RNA degradation in yeast and human mitochondria
Roman J. Szczesnya,b, Lukasz S. Borowskia, Magdalena A. Wojcika, Piotr P. Stepiena,b, Pawel
Golika,b
a
Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw,
Pawinskiego 5a, 02-106 Warsaw, Poland;
b
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02106 Warsaw, Poland
Corresponding Author:
Roman J. Szczesny, Institute of Genetics and Biotechnology, Faculty of Biology, University
of Warsaw, Pawinskiego 5a, 02-106 Warsaw, Poland, Tel: (+48) 225922024, Fax: (+48)
226584176, e-mail: rszczesny@ibb.waw.pl
Abstract
Proper function of mitochondria requires that expression of mitochondrially encoded genes be
maintained at the right level for the cell’s present conditions, and changed in response to
stimuli. To that end the levels of mitochondrial RNAs must be kept under strict, dynamic
control. Since yeast and human mitochondria have limited possibilities to regulate gene
expression by altering the transcription initiation rate, posttranscriptional processes, including
RNA degradation, are of great importance. In both organisms mitochondrial RNA
degradation seems to mostly depend on the RNA helicase Suv3. Yeast Suv3 functions in
cooperation with Dss1 ribonuclease by forming a two-subunit complex called the
mitochondrial degradosome. The human ortholog of Suv3 (hSuv3, hSuv3p, SUPV3L1) is also
indispensable for mitochondrial RNA decay but its ribonucleolitic partner has so far escaped
identification. In this review we summarize the current knowledge about RNA degradation in
human and yeast mitochondria.
Key words: mitochondrial degradosome / RNA degradation / Suv3 helicase (SUPV3L1,
hSuv3p, hSuv3, Suv3p) / Dss1 ribonuclease / polynucleotide phosphorylase (PNPase)
1. Introduction
During the first decades of molecular biology the process of RNA degradation
attracted much less attention than RNA synthesis. Yet RNA decay is one of the key elements
in cell metabolism; it enables regulation of gene expression by destroying transcripts thus
allowing changes in their abundance, it also removes aberrant RNA molecules and
intermediates of transcription or processing, which might interfere with the translation
machinery.
In order to achieve these functions RNA degradation must be a tightly regulated
process, responding to the intracellular milieu and signals concealed within RNA molecules.
Therefore RNA decay is performed by multi-protein complexes: degradosomes in bacteria,
exosomes in the cytosol or nuclei of eukaryotes. Mitochondria contain their own genome, so
the regulation of mitochondrial gene expression is partly achieved by RNA degradation
systems, called mitochondrial degradosomes. In this review we shall focus on the two most
studied: the yeast and the human mitochondrial degradosomes, both sharing an evolutionarily
conserved RNA helicase Suv3.
2. The yeast mitochondrial degradosome
2.1. The mitochondrial degradosome of Saccharomyces cerevisiae
Earliest data suggesting the existence of a mitochondrial degradosome complex
resulted from several genetic and molecular studies performed in Saccharomyces cerevisiae.
Yeast mitochondrial mRNA transcripts are processed at their 3’ ends at a conserved 12nucleotide sequence (the dodecamer) [1, 2] by a mechanism whose exact molecular nature
still remains to be elucidated. A nuclear suppressor of a deletion in the dodecamer sequence
of the mitochondrial VAR1 gene was found to be a hypomorphic mutation in the SUV3
(YPL029W) gene, termed SUV3-1 [3, 4]. The SUV3 gene was then found to encode a protein
from the large superfamily of ATP-dependent RNA helicases [5]. Margossian et al. [6]
demonstrated that the Suv3 protein was a component of the 3’-5’ exoribonucleolytic activity
previously isolated and described by Min et al. [7]and termed mtEXO.
In the early work on the function of SUV3 and the mtEXO complex, a lot of stress was
put on its involvement in splicing and the degradation of excised introns [5, 6, 8], but it soon
became apparent that its role was not limited to processing of transcripts containing any
particular intron [9] and was of a more general nature, as the phenotype of nullomorphic
suv3Δ strains included a strong respiratory deficiency even in the strains devoid of introns in
mtDNA [10].
