The Yeast Mitochondrial Degradosome

ITS COMPOSITION, INTERPLAY BETWEEN RNA HELICASE AND RNase ACTIVITIES AND THE ROLE IN MITOCHONDRIAL RNA METABOLISM*

Andrzej DziembowskiDagger §, Jan PiwowarskiDagger , Rafal HoserDagger , Michal MinczukDagger , Aleksandra DmochowskaDagger , Michel Siep||, Hans van der Spek, Les Grivell**, and Piotr P. StepienDagger DaggerDagger

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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' right-arrow 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.

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 omega  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 Delta SUV3 and Delta 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.

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Strains Used and Constructed-- For an analysis of phenotypes of SUV3 and DSS1 disruptions we used the previously described strains: BWG Delta i (wt) (MATa, his1, ade1, leu2, ura3), Delta SUV3 Delta i (MATa, his1, ade1, leu2, Suv3Delta ::ura3), and Delta DSSDelta i (MATa, his1, ade1, leu2, Dss1 Delta ::ura3) (10, 14).

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 [gamma -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).

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, 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (19K):
[in this window]
[in a new window]
 
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.


View larger version (28K):
[in this window]
[in a new window]
 
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.

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.


View larger version (45K):
[in this window]
[in a new window]
 
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.

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).


View larger version (25K):
[in this window]
[in a new window]
 
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).

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.


View larger version (18K):
[in this window]
[in a new window]
 
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.

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 Delta SUV3 and Delta DSS1 strains with intronless mtDNA.

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.


View larger version (67K):
[in this window]
[in a new window]
 
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), Delta SUV3, and Delta 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.

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.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7.   Inactivation of degradosome components causes accumulation of precursors not processed at the 5'-end. Analysis of 5'-end processing of RNA in Delta SUV3 and Delta DSS1 strains by primer extension using oligonucleotides labeled with [gamma -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.

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 Delta DSS1 and Delta 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.


View larger version (46K):
[in this window]
[in a new window]
 
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.

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 Delta DSS and Delta 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 Delta SUV3 and Delta 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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 9.   There are no changes at the 3'-end or polyadenylation of mature COB mRNA in Delta SUV3 or Delta DSS1 strains. Analysis of the 3'-end of COB mRNA in Delta SUV3 and Delta 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

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' right-arrow 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.

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 Delta SUV and Delta 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.

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 Delta SUV and Delta 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.

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 Delta SUV or Delta 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).

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' right-arrow 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.

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.

    ACKNOWLEDGEMENTS

We thank Ewa Bartnik and Piotr Słonimski for critical reading of the manuscript and Anna Dziembowska for editing the manuscript, Bertrand Seraphin for plasmids containing the TAP tag.

    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.

Dagger Dagger 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

    ABBREVIATIONS

The abbreviation used is: TAP, tandem affinity purification method.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
36. Coburn, G. A., Miao, X., Briant, D. J., and Mackie, G. A. (1999) Genes Dev. 13, 2594-2603[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.