in tRNA processing we analyzed
the maturation of tRNA in Inactivation of Degradosome Components Does Not Cause Aberrations
at the 3'-End of Mature COB mRNA and There Is No Polyadenylation of
this Transcript--
Mature COB mRNA is unstable in
The Mitochondrial Degradosome Contains Only Two Large Subunits
(Suv3p and Dss1p) and Co-purifies with Mitochondrial
Ribosomes--
The major enzymatic complex responsible for RNA
turnover in mitochondria, the mitochondrial degradosome, was identified
more than 10 years ago but there were still many unanswered questions about its composition and function.
In the present study we have shown that the purified degradosome
contains only two large subunits: Dss1p migrating at 105 kDa and Suv3p
migrating at 75 kDa. It has been proposed that the 90-kDa protein
previously seen in degradosome preparations (4, 31) corresponds to the
SUV3-encoded protein but direct proof was lacking (12). In contrast to
this, the data presented in this paper show that in degradosome
preparations from mitochondrial extracts obtained in conditions
minimizing degradation of Suv3p and Dss1p, the 90-kDa protein is
absent. The procedure described, yielding the pure, active degradosome
has been repeated 5 times and only 2 large (75 and 105 kDa) proteins
were present. Migration of Suv3p as a 75-kDa protein is not surprising
because the calculated molecular weight of this protein after cleavage
of the leader is 81,000. We think that the most probable
explanation the discrepancy between our present results and previously
published data is that the third protein band of 90 kDa was a product
of Dss1p degradation. Indeed, immunoblot analysis revealed that in
total cell extracts Dss1p is unstable and produces a band migrating
between Dss1p and Suv3p on SDS-PAGE. Finally, our present data are in a
good agreement with the molecular mass of the native, enzymatically active complex, estimated as 160 kDa (31).
Mass spectrometry identification of several of the additional proteins
present in degradosome preparations has shown that they are of
ribosomal origin. We identified proteins from both small and large
ribosomal subunits, suggesting that the degradosome is associated with
intact ribosomes. The ribosomal proteins do not seem to be a
contamination because mock purification has shown no proteins. In
addition, in contrast to cytoplasmic ribosomal proteins, mitochondrial
ribosomal proteins were not found as purification artifacts in
high-throughput yeast protein complexes purified by the TAP method
(32). Ribosome purification and subsequent immunological analysis
confirmed that degradosome complexes are associated with mitochondrial
ribosomes. The ribosome purification method used in this study pellets
only very large complexes, and the mitochondrial degradosome of native
molecular weight estimated as 160,000 could not be pelleted without
association with another large complex (31). Association of the
degradosome with ribosomes is probably not dependent on active
translation and/or mediated by mRNA bridging because ribosome
purification was performed in buffers containing high concentrations of
EDTA, which causes dissociation of ribosome subunits. In our
experiments all degradosome particles were associated with ribosomes
but in vivo the degradosome concentration may be lower than
that of ribosomes because there are no published data on the presence
of 75- and 110-kDa proteins in mitochondrial ribosome preparations
analyzed so far (33, 34). Our data do not allow us to identify which
domains of the degradosome and of the mitochondrial ribosome are
responsible for the binding. In Escherichia coli the
degradosome complex was found to be membrane-associated, but the
physiological significance of this fact is not known (35). It is worth
noting that in the null mutants for the SUV3 or
DSS1 genes a strong inhibition of mitochondrial translation
was detected (13). Further research is needed to understand the
physiological role and mechanism of this interaction.
The Mitochondrial Degradosome Has RNA Helicase and Exoribonuclease
Activity, Which Are Both Essential for Mitochondrial
Biogenesis--
Suv3p and Dss1p proteins exist in vivo
exclusively as a protein complex, which has both RNA helicase and 3'
To the best of our knowledge the interplay between these two helicase
and exoribonuclease activities is unique. The bacterial degradosome,
which also has RNA helicase and exoribonuclease subunits, is able to
degrade RNA without ribonucleotide triphosphates. RNA helicase activity
of this complex is only essential for degradation of RNA containing
double-stranded regions (36). Dss1p contains an exoribonuclease domain
but does not possess any RNA binding domain present in bacterial
exoribonucleases so it is possible that Suv3p is responsible for
interactions with RNA and delivers unwound RNA to Dss1p.
