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Title: The yeast mitochondrial degradosome: its composition, interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism.

Running title: Functional and biochemical analysis of the yeast mitochondrial

degradosome.

Andrzej Dziembowski, Department of Genetics, Warsaw University and Institute of Biochemistry

and Biophysics, Polish Academy of Sciences Pawinskiego 5A, 02-106 Warsaw, Poland; E-mail:

[email protected]

Jan Piwowarski, Department of Genetics, Warsaw University and Institute of Biochemistry and

Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland; E-mail:

[email protected]

5DIDá +RVHU, Department of Genetics, Warsaw University, Pawinskiego 5A, 02-106 Warsaw,

Poland;

0LFKDá 0L�F]XN, Department of Genetics, Warsaw University and Institute of Biochemistry and

Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland; E-mail:

[email protected]

Aleksandra Dmochowska, Department of Genetics, Warsaw University and Institute of

Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw,

Poland;E-mail: [email protected]

Michel Siep, current address: Department of Endocrinology and Reproduction, Faculty of

Medicine and Health Sciences, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands; E-mail:

[email protected]

Hans van der Spek, Section for Molecular Biology, Swammerdam Institute for Life Sciences,

University of Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands; E-mail:

[email protected]

Les Grivell, current address: EMBO, Meyerhofstrasse 1, 69117 Heidelberg, Germany; E-mail:

[email protected]

3LRWU 3� 6WSLH� , Department 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: [email protected].

* To whom correspondence should be addressed,

9 figures

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 7, 2002 as Manuscript M208287200 by guest on January 7, 2020

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Summary

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 which 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.

Keywords:

yeast/mitochondria/RNA turnover/mtEXO/mitochondrial degradosome

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Introduction

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)

(4) (5).

We use the yeast Saccharomyces cerevisiae as a model to study mitochondrial

RNA metabolism. There are only 3 known ribonucleases which are involved in RNA

turnover in yeast mitochondria: Ynt20 (6), Nuc1 (7) and the multiprotein complex known

as the mitochondrial degradosome or MtEXO (4). Since YNT20 and NUC1 are not

essential for mitochondrial gene expression, their function is redundant (8); (6). 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’:�¶ H[RULERQXFOHDVH DQG LQ DGGLWLRQ ZDV VKRZQ WR KDYH 51$-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 which was shown to be a bona fide

degradosome component (9,12) and DSS1 encoding a 105 kDa putative hydrolytic

exoribonuclease homologous to bacterial RNaseII (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


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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 gene gave similar phenotypes: respiratory

incompetence, very strong inhibition of mitochondrial translation, 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 21S 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

15S rRNA and COB mRNA were found to be unstable, moreover precursors of 21S 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


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shown that the lack of degradosome activity causes aberrations of all processing steps of

rRNA and mRNA, but not tRNA.


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Materials and methods

Strains used and constructed. For an analysis of phenotypes of SUV3 and DSS1

disruptions we used the previously described strains: BWG ∆i (wt) [MATa, his1, ade1,

leu2, ura3] , ∆SUV3 ∆i [MATa, his1, ade1, leu2, Suv3∆::ura3], ∆DSS∆i [MATa, his1,

ade1, leu2, Dss1 ∆::ura3], (10,14)

Strains used for degradosome purifications were derived from W303. SUVTAP and

DSSTAP were constructed by in vivo recombination as described in (17) using plasmid

pBS1539 as template. Strain DSSSER was constructed by in vivo recombination. Strain

W303 was transformed by the PCR product obtained on template of total DNA from

DSSTAP strain and primers: SER (TTA TAC AGG TCG ACC TTT TAG ACA TGA

AAT GAT TGG AGC TAA ACA ATC TTT GAC AGT AAC) CON (AAC ATG AAT

TAA CAC CAT CGC AGC AAC GAG). 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 by 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: TAP-tag -

PAP (peroxidase-anti-peroxidase, Sigma), NAM9 (19) and visualized using ECL from

Amersham.