The second component of the mitochondrial degradosome (mtEXO) was identified in
a genetic screen for multicopy suppressors of the suv3Δ phenotype, and shown to be an
RNase II-like protein encoded by the DSS1 (YMR287C) gene [11, 12]. In the landmark study,
Dziembowski et al. [13] demonstrated that the yeast mitochondrial degradosome is a complex
composed of only two subunits: an RNR (RNase II-like) family exoribonuclease encoded by
the DSS1 gene and an NTP-dependent RNA helicase related to the DExH/D (Ski2p)
superfamily, encoded by the SUV3 gene, and that its function is related to multiple aspects of
RNA processing, degradation and surveillance.
Both subunits of the degradosome are essential for its function, and nullomorphic
mutations in either SUV3 or DSS1 lead to a severe pleiotropic dysfunction of the
mitochondrial gene expression system, with observable overaccumulation of excised intronic
sequences and high-molecular-weight precursors and depletion of mature transcripts [9-13].
Defects in processing of rRNA, tRNA and VAR1 transcripts also lead to an impairment in
translation, which in turn results in the loss of genome stability and conversion to ρ-/ρ0
cytoplasmic petites [14-16]. This effect is particularly pronounced in strains containing the ω
intron of the LSU rRNA (21S rRNA) gene, whereas the intronless mitochondrial DNA (and
variants thereof with a limited number of introns) can be, to a certain extent, maintained in
degradosome-deficient mutants [8-12].
The degradosome is the main general ribonuclease in yeast mitochondria (which lack
the phosphorolytic polynucleotide phosphorylase activity) and is responsible for the bulk of
RNA turnover and surveillance activity. It plays an important role in the degradation of
excised introns, thus permitting the recycling of protein factors, such as Mrs1p, critical for
splicing [17]. As the polycistronic primary transcripts in yeast mitochondria undergo
extensive and complex posttranscriptional processing (reviewed in [15, 18]), the degradosome
activity is also necessary for degradation of the processing by-products and aberrant or
defective molecules (RNA surveillance). Turnover of RNAs, including mature transcripts, is
also an obvious prerequisite for the control of gene expression, which is always determined
through an interplay of RNA synthesis and degradation. This is particularly evident in
mitochondria, where the transcriptional control is relatively simple compared to the nuclear
genome [15, 19, 20]. Functioning of the mitochondrial genetic system depends therefore on
the balance between RNA synthesis and degradation by the degradosome complex. This
balance was experimentally demonstrated by the suppression of a degradation-deficient
phenotype of the suv3Δ mutant by hypomorphic mutations in genes encoding the
mitochondrial RNA polymerase (RPO41) and its transcription factor (MTF1) that strongly
decreased the rate of transcription, thus restoring the balance lost due to the degradosome
deficiency [10].
Other identified or putative ribonucleases known so far play only minor, if any, roles
in RNA turnover in yeast mitochondria. The 5’-3’ exoribonuclease activity dependent on the
Pet127 protein [21, 22] is required for 5’ end processing of several transcripts, but its deletion
does not result in a loss of mitochondrial gene expression (and respiratory competency) at
normal temperatures [22]. Interestingly, overexpression of PET127 can partially complement
the phenotype resulting from the degradosome deficiency [23]. The protein encoded by the
NUC1 gene, once considered as the candidate for a general mitochondrial ribonuclease [2426] is now known to be a DNA endonuclease involved in mtDNA recombination and in cell
cycle regulation [27-30], and plays no discernible role in RNA degradation [13]. For other
putative ribonucleases that localize in the mitochondrion according to proteomic studies and
high-throughput screens [31, 32]], such as Rex2p and Ngl1p, no evidence of a significant
involvement in general mitochondrial RNA turnover was found (authors’ unpublished data
and [17]). Notably, of all the mitochondrial ribonucleases, only the degradosome was found to
be efficient in releasing intron-bound splicing cofactors by degradation of intronic RNA [17].