Is There No Polyadenylation of RNA in Yeast
Mitochondria?--
RNA polyadenylation exists in nearly all
known genetic systems. In the cytoplasm of eukaryotes polyadenylation
stabilizes mRNA. In prokaryotes (37) and plant organelles (30, 38) polyadenylation destabilizes transcripts and steady state levels of
mRNAs containing poly(A) are very low. In human mitochondria polyadenylation is abundant and rather stabilizes than destabilizes mRNAs (39). In yeast mitochondria polyadenylation is not abundant and mRNAs are processed at the 3'-end exactly 2 nucleotides after a
conserved dodecamer sequence (24) but it was possible that as in
prokaryotes polyadenylation directs RNA to degradation and that only a
very small fraction of RNA is polyadenylated in vivo. In
bacteria, RNase II, which is homologous to Dss1p, can stabilize mRNA by poly(A) trimming (40). In strains devoid of Dss1p and Suv3p
mature 15 S rRNA and COB mRNA are unstable so it was
possible that this instability was mediated by polyadenylation of these RNAs and other mitochondrial RNases were responsible for degradation. In this case polyadenylated COB mRNA should accumulate
in degradosome-deficient strains. Our experiments have shown that there
are no differences at the 3'-end of mature COB mRNA
between wild type and The Mitochondrial Degradosome Is a Part of the Mitochondrial RNA
Surveillance System--
It has been suggested previously that the
main function of the mitochondrial degradosome is intron turnover and
splicing factor recycling (3, 12). Our results do not support this
hypothesis as the introduction of intronless mitochondrial genomes to
Lack of degradosome activity causes accumulation of unprocessed
mRNA and rRNA but the important fact is that at the same time mature RNAs of proper size are abundant. Therefore the processing of
intronless RNA maturation is not abolished in the
It is not known what discriminates between normal stable mRNA and
aberrant mRNA, which should be promptly degraded. Cis
stability elements of mitochondrial mRNAs are located at
5'-untranslated regions to which stabilizing proteins and translation
activators bind (26, 44). RNA turnover has a 3'
Another protein, which may also be involved in mitochondrial RNA
surveillance, is the PET127 gene product (48), which was found to suppress SUV3 and DSS1 disruptions when
overexpressed (49). This fact prompted us to speculate that the 3'- and
5'-end of yeast mitochondrial mRNA interact (49). Just like the
SUV3 and DSS1 genes, mutations in the
PET127 gene stabilize mRNAs lacking cis
stability elements located in their 5'-untranslated regions (42, 48).
In addition Pet127p is involved in 5'-end processing of mitochondrial
RNA. The same mutation in the COB 5'-untranslated region is
suppressed by mutations in DSS1 and PET127 genes
suggesting that both proteins may function in the same pathway. The
ability of PET127 overexpression to suppress the effects of
DSS1 disruption suggests that it could also activate an
alternative degradosome-independent RNA turnover pathway. Further
research is needed to understand the interplay between Pet127p and the degradosome.
It would be interesting to find out if similar RNA surveillance
mechanisms exist in mitochondria of other organisms.
SUV3-encoded protein is highly conserved through evolution
and there are orthologs of Suv3p in all eukaryotes, but except for
fungi there are no orthologs of Dss1p. We have analyzed the human
ortholog of Suv3p and found that it is localized in mitochondria and
possesses both RNA and DNA helicase activities (50,
51).2 Research is in progress
to identify the function and the putative partners of human
SUV3 RNA helicase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5' exoribonuclease and in addition was shown to have RNA-dependent NTPase activity
(11). SDS-PAGE analysis of the purified degradosome has shown 3 protein bands migrating at 110, 90, and 75 kDa. However, in
contrast to this the native molecular weight of the complex was
estimated by size exclusion chromatography as 160 kDa (11). So far only 2 genes encoding degradosome subunits have been identified:
SUV3 encoding an 84-kDa putative RNA helicase that was shown
to be a bona fide degradosome component (9, 12) and
DSS1 encoding a 105-kDa putative hydrolytic exoribonuclease
homologous to bacterial RNase II (10). In the case of Dss1p there was
no direct proof that it is indeed part of the degradosome, but
inactivation of the DSS1 gene resulted in a complete loss of
degradosome activity in mitochondria (13). The putative third (75 kDa)
component remained to be identified.