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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 l 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, 2X complete protease

inhibitor (Boehringer ), 1 mM PMSF) incubated with rotation for 15 min in 40C,

centrifuged 20,000 g at 4 0C and the supernatant was collected. TAP purification

procedure was performed exactly as described (17) with the exception that 5 ml of

mitochondrial extract was mixed with 5ml of IPP150 buffer (10mM Tris pH8, 150mM

NaCl, 0.1% NP-40) and directly loaded on a IgG affinity column without dialysis. For

SDS-PAGE proteins were precipitated by TCA. For biochemical analysis protein

complexes were dialyzed to buffer containing 50% glycerol 10mM Tris pH 8.0 50 mM

KCl, 10 mM MgCl2 and 1mM DTT.

Protein identification. Proteins were identified by Mass Spectrometry Laboratory at

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-Tof 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 protein 18%. 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 SPVFIVHV FSGEETLGEG YGSSLK);

Mrpl40p (GQPDL IIPWPKPDPI DVQTNLATDP, VIAREQTFWV DSVVR, VFEFLEK)

Mrp1p(GLFSIEGLQK, EVSYIPL LAIDASPK);


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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 (21)

with the exception that substrates after annealing were purified by 15% native acrylamide

gel electrophoresis. The RNA helicase activity assay was performed in 20 µl reaction

volume containing 10 mM Tris-Cl pH 8.0, 25 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1

mg/ml BSA, 1mM CTP , and 100 fM 32P-labelled 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, USA). 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 1X NBC buffer (0.5 M boric acid, 10 mM sodium

citrate, 50mM 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 trans-Blot Cell apparatus (Bio-Rad). Blots were washed 2

times with 2X SSC. RNA was immobilized by UV crosslinking and incubation at 800C in


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vacuum, hybridized at 550C for oligonuclotide probes or at 65 0C for DNA fragment probes

in : 7% SDS, 0.5M Na3P04 pH 7.4, 1mM EDTA, 1% BSA buffer.

Oligonucleotides used for hybridization were as follows: COB mRNA(TAT CTA TGT

ATT AAT TTA ATT ATA TAT TAT TTA TTA ACT CTA CCG AT) COB 3’- precursor

(TAT TAT TAT TAT TTA ATT TTT ATA AAT TAG AGA TAT), VAR1 mRNA( AAA

TAT AAT AGA AAA AAG AGT ATT ATA TAT TAA TAT AAA ATA TAT TAA

TAT) VAR1 precursor (GAA GGA GTT TGG TTA AAG AAG ATA AAG AAT AAA

A),15S rRNA (TAT AAG CCC ACC GCA GGT TCC CCT ACG GTA ACT GTA) 15S

rRNA 3’- precursor (ATA TAA TAT TAA TAT TTA TAT TAA TAT TTA GAT TAA

TAT TTA)

Probes for tRNA detection were the products of PCR reaction using radiolabelled [P32]

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 (AAA TAA ATA TTA TTA TAT TAA GAT

AG) and Valth L + 5’-biotin (AAT AAT AT TAT AAT AAA TTTA TAA ATG) and both

strands were used as separate probes. For tRNA Ala I Ile primers were tRANL1 (TAT

AAT TTT ATA TTT TAA TAT AGG) and TRNAP+5’-biotin (GAA ACT AAC AGG

GAT TGA ACC) and as a probe was used the strand containing biotin.

S1 nuclease mapping. 3’- ends of COX1 and COB mRNAs were mapped by classical S1

mapping method using single stranded probes (22) 3’- ends for 21S rRNA and 15S rRNA

were mapped by solid phase S1 nuclease mapping (23). The probes:

For COX1 mRNA, the DNA fragment excised by EcoR1 and BstY1 restriction

enzymes from pUCCOX1 plasmid containing 3’- end of COX1 gene labeled at the 3’- end

with [P32] dATP using the Klenow fragment (Fermentas) were used as a probe. Single

stranded RNA was purified by denaturing acrylamide gel electrophoresis. Plasmid


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pUCCOX1 was constructed by cloning of the PCR product obtained using primers:

MWG1 (TAA AAT GGA ACT AAT) MWG2 (TGT GGC TTC CCA ATG CAT TTC

TTA GG) and mitochondrial DNA as a template, digested with NsiI and BstY1 into

pUC18 vector digested with NsiI and BamH1.

For COB mRNA, the DNA fragment excised by Csp61 and BamHI restriction

enzymes from pUCCOB plasmid containing the 3’- end of COB, labeled at the 3’- end

with [P32] 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 (CGG

GAT CCT AGG ACA AAT TGG AGG TGCC) MWG 24 (CGG AAT CAA ATC TCC

TTG CGG GGT CC) and the template of mitochondrial DNA and digested with EcoRI and

BstY1 into pUC18 vector digested with Eco RI and BamH1.