The analysis of the enzymatic properties of the yeast mitochondrial degradosome
reconstituted in vitro from Suv3 and Dss1 proteins expressed in E. coli [33-35] provided
significant insights into its function, revealing a remarkable degree of functional
interdependence of the two subunits. The complex purifies as a heterodimer with the apparent
molecular weight of ~190 kDa, which corresponds to a 1:1 Suv3p:Dss1p stoichiometry. The
functional relationships between the subunits of the degradosome involve all its enzymatic
functions – ATP hydrolysis, nucleic acid duplex unwinding and RNA degradation (Fig. 1).
The Suv3 protein alone, in the absence of Dss1p, does not display any RNA-unwinding
activity in vitro. Its 3’ to 5’ directional helicase activity, requiring a free 3’ single-stranded
substrate, is detectable only when Suv3p is in complex with Dss1p. Interestingly, this
dependence is not directly related to the degradation of substrate, as it is also observed when
an RNA/DNA heteroduplex with the loading strand composed of DNA, which is not degraded
by Dss1p, is used as the substrate [34]. When the substrate is an RNA/RNA duplex, the
helicase activity precedes the exoribonuclease – the loading strand (with the 3’ singlestranded protruding end) is degraded first as the duplex is being unwound, and the
complementary strand is released, and subsequently degraded from its 3’ terminus.
The ATPase activity of Suv3p, with a Vmax of about 3 μmol·min−1·mg−1, corresponding
to a kcat of about 250 min−1, and Km of about 100-200 μM, does not depend on the presence of
Dss1p. In complex with Dss1p, however, the ATPase activity of Suv3p is clearly dependent
on the presence of a single-stranded nucleic acid substrate and is decreased by about six-fold
when the substrate is absent. In the absence of Dss1p, the Suv3 protein in vitro displays its
maximum ATPase activity even in the absence of a nucleic acid substrate [34].
The exoribonuclease activity of the degradosome complex is conferred by the Dss1p
subunit, which is an exoribonuclease from the RNR (RNase II) family. The activity of the
Dss1 protein outside the complex is, however, only modest, with a Vmax of about 6
nmol·min−1·mg−1, corresponding to a kcat of about 0.6 min−1. In the presence of the Suv3p
subunit, the exoribonuclease activity of the entire complex is significantly enhanced by about
10-fold, with a Vmax of about 55 nmol·min−1·mg−1, corresponding to a kcat of about 6 min−1
[34]. Unlike the basal activity of Dss1p alone, the RNase activity of the entire degradosome
complex is completely dependent on ATP, and becomes undetectable in the absence of NTPs,
in accordance with the observations made for the activity purified from yeast mitochondria [7,
13].
The RNase activity of the degradosome complex is an ATP-dependent progressive 3’
to 5’ exoribonuclease that releases nucleoside monophosphates and leaves a short residual
core of four nucleotides [34]. Such activity is generally expected of the RNases belonging to
the RNR family, and the size of the residual core makes mtEXO more similar to bacterial
RNase II, which leaves residual cores of 3–5 nt [36, 37] than to bacterial RNase R [38] or
yeast exosome subunit Dis3p [39] which leave slightly shorter cores of about 2–3 nt.