intron from the 21 S rRNA
transcript was accumulated up to 90-fold, other group I introns
accumulated as well, but to a smaller extent (3, 14-16). On the basis
of these results it has been suggested that the physiological function of the degradosome is to protect mitochondria from a toxic effect of
undegraded introns (12). The function of the mitochondrial degradosome
is, however, not exclusively intron-related: we have shown that
introduction of intronless mitochondrial genomes to
SUV3 and
DSS1
strains does not restore respiratory competence (13, 14). In such
strains 15 S rRNA and COB mRNA were found to be
unstable, moreover precursors of 21 S rRNA and VAR1 mRNA were present (13). The above results indicated that more detailed analysis of the involvement of the degradosome in mitochondrial RNA
metabolism is required.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
i (wt)
(MATa, his1, ade1, leu2, ura3),
SUV3
i (MATa, his1, ade1, leu2, Suv3
::ura3), and
DSS
i
(MATa, his1, ade1, leu2, Dss1
::ura3) (10, 14).
-32P]ATP-labeled
primer in 200 mM KCl, 10 mM Tris, pH 8.3. The
reaction mixture was heated at 85 °C for 5 min and slowly cooled to
42 °C and then 10 µl of elongation mixture was added (2× buffer
for avian myeloblastosis virus, dNTP 2 mM, 7 units
of avian myeloblastosis virus (Roche Molecular Biochemicals). Reactions
were stopped by addition of 12 µl of formamide and products were
resolved in 6% acrylamide, 7 M urea gels. Accompanying
sequencing reactions were used as molecular weight markers. Primers
were used as follows: COX1 (TATATTTAATGATATTAATACTCTC),
VAR1 (GAAATATATATATATATATAATATGCATCC), COB
(CAATTATTATTATTATTATTATACATAAA), 15 S rRNA (CGTATGACTCGTATGCGTCATGTCC), 21 S rRNA (TTTAATTATTATACTCCATGTTATCT), COX3
(ATTAATATAAATCATTGATAATATCTT), and ATP6/8 (ATATTAGTATTTATTTATATAGTTCCC).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dss1p is partially degraded in total native
yeast extracts but is more stable in native mitochondrial
extracts. Stability of Dss1p protein containing a TAP tag was
analyzed by Western blotting using anti-TAP antibodies. As a control we
used the same procedure for a yeast strain containing TAP-tagged Suv3
protein that was more stable in both types of extracts.
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Fig. 2.
The degradosome is composed of Suv3 and Dss1
proteins. The degradosome was purified from TAP-tagged yeast
strains DSSTAP and SUVTAP and from wild-type strain W303 as a control.
After purification all eluted fractions were pooled and divided into
equal portions. The first portion was dialyzed against the appropriate
buffer and assayed for exoribonuclease activity. The other was
trichloroacetic acid-precipitated and subjected to SDS-PAGE.
A, NTP-exoribonuclease activity assay. Protein samples
were incubated with internally (32P-labeled) UTP-labeled
RNA with and without NTPs and subjected to polyethyleneimine-cellulose
TLC under conditions in which the substrate (RNA) remains at the bottom
of the chromatogram and reaction products (UMP) migrate with the front.
B, SDS-PAGE analysis of the purified degradosome using
silver staining. Proteins identified by mass spectrometry are indicated
by arrows. Molecular weight markers are indicated on the
left.
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Fig. 3.
The degradosome is associated with
mitochondrial ribosomes. 100 µg of mitochondrial extract
proteins from the DSSTAP strain was separated into ribosomal and
soluble fractions by centrifugation through a sucrose cushion. All
fractions (total extract, soluble, and ribosomal) were analyzed for the
presence of ribosomal protein Nam9p and the degradosome component Dss1p
by Western blotting using anti-TAP and anti-Nam9 antibodies.
A, SDS-PAGE. B, Western blot. Dss1 protein was
present exclusively in the ribosomal fraction.
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Fig. 4.
Mitochondrial degradosome has RNA helicase
activity that precedes RNA hydrolysis by this complex.
A, RNA helicase substrate in which the shorter RNA strand
was radioactively labeled at the 5'-end. B, analysis of RNA
helicase activity by the strand displacement method. 100 fmol of
substrate were incubated in the presence of UTP with increasing
concentrations of TAP-purified degradosome from the DSSTAP strain.
Reaction products were resolved in native acrylamide gels in a 10-min
time course. The amount of degradosome was estimated by SDS-PAGE and
was about 0.1, 0.3, and 0.5 ng of Suv3p protein, respectively. An
aliquot of the duplex substrate was heat-denatured at 90 °C for 5 min and served as a marker for the position of single-stranded RNA
(indicated as 90 °C).