For 15S rRNA, the PCR product obtained using primers 15SL (ACA GGC GTT

ACA TTG TTG TC) 15SP( CCC TTA TTT ATT TAA AGA AGA TG) digested with

Sau3A restricton enzyme was used as a probe.

For 21S rRNA, the PCR product obtained using the primers 21SNL2 (AGT ACG

CAA GGA CCA TAA TG) 21SR2 (TTT CGA TTA CAA AAC GAA TGC) digested with

NcoI restriction enzyme was used as a probe.

Primer extension. 5 µg of mitochondrial RNA were hybridized with 2pM of γ[32P]ATP

labeled primer in 200 mM KCl, 10 mM Tris pH 8.3. The reaction mixture was heated at

850C for 5 min and slowly cooled to 420C and then 10 µl of elongation mix was added (2x

buffer for AMV, dNTP 2mM, 7u. AMV Boehringer) Reactions were stopped by addition

of 12 µl formamide and products were resolved in 6% acrylamide 7M urea gels.

Accompanying sequencing reactions were used as molecular weight markers. Primers were


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used as follows: COX1(TAT ATT TAA TGA TAT TAA TAC TCTC); VAR1 (GAA ATA

TAT ATA TAT ATA TAA TAT GCA TCC) COB (CAA TTA TTA TTA TTA TTA TTA

TAC ATA AA); 15S rRNA (CGT ATG ACT CGT ATG CGT CAT GTC C); 21S rRNA

(TTT AAT TAT TAT ACT CCA TGT TAT CT); COX3 (ATT AAT ATA AAT CAT

TGA TAA TAT CTT); ATP6/8 ( ATA TTA GTA TTTA TTT ATA TAG TTC CC)

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 (TAT CTA TGT ATT AAT TTA ATT

ATA TAT TAT TTA TTA ACT CTA CCG AT) in 20 µl by heating at 650C for 5 min, and

slow renaturation to 370C (90-120 min). Heteroduplexes were ethanol precipitated,

resuspended in 5µl of digestion buffer (20mM sodium citrate pH 5.0, 10mM EDTA, 5 u

RNase T1 (Gibco-BRL)) and incubated at 37 0C for 30 min. 5µl of formamide were added

to the samples. The samples were heated at 750C for 3 min and resolved in 8% acrylamide

7M urea gel, transferred to nylon membrane by electrophoresis transfer in 0.5 TBE buffer

in trans-Blot Cell (Bio-Rad) and hybridized using [32P] end labeled CobT1

oligonucleotide. The accompanying DNA sequencing reactions were used as molecular

weight markers.


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Results

The mitochondrial degradosome contains only 2 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 tandem affinity purification method (TAP) (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 which 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 75kDA and

105 kDa which 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 due to 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.


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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 immunobloting 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 NTP-ase 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

which 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).


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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

which 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 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 non-respiring 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 Figure 5 show that the mutation Tyr814Ser 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


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mitochondrial genomes were introducted into mutant strains (13). 15S rRNA and COB

mRNAs were unstable, in addition in the mutants we detected several precursors of 21S

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 ∆SUV3

and ∆DSS1 strains with intronless mtDNA.

We analyzed processing of 3’- ends of 21S rRNA, 15S 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 21S rRNA is strongly inhibited

and the precursor accumulates. In the case of 15S 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 15S 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.


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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: 15S rRNA and COB, VAR1 and ATP6/8 mRNA. As a control we

used 21S rRNA, COX1 mRNA not requiring 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 which 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).

In order to check if the degradosome is involved in tRNA processing we analyzed

the maturation of tRNA in ∆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.


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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.

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 ∆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 no reports showing conclusively 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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Discussion.

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 since the calculated

molecular weight of this protein after cleavage of the leader is 81 kDa. We think that the

most probable explanation the discrepancy between our present results and previously

published data is that the third protein band of 90kDa 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).


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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 since 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 purification 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 kDa 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 concentration 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 E.

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.