Point mutations in the Suv3 protein that abolish its ATPase enzymatic activity result in
a loss of the RNase activity of the degradosome, even though the mutated protein still can
form a complex with Dss1p (authors’ unpublished data). The dependence of the
mitochondrial degradosome ribonucleolytic activity on the ATP-dependent RNA-helicase is
superficially similar to the situation observed in the bacterial degradosome [40, 41], where the
helicase unwinds the secondary-structure elements in the RNA substrate that impede the
enzyme’s progress. In the case of the mitochondrial degradosome, however, the helicase
strongly stimulates the ribonuclease activity of the complex in an ATP-dependent manner
even when short single-stranded substrates devoid of any secondary structure are used in the
assay [34], which suggests a different mechanism underlying the cooperation of the helicase
and RNase activities. In a proposed model, the Suv3p helicase acts as a molecular motor
feeding the substrate to the catalytic centre of the Dss1p RNase [34]. A similar role was
proposed for the human exosome-associated proteins Ski2p and RHAU [42, 43]. Structural
and mechanistic data obtained for DExH/D helicases NS3 and NPH-II [44-46] suggest a
common mechanism for this class of enzymes, involving active movement along a single
strand of a nucleic acid by conformational changes driven by ATP hydrolysis. Such activity
can assure both secondary structure unwinding (as in the bacterial degradosome), and
translocation of the single stranded substrate towards the active centre of the RNase (as
proposed for the mitochondrial degradosome). Known structures of RNR family RNases, like
RNase II [47] and Rrp44 [48], suggest that the substrate is threaded through a channel to the
active centre located at its end. In the proposed model, the Suv3 protein would move along
the RNA substrate, pushing it into the channel of the Dss1p RNase, using energy obtained
from ATP hydrolysis.
This model explains the properties of the exoribonucleolytic activity of the
degradosome complex observed in vitro by Malecki et al. [34]. In the absence of the helicase
subunit, the Dss1p enzyme is capable of binding and hydrolysing the substrate, but without
additional active substrate transport the process is inefficient. Interestingly, genetic studies
demonstrated that another mitochondrial DExH/D RNA helicase, encoded by the MSS116
gene can partially substitute for Suv3p when overexpressed [49]. The Suv3p helicase greatly
increases the rate of RNA substrate degradation by actively threading the substrate to the
catalytic centre of the helicase, and the entire degradosome complex becomes a much more
efficient RNA degrading molecular machine. Yet, when the molecular motor activity of the
Suv3 protein is arrested by the lack of ATP (or by mutations affecting its ATPase activity),
the movement of substrate ceases altogether and no RNA degradation is possible, hence the
entire complex is completely dependent on ATP. This ATP dependence is, obviously, not
observed when the substrate is degraded by Dss1p alone, in the absence of Suv3p. This model
is also consistent with the experiments on RNA/RNA duplex substrates [34], with the loading
strand degraded concomitantly with the duplex unwinding, and the complementary strand
released in its entirety before being, in turn degraded from the 3’ end.
The model presented above is consistent with biochemical and genetic observations of
the mitochondrial degradosome function, but will remain speculative until definite structural
data are available. Obtaining the structural data for the degradosome complex is therefore a
significant and potentially fruitful research avenue. The yeast mitochondrial degradosome,
with its simple composition can serve as a good model for understanding the fundamental
principles of RNA degradation by molecular machines combining the helicase and
exoribonuclease activities, common in all the domains of life.
2.2. The mitochondrial degradosome in other yeasts
The bulk of research on the yeast mitochondrial degradosome was performed in S.
cerevisiae – the standard model for studying mitochondrial genetics and function.
Bioinformatic analysis indicates that similar complexes, comprising a helicase orthologous to
Suv3p and an RNR family hydrolytic exoribonuclease are found in most, if not all lineages of
Fungi. Given their general relatedness, it is of little surprise that the mitochondrial
degradosome of other Saccharomyces sensu stricto species is significantly conserved and, at
least for closely related species, functionally interchangeable [50]. Sequences orthologous to
Suv3p and Dss1p are also found in more distant members of Saccharomycotina, such as
Candida sp., little is known, however, of their function. Deletion of the gene encoding the
Suv3p ortholog in Candida albicans leads to an impairment of biofilm formation, formation
of chlamydospores and pathogenicity, and increased sensitivity to oxidative stress [51-54].
Such phenotypes are commonly linked to defects in the mitochondrial respiratory function in
this species.