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Fig. 5.
Change of Y814S in Dss1p causes the loss of
degradosome exoribonuclease activity but has no influence on RNA
helicase activity. The degradosome purified from mitochondria from
W303/DSSSER or W303/DSSTAP (Wt) diploid yeast strain was
assayed for exoribonuclease and RNA helicase activities as described in
the legends to Figs. 2 and 4, respectively. For both experiments the
same amounts of purified degradosome were used.
SUV3 and
DSS1 strains with intronless mtDNA.
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Fig. 6.
Inactivation of degradosome components causes
accumulation of precursors not processed at the 3'-end but does not
inhibit the processing completely. A, analysis of the
3'-ends of 21 S rRNA, 15 S rRNA, and COB and COX1
mRNAs from BWG (wt), SUV3, and
DSS1 strains by S1 nuclease
mapping. Reaction products for 15 S rRNA and COB and
COX1 were resolved in 6% acrylamide, 7 M urea
gels, and for 21 S rRNA in denaturing agarose-formaldehyde gels. The
position of mature RNA is indicated on each gel. B, analysis
of 3'-end processing by Northern blot hybridization using
oligonucleotide probes specific for the mature RNA and 3'-precursor.
The positions of the mature RNA and RNA size markers are indicated on
each gel.
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Fig. 7.
Inactivation of degradosome
components causes accumulation of precursors not processed at the
5'-end. Analysis of 5'-end processing of RNA in SUV3 and
DSS1 strains by primer extension using oligonucleotides labeled with
[
-32P]ATP. We analyzed all specifically processed
RNAs: 15 S rRNA and COB, VAR1, ATP6/8
mRNAs. As a control we used the unprocessed RNAs: 21 S rRNA,
COX1 mRNA, and COX3 as an example of an
mRNA with its 5'-end released by tRNA excision. An analysis was
also performed for tRNAVal. On each gel positions of the
mature RNA and precursor are indicated. Numbers in
brackets indicate the difference in length of precursor and
mature RNA.
DSS1 and
SUV3 strains by Northern
blotting. Four tRNAs were tested. (a) tRNAVal
and tRNAThr, which are encoded by complementary strands.
Their transcripts partially overlap. Single-stranded DNA fragments
complementary to tRNA and overlapping regions were used as probes. As
can be seen, there are no aberrancies in tRNA processing, and there is no accumulation of transcribed intergenic regions (Fig.
8). (b) tRNAAla
and tRNAIle, which are produced from one transcript (Fig.
8). The processing of these tRNAs was also found to be correct and no
accumulation of precursors or intergenic regions was observed. In all
cases the amount of mature tRNAs was 2-3 times lower in our mutant
strain in comparison to respiring wild-type yeast, but no effect on
processing was detected.
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Fig. 8.
The mitochondrial degradosome is not involved
in tRNA maturation. Northern blot analysis of mitochondrial tRNAs
in wild-type and SUV3 and DSS1-disrupted strains.
RNA was resolved in 6% acrylamide, 7 M urea gels. Val and
Thr tRNAs are transcribed from complementary strands and their
transcripts partially overlap. Single-stranded DNA fragments derived
from the same PCR product were used as probes. Ala and Ile tRNAs are
derived from the same transcript, so a single-stranded DNA fragment
complementary to both tRNAs including the transcribed intergenic region
was used as a probe for hybridization. Single-stranded DNA size markers
are shown on the left.
DSS and
SUV3 mutant stains (13). We asked if this instability is
caused by small changes at the 3'-end of mRNA or by possible
polyadenylation. So far there were two early reports addressing the
question whether polyadenylation occurs in yeast mitochondria (28, 29).
In plant mitochondria as in bacteria polyadenylation destabilizes
transcripts (30). To analyze the mature 3'-end with high resolution we
used Northern blot analysis of RNA, which was cut by ribonuclease T1.
To protect some guanylate residues from cleavage we hybridized
synthetic DNA oligonucleotides to the 3'-end of mature COB
mRNA and cut the mRNA by G-specific ribonuclease T1 (Fig.
9A). RNA was resolved on
sequencing gels and analyzed by Northern blot using as a probe the same
oligonucleotide as for protection. On the autoradiograms (Fig.