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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’-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


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polyadenylation directs RNA to degradation and that only a very small fraction of RNA is

polyadenylated in vivo. In bacteria RNaseII which is homologous to Dss1p can stabilize

mRNA by poly(A) trimming (40). In strains devoid of Dss1p and Suv3p mature 15S 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 ∆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.

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 ∆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.

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

∆SUV or ∆DSS1 strains. The degradosome is most probably not involved in RNA

processing directly because processing is endonucleolytic and the degradosome does not


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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’-UTR 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 since 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’-UTRs to which stabilizing proteins and translation activators bind

(26,44). RNA turnover has a 3’→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


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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’-UTRs

(42,48). In addition Pet127p is involved in 5’-end processing of mitochondrial RNA. The

same mutation in the COB 5’-UTR region is suppressed by mutations in DSS1 and PET127

genes suggesting that both proteins may function in the same pathway. The ability of

PET127 overexpresion 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);

Minczuk et al in preparation). Research is in progress to identify the function and the

putative partners of human SUV3 RNA helicase.


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Acknowledgments

:H WKDQN (ZD %DUWQLN DQG 3LRWU 6áRQLPVNL IRU FULWLFDO UHDGLQJ RI WKH PDQXVFULSW DQG $QQD

Dziembowska for editing the manuscript, Bertrand Seraphin for plasmids containing the

TAP-tag, Andrzej Dziembowski was the recipient of an EMBO short-term fellowship for

work carried out in the Section for Molecular Biology, University of Amsterdam. In the

years 2001 and 2002 Andrzej Dziembowski received FNP (Foundation for Polish Science)

fellowship for young scientists. This work was supported by the State Committee for

Scientific Research, Grants No. 6P04 00319 and 6P04 01818, by the Polish-French Center

for Biotechnology of Plants, and by the Faculty of Biology grant BW.


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Figure legends

Figure 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 which was more stable in both types of

extracts.

Figure 2 The degradosome is composed of Suv3 and Dss1 proteins. The degradosome was

purified from Tap-tagged yeast strains DSSTAP and SUVTAP and from the 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 TCA-precipitated and subjected to SDS-PAGE. A)

NTP-exoribonuclease activity assay. Protein samples were incubated with internally ([32P]-

labelled) UTP labeled RNA with and without NTPs and subjected to PEI-Cellulose TLC

under conditions in which the substrate (RNA) remains at the bottom of the chromatogram

and reaction products (UMP) migrates 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.

Figure 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


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antibodies. A)SDS-PAGE B) Western blot. Dss1 protein was present exclusively in the

ribosomal fraction.

Figure 4 Mitochondrial degradosome has RNA helicase activity which 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 fmoles 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).

Figure 5 Change of tyrosine 814 to serine in Dss1p causes the loss of degradosome

exoribonuclease activity but has no influence on RNA helicase activity. The degradosome

purified from mitochondria from a W303/DSSSER W303/DSSTAP diploid yeast strain was

assayed for exoribonuclease and RNA helicase activities as described in Figs 2 and 4,

respectively. For both experiments the same amounts of purified degradosome were used.

Figure 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 21S rRNA, 15S rRNA and COB and COX1 mRNAs from

BWG (wt), ∆SUV3 and ∆DSS1 strains by S1-nuclease mapping. Reaction products for 15S

rRNA and COB and COX1 were resolved in 6% acrylamide 7M urea gels, and for 21S rRNA

in denaturing agarose formaldehyde gels. The position of mature RNA is indicated on each

gel.


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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.

Figure 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 γ[32P]-labeled ATP. We analyzed all

specifically processed RNAs: 15S rRNA and COB, VAR1, ATP6/8 mRNAs. As a control we

used the unprocessed RNAs: 21S 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.

Figure 8 The mitochondrial degradosome is not involved in tRNA maturation. Northern blot

analysis of mitochondrial tRNAs in wt and SUV3 and DSS1-disrupted strains. RNA was

resolved in 6% acrylamide/7M 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.

Figure 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) which 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.


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Dmochowska, Michel Siep, Hans van der Spek, Les Grivell and Piotr P. StepienAndrzej Dziembowski, Jan Piwowarski, Rafal Hoser, Michal Minczuk, Aleksandrahelicase and RNase activities and the role in mitochondrial RNA metabolism

The yeast mitochondrial degradosome: its composition, interplay between RNA

published online November 7, 2002J. Biol. Chem.

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