Orthologs of both subunits of the mitochondrial degradosome were also identified in a
Taphrinomycotina member – Schizosaccharomyces pombe [55], a very distant relative of
budding yeasts, which exhibits significant differences in the organization and expression of
the mitochondrial genome when compared to S. cerevisiae [18, 56]. The S. pombe orthologs
of Suv3p and Dss1p share 44% and 34% protein sequence similarity to their S. cerevisiae
counterparts, respectively. The S. pombe complex was termed “processosome”, and its
helicase and RNase subunits were named Pah1p and Par1p, respectively [55]. Deletion of the
par1 gene results in a complete mitochondrial dysfunction, while the deletion of pah1 has a
slightly less pronounced respiratory phenotype. The function of the helicase component is
partially conserved between S. cerevisiae and S. pombe, as the overexpression of Suv3p
partially rescues the phenotype of pah1 deletion. On the other hand, Dss1p of S. cerevisiae is
unable to compensate the phenotype of the par1 deletion, even when overexpressed. The
molecular defects observed in par1Δ or pah1Δ mutants suggest that the main function of the
fission yeast processosome is related to the processing of the 3’ ends of transcripts at the
conserved C-rich sequence (C-core), functionally equivalent to the dodecamer of S.
cerevisiae, hence the name given to the complex. Hoffmann et al. [55] suggest that the main
RNA degradation activity of S. pombe mitochondria is the 5’-3’ exoribonuclease governed by
the ortholog of Pet127p [57], while the 3’-5’ exoribonucleolytic activity of the processosome
(degradosome) is required for the processing of 3’ ends, whereas in S. cerevisiae it’s the
degradosome that provides the main RNA turnover activity, whereas the function of Pet127p
is limited to the processing of 5’ ends. This may be related to pronounced differences in the
organization and processing of mitochondrial transcripts in the two species. The mtDNA of S.
pombe is more compact, with short intergenic regions and encodes only two primary
polycistronic transcripts that are processed by tRNA excision and C-core processing [18, 56].
The effect of the par1 or pah1 deletion on S. pombe mitochondrial transcripts is clearly
dependent on their position in the primary transcripts relative to the 5’ end. Yet some aspects
of the S. pombe processosome deficiency phenotype, such as the overaccumulation of
unprocessed precursors and decreased levels of mature transcripts, resemble the phenotypes
observed in degradosome-deficient strains of S. cerevisiae. Despite significant changes in the
organization and expression of the mitochondrial genome of these distantly related yeast
species, the fundamental function of the degradosome seems therefore to be at least partially
maintained.
3. RNA degradation in human mitochondria
In contrast to yeast, RNA degradation in human mitochondria remains elusive. The
hSuv3p helicase is by far the best-known component of this machinery in humans. A
ribonuclease, which would be responsible for degradation of mitochondrial RNA under
normal conditions, has not yet been identified. A bioinformatic analysis only showed that the
human genome encodes no close homologs of Dss1p. Current knowledge about mitochondrial
RNA decay derives mostly from research on the function of the hSuv3p helicase.
3.1. Human Suv3 helicase (hSuv3p)
The human nuclear gene encoding the hSuv3p helicase was identified by its homology
to the yeast gene [58]. The mRNA for hSuv3p was detected in all tissues but the highest level
was observed in tissues with increased energy requirements [58]. Further research showed
that pre-mRNA of hSuv3p undergoes alternative splicing which results in formation of two
messenger RNA [59]. As one isoform of the mRNA encodes a protein lacking the catalytic
domains it was suggested that the activity of helicase hSuv3p in cell could be regulated by
differential splicing [59].
hSuv3p is localized in the mitochondrial matrix, and its N-terminal fragment contains
a mitochondrial localisation signal [60]. The protein was found to be a component of the
mitochondrial nucleoid [61, 62]. Moreover, in some cell types hSuv3p is also localized in the
nucleus [63, 64].
The hSuv3p helicase has all catalytic domains found in its yeast ortholog [58].
Biochemical studies of hSuv3p protein purified from heterologous system showed that it has
ATPase activity, which is modestly stimulated by polynucleotides [60]. Hydrolysis of ATP is
necessary for hSuv3p to unwind double-stranded nucleic acids [60, 65]. Although it was
claimed initially that this protein acts in the 5’-3’ direction [65] our studies (unpublished data)
and experiments performed by others [64] clearly showed that the hSuv3p helicase moves
along the substrate from the 3' to the 5' end. Additionaly, a second study performed by the
group of Lee also showed hSuv3p function in the 3’ to 5’ direction [66]. The human protein,
like its yeast ortholog, does not unwind blunt-ended substrates and requires a single-stranded
3’ end for its activity. hSuv3p is a multisubstrate enzyme capable of unwinding both RNA
and DNA duplexes as well as RNA/DNA heteroduplexes, with highest efficiency for dsDNA
[60, 65]. The latter feature was surprising because based on previous studies of yeast Suv3p
the human homolog had been expected to prefer RNA substrates [60].
3.2. Role of the hSuv3p helicase in mitochondrial RNA metabolism
3.2.1. Degradation of mitochondrial RNA
Expression of genetic information at the right level is crucial for the functioning of
biological systems. The steady-state level of each transcript is achieved by balancing its
synthesis and degradation rates. The human mitochondrial genome contains 37 genes which
are transcribed as three policistronic RNAs that later undergo endonucleolytic processing [6769]. In contrast to S. cerevisiae, mammalian mitochondrial mRNAs are polyadenylated [70,
71]. Human mitochondria have scarce possibilities to regulate gene expression at the
transcription initiation level compared to the nuclear system. For this reason, degradation of
RNA is of importance.
The function of human Suv3 helicase was shown by RNA interference [72, 73] and by
overexpression of a dominant-negative, catalytically inactive mutant version of the protein
[73]. Both approaches revealed that the hSuv3p protein participates in turnover of functional
RNA (mRNA, rRNA) as well as removal of non-coding RNA, making it a key component of
the RNA control system in human mitochondria.
A disruption of hSuv3p function leads to a significant accumulation of mitochondrial
RNA degradation intermediates [72, 73] and causes stabilization of the ND3 mRNA [73]. At
the same time, long-term expression of the gene encoding the inactive version of hSuv3p
results in decrease of mature mRNA ND3 [73]. The reason for this is currently unknown,
perhaps it is the secondary effect of a general disturbance in the metabolism of mitochondrial
nucleic acids, which may be caused by the accumulation of unnecessary and aberrant RNA.
The amount of RNA present in the cell is lower than the outcome of its synthesis.
Some newly formed RNAs are removed because they were processed improperly or
correspond to non-coding regions [74]. A special feature of the human mitochondrial genome
is the uneven distribution of genetic information between the two strands of mtDNA. Coding
sequences mostly occure on the L strand. Thus, the majority of functional RNAs are produced
by H-strand transcription, whereas synthesis of RNA from the L strand leads mainly to the
formation of non-coding RNA. Studies in the early 1970's have shown that transcriptional
products of the non-coding regions are rapidly removed [75]. However, the molecular
mechanism of this process has not been discovered for a very long time. Recently, it was
shown that it depends on the hSuv3p helicase. Szczesny et al. found that hSuv3p dysfunction
leads to significant accumulation and stabilization of non-coding RNA originating either from
L- or H-strand transcription [73]. This suggests a hSuv3p-dependent mechanism responsible
for the removal of non-coding RNA in human mitochondria. This mechanism need not be the
only one, but apparently its impairment cannot be compensated by other, as yet undescribed
RNA degradation pathways.
There are three basic modes of RNA degradation – catalyzed by exonucleases acting
processively, distributively or performed by endonucleases [74]. Sequence analysis of
degradation intermediates, which accumulate in response to hSuv3p dysfunction, showed that
the majority of truncated transcripts were shortened at the 3' end [73]. This indicates that
hSuv3p-dependent RNA degradation occurs in the 3'-5' direction and is consistent with the
biochemical studies of the hSuv3p helicase, which showed that it unwinds double-stranded
substrates from the 3’- to 5’ end [64, 66]. It is worth nothing that mitochondrial RNA decay
intermadiates observed in cells with inhibited hSuv3p function have various length (with the
exception of mirror RNAs, see below). This may suggest that the hSuv3p-dependent
mechanism of RNA degradation involves processive degradation.
The role of helicases in RNA degradation may vary. They may unwind double
stranded RNA making it susceptible to single-strand specific RNAses, or they may catalyze
dissociation of RNA-protein complexes which in turn facilitates RNA degradation [74]. In
case of the yeast mitochondrial degradosome the helicase Suv3p moves along the substrate
and delivers it to the catalytic center of Dss1p [34]. There is no data on whether human
hSuv3p acts the same way. However, the fact that degradation intermediates of the 12S rRNA
are mainly truncated on the 3' side of double-stranded regions may suggest that hSuv3p is
required for substrate unwinding [73]. Identification of a nuclease cooperating with hSuv3p in
RNA decay would allow biochemical studies necessary to determine the mechanism of
degradation and would be of great help to narrow down the role of hSuv3p in this process.
It is interesting that detailed analysis of truncated transcripts formed in response to
inhibition of hSuv3p activity showed a presence of RNA molecules truncated at the 5’ end
[73]. As it seems less likely that hSuv3p could play any direct role in 5’-3’ RNA degradation,
it may suggest that in human mitochondria there is another RNA degradation pathway which
acts from the 5’- to the 3’ end.
3.2.2. Processing of the primary L-strand trancript
Perturbation of the hSuv3p function leads to a strong accumulation of so-called mirror
RNA [73]. These transcripts arise from transcription of the opposite DNA strand and they
have a similar size to their corresponding mRNAs, are complementary to them at almost the
entire length, and are adenylated at the 3’ end [73]. This is in agreement with previous studies
showing existence of adenylated L-strand trancripts [76]. In contrast to messenger RNA,
mirror RNA species can not be clearly divided into oligo- and poliadenylated molecules. It
has been observed, however, that in cells with unaffected RNA degradation systems the
length of the 3’ tails ranges from 0 to 35 nucleotides [73].
In physiological conditions mirror RNAs are almost undetectable but they can be
studied when mtRNA degradation is impaired. These transcripts in most cases arise by
excision of the tRNAs precursor from the primary L-strand RNA molecule [73]. Thus, their
detection confirms that the L-strand transcript, like the policistronic H-strand transcript, is
processed by the tRNA punctuation mechanism [69]. However, some of the mirror RNAs are
not flanked on both sides by tRNA sequences. For example, there is no tRNA on the 3’ side
of the mirror ND1. Instead there is a binding site for the mTERF protein which is suggested to
terminate the transcription of the L strand [77]. Thus, most likely the 3' end of the mirror ND1
is generated by the transcription termination.
Inhibition of hSuv3p activity does not affect excision of tRNA molecules from the
policistronic transcripts but it blocks RNA degradation. As a consequence, the mirror
transcripts are stabilized and strongly accumulated.
An unresolved question remains whether mirror RNAs or other non-coding RNAs
have any function in human mitochondria, or whether they are merely a by-product of the
precursor RNA processing. The fact that normally they have a very short half-life and are
hardly detectable in normal cells suggests they are not neutral to the function of the
mitochondrial genetic system. It is believed that degradation of nuclear and cytoplasmic RNA
(functional and non-coding) is a very efficient process because it protects the cell from the
presence of short fragments of RNA. Such fragments could saturate RNA-binding proteins
blocking their function or enter into small RNA pathways which regulate gene expression
[74]. Due to the organization of human mitochondrial genome and the peculiarities of its
expression, the mitochondrial non-coding RNA is fully complementary to the functional one.
Hybridization of both RNA species could lead to formation of double-stranded RNA, which
might inhibit mitochondrial translation.
Indeed, Khidr et al. [72] showed that silencing of hSuv3p decreases mitochondrial
translation. This may be due to the presence of large amounts of non-coding RNA which
titrates ribosomes or due to the fact that noncoding RNA hybridizes with the mRNAs leading
to inhibition of its translation.
Accumulation of some non-coding RNAs is concomitant with decreasing of the levels
of their functional counterparts [73]. Studies performed by Mukherjee et al. [78] indicate that
in human mitochondria there is an RNA decay pathway which uses antisense RNA
mechanism to degrade specific transcripts. Hence, it is tempting to speculate that the
regulation of levels of non-coding RNA, for example by control of its degradation, could be
one of the means of regulating human mitochondrial genome expression. If such a hypothesis
could be proved, difficult though it may be, it would be of great importance for understanding
of the mitochondrial genetic system in humans.
3.2.3. Potential role of uridylation
One of the functions of the RNA quality control system (RNA surveillance) is
removing abnormally processed transcripts. There is no evidence for involvement of hSuv3p
in processing of primary transcripts. However, detailed studies of RNA isolated from cells
expressing the inactive form of hSuv3p did reveal some improperly processed transcripts.
Interestingly, these RNA molecules were uridylated at the 3’ end [73]. Although currently it is
not possible to clearly determine whether hSuv3p is directly involved in the degradation of
uridylated transcripts, it is likely that in the human mitochondria addition of poly(U) or
oligo(U) tails marks an incorrectly processed transcripts destined for degradation.
Notably, in mitochondria of Trypanosoma brucei the by- products of 12S rRNA
processing and aberrantly processed 12S rRNA molecules have oligo(U) tails. Such
transcripts are degraded by the TbDSS-1 ribonuclease, which is a homologue of yeast Dss1p
[79]. Moreover, in human mitochondria silencing of polynucleotide phosphorylase (PNPase)
leads to increase of the aberrantly proccessed COX1 transcripts. Most importantly, these
transcripts were uridylated [80].
3.3. Human mitochondrial ribonuclease - a missing player
RNases are master regulators of steady state levels and decay of RNA. Despite many
years of investigation the identity of the enzyme responsible for RNA turnover in human
mitochondria remains unknown.
Until now, the only known ribonuclease implicated in mitochondrial RNA degradation
is endoribonuclease L (RNase L). However, this enzyme is activated only under stress
conditions. RNase L was shown to degrade mtRNA in response to interpheron alpha and is
indispensable for induction of apoptosis [81, 82]. This pathway of mtRNA degradation seems
to depend on mitochondrial translation [82]. It was also reported that RNase L is capable to
degrade mitochondrial mRNA when mouse cells were treated with the ionophore monensin
[83]. Nevertheless, there is no evidence that RNAse L is involved in mitochondrial RNA
decay under normal conditions.
Another enzyme which could play a role in mitochondrial RNA degradation is
polynucleotide phosphorylase. The gene encoding PNPase is evolutionarily conserved, and is
present in species ranging from bacteria through plants, worms, flies, mice to humans [84,
85]. Polynucleotide phosphorylase is able to perform processive 3’-5’ phosphorolytic
degradation of RNA; interestingly, it can also catalyze the reverse reaction in which adds
hetereopolymeric adenine-rich rich tails to RNA [86-90]. In bacteria PNPase is part of the
degradosome, a complex degrading polyadenylated RNAs and consisting of Rnase E,
PNPase, the helicase RhlB and the glycolytic enzyme enolase [91].
Two forms of PNPase exist in plants, one targeted into mitochondria, the other located
in plastids [92]. Chloroplast polynucleotide phosphorylase is involved in the metabolism of
all classes of chloroplast RNAs, however, depletion of this protein has minor effect on mRNA
levels [93]. The mitochondrial enzyme takes part in degradation of rRNA and tRNA
processing intermediates, non-coding RNA transcribed from intergenic regions and antisense
RNA [94].
Human PNPase was identifided during a screen for genes upregulated in the process of
terminal cellular differentation and senescence [95]. The protein was shown to localize to
mitochondria [96] and its phosphorolytic activity was confirmed in vitro [66, 97]. Because a
human orthologe of the