9B) we were able to see two bands specific for complete digestion of the mature 3'-end and the precursor. In the
SUV3 and
DSS1 strains there was accumulation of precursors, but there were no
changes at the mature 3'-end of COB mRNA. Therefore, the instability of COB mRNA is not caused by changes at the
3'-end of mRNA and there is no polyadenylation of this
mRNA.
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Fig. 9.
There are no changes at the 3'-end or
polyadenylation of mature COB mRNA in
SUV3 or
DSS1 strains.
Analysis of the 3'-end of COB mRNA in
SUV3 and
DSS1 strains by ribonuclease T1 digestion and Northern blot
hybridization. Two bands (143 and 223 nucleotides) that represent
complete digestion products of mature mRNA and a precursor not
processed at the 3'-end are visible on autoradiograms.
A, mapping strategy. B, Northern
blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5' exoribonuclease activities. Exoribonuclease activity is
absolutely dependent on RNA helicase, but in vitro the
degradosome without RNase activity is still able to unwind RNA.
Nevertheless RNase activity of the degradosome is essential for
mitochondrial biogenesis.
SUV and
DSS1 strains.
This is a suggestion that in yeast mitochondria there is no
polyadenylation of RNA or at least that the COB mRNA is
not polyadenylated.
SUV and
DSS1 strains does not restore respiratory
competence. Therefore the degradosome must have other functions
essential for mitochondrial biogenesis and the effects observed on
intron metabolism of SUV3 mutants are rather the consequence
of general perturbations in RNA turnover.
SUV or
DSS1 strains. The degradosome is most probably not involved in RNA processing directly because processing is endonucleolytic and the
degradosome does not possess such activity at least in
vitro. The most plausible explanation is that the accumulation of
precursors is not caused by impairment of RNA processing but rather by
the lack of RNA degradation. We suggest that the degradosome is a central part of the mitochondrial RNA surveillance system, which degrades aberrant and unprocessed RNAs. This hypothesis is supported by
the fact that mutations in degradosome components stabilize and restore
expression of unstable mutated mRNAs. Mutation in SUV3
stabilizes VAR1 mRNA containing a deletion of 206 nucleotides in the 3'-end region (41). Mutations in DSS1
suppress deletions in the 5'-untranslated region of the COB
gene, which is necessary for mRNA stability (42). Association of
the degradosome particles with ribosomes also suggests the function of
the degradosome in RNA surveillance because in mammals many cytoplasmic
RNA surveillance factors can be found in ribosomal fractions (43).
5' direction
(45) so 3'- and 5'-ends of mRNA must interact physically or
functionally. At the 3'-end of all mature mitochondrial mRNAs there
is a conserved dodecamer sequence to which the dodecamer binding
protein binds (46, 47). Interaction between proteins binding to both
ends of RNA could stabilize mRNA, similarly to cytoplasmic
mRNAs. When RNA molecules are not processed, the 3'-end is free and
RNA is recruited for degradation. Unfortunately the gene coding for the dodecamer binding protein has not been identified and more research is
needed to identify putative interactions of the degradosome with RNA
regions or other RNA binding proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Ewa Bartnik and Piotr S
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FOOTNOTES |
---|
* This work was supported in part by State Committee for Scientific Research Grants 6P04 00319 and 6P04 01818, the Polish-French Center for Biotechnology of Plants EU Centre of Excellence in Molecular Biology Grant ICA-CT-2000-70010, and Faculty of Biology Grant BW.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.
§ Supported by a Foundation for Polish Science fellowship for young scientists and an EMBO short-term fellowship for work at University of Amsterdam.
Current address: Dept. of Endocrinology and Reproduction,
Faculty of Medicine and Health Sciences, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: siep@endov.fgg.eur.nl.
** Current address: EMBO, Meyerhofstrasse 1, 69117 Heidelberg, Germany. E-mail: grivell@embl-heidelberg.de.
To whom correspondence should be addressed: Dept. of Genetics,
Warsaw University and Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland. Tel.:
48-22-659-70-72 (ext. 22 40); Fax: 48-22-658-47-54; E-mail: stepien@ibb.waw.pl.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M208287200
2 Minczuk, M., Piwowarski, J., Papworth, M. A., Awiszus, K., Schalinski, S., Dziembowski, A., Dmochowska, A., Bartnik, E., Tokatlidis, K., Stepien, P. P., and Borowski, P. (2002) Nucl. Acids. Res. 30, 5074-5086
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ABBREVIATIONS |
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The abbreviation used is: TAP, tandem affinity purification method.
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REFERENCES |
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1. | Carpousis, A. J., Vanzo, N. F., and Raynal, L. C. (1999) Trends Genet. 15, 24-28[CrossRef][Medline] [Order article via Infotrieve] |
2. | Butler, J. S. (2002) Trends Cell Biol. 12, 90-96[CrossRef][Medline] [Order article via Infotrieve] |
3. | Margossian, S. P., and Butow, R. A. (1996) Trends Biochem. Sci. 21, 392-396[CrossRef][Medline] [Order article via Infotrieve] |
4. | Dziembowski, A., and Stepien, P. P. (2001) Methods Enzymol. 342, 367-378[Medline] [Order article via Infotrieve] |
5. | Gagliardi, D., Perrin, R., Marechal-Drouard, L., Grienenberger, J. M., and Leaver, C. J. (2001) J. Biol. Chem. 13, 1803-1818 |
6. | Hanekamp, T., and Thorsness, P. E. (1999) Curr. Genet. 34, 438-448[CrossRef][Medline] [Order article via Infotrieve] |
7. | Vincent, R. D., Hofmann, T. J., and Zassenhaus, H. P. (1988) Nucleic Acids Res. 16, 3297-3312[Abstract] |
8. | Zassenhaus, H. P., Hofmann, T. J., Uthayashanker, R., Vincent, R. D., and Zona, M. (1988) Nucleic Acids Res. 16, 3283-3296[Abstract] |
9. | Stepien, P. P., Margossian, S. P., Landsman, D., and Butow, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6813-6817[Abstract] |
10. | Dmochowska, A., Golik, P., and Stepien, P. P. (1995) Curr. Genet. 28, 108-112[Medline] [Order article via Infotrieve] |
11. | Min, J. J., and Zassenhaus, H. P. (1991) SAAS Bull. Biochem. Biotechnol. 4, 1-5[Medline] [Order article via Infotrieve] |
12. | Margossian, S. P., Li, H., Zassenhaus, H. P., and Butow, R. A. (1996) Cell 84, 199-209[Medline] [Order article via Infotrieve] |
13. | Dziembowski, A., Malewicz, M., Mínczuk, M., Golik, P., Dmochowska, A., and Stepien, P. P. (1998) Mol. Gen. Genet. 260, 108-114[CrossRef][Medline] [Order article via Infotrieve] |
14. | Stepien, P. P., Kokot, L., Leski, T., and Bartnik, E. (1995) Curr. Genet. 27, 234-238[CrossRef][Medline] [Order article via Infotrieve] |
15. | Golik, P., Szczepanek, T., Bartnik, E., Stepien, P. P., and Lazowska, J. (1995) Curr. Genet. 28, 217-224[Medline] [Order article via Infotrieve] |
16. | Conrad-Webb, H., Perlman, P. S., Zhu, H., and Butow, R. A. (1990) Nucleic Acids Res. 18, 1369-1376[Abstract] |
17. | Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) Nat. Biotechnol. 17, 1030-1032[CrossRef][Medline] [Order article via Infotrieve] |
18. | Daum, G., Sperka-Gottlieb, C. D., Hermetter, A., and Paltauf, F. (1988) Biochim. Biophys. Acta 946, 227-234[Medline] [Order article via Infotrieve] |
19. |
Chacinska, A.,
Boguta, M.,
Krzewska, J.,
and Rospert, S.
(2000)
Mol. Cell. Biol.
20,
7220-7229 |
20. | Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Gallinari, P.,
Brennan, D.,
Nardi, C.,
Brunetti, M.,
Tomei, L.,
Steinkuhler, C.,
and De Francesco, R.
(1998)
J. Virol.
72,
6758-6769 |
22. | Berk, A. J. (1989) Methods Enzymol. 180, 334-347[Medline] [Order article via Infotrieve] |
23. | Dziembowski, A., and Stepien, P. P. (2001) Anal. Biochem. 294, 87-89[CrossRef][Medline] [Order article via Infotrieve] |
24. | Hofmann, T. J., Min, J., and Zassenhaus, H. P. (1993) Yeast 9, 1319-1330[Medline] [Order article via Infotrieve] |
25. |
Mian, I. S.
(1997)
Nucleic Acids Res.
25,
3187-3195 |
26. | Grivell, L. A. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 121-164[Abstract] |
27. |
Reichert, A. S.,
and Morl, M.
(2000)
Nucleic Acids Res.
28,
2043-2048 |
28. | Hendler, F. J., Padmanaban, G., Patzer, J., Ryan, R., and Rabinowitz, M. (1975) Nature 258, 357-359[Medline] [Order article via Infotrieve] |
29. | Groot, G. S., Flavell, R. A., Van Ommen, G. J., and Grivell, L. A. (1974) Nature 252, 167-169[Medline] [Order article via Infotrieve] |
30. | Gagliardi, D., and Leaver, C. J. (1999) Eur. J. Biochem. 18, 3757-3766 |
31. |
Min, J.,
Heuertz, R. M.,
and Zassenhaus, H. P.
(1993)
J. Biol. Chem.
268,
7350-7357 |
32. | Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M. A., Copley, R. R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G., and Superti-Furga, G. (2002) Nature 415, 141-147[CrossRef][Medline] [Order article via Infotrieve] |
33. | Graack, H. R., and Wittmann-Liebold, B. (1998) Biochem. J. 329, 433-448[Medline] [Order article via Infotrieve] |
34. |
Saveanu, C.,
Fromont-Racine, M.,
Harington, A.,
Ricard, F.,
Namane, A.,
and Jacquier, A.
(2001)
J. Biol. Chem.
276,
15861-15867 |
35. |
Liou, G. G.,
Jane, W. N.,
Cohen, S. N.,
Lin, N. S.,
and Lin-Chao, S.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
63-68 |
36. |
Coburn, G. A.,
Miao, X.,
Briant, D. J.,
and Mackie, G. A.
(1999)
Genes Dev.
13,
2594-2603 |
37. | Coburn, G. A., and Mackie, G. A. (1999) Prog. Nucleic Acids Res. Mol. Biol. 62, 55-108[Medline] [Order article via Infotrieve] |
38. |
Lupold, D. S.,
Caoile, A. G.,
and Stern, D. B.
(1999)
Plant Cell
11,
1565-1578 |
39. | Ojala, D., Montoya, J., and Attardi, G. (1981) Nature 290, 470-474[Medline] [Order article via Infotrieve] |
40. | Marujo, P. E., Hajnsdorf, E., Le Derout, J., Andrade, R., Arraiano, C. M., and Regnier, P. (2000) RNA (N.Y.) 6, 1185-1193[CrossRef] |
41. | Butow, R. A., Zhu, H., Perlman, P., and Conrad-Webb, H. (1989) Genome 31, 757-760[Medline] [Order article via Infotrieve] |
42. |
Chen, W.,
Islas-Osuna, M. A.,
and Dieckmann, C. L.
(1999)
Genetics
151,
1315-1325 |
43. | Mangus, D. A., and Jacobson, A. (1999) Methods 17, 28-37[CrossRef][Medline] [Order article via Infotrieve] |
44. | Fox, T. D. (1996) Experientia (Basel) 52, 1130-1135 |
45. | Min, J., and Zassenhaus, H. P. (1993) J. Bacteriol. 175, 6245-6253[Abstract] |
46. | Li, H., and Zassenhaus, H. P. (2000) Curr. Genet. 37, 356-363[CrossRef][Medline] [Order article via Infotrieve] |
47. | Li, H., and Zassenhaus, H. P. (1999) Biochem. Biophys. Res. Commun. 261, 740-745[CrossRef][Medline] [Order article via Infotrieve] |
48. | Wiesenberger, G., and Fox, T. D. (1997) Mol. Cell. Biol. 17, 2816-2824[Abstract] |
49. | Wegierski, T., Dmochowska, A., Jablonowska, A., Dziembowski, A., Bartnik, E., and Stepien, P. P. (1998) Acta Biochim. Pol. 45, 935-940[Medline] [Order article via Infotrieve] |
50. | Dmochowska, A., Kalita, K., Krawczyk, M., Golik, P., Mroczek, K., Lazowska, J., Stepien, P. P., and Bartnik, E. (1999) Acta Biochim. Pol. 46, 155-162[Medline] [Order article via Infotrieve] |
51. | Dmochowska, A., Stankiewicz, P., Golik, P., Stepien, P. P., Bocian, E., Hansmann, I., and Bartnik, E. (1998) Cytogenet. Cell Genet. 83, 84-85[CrossRef][Medline] [Order article via Infotrieve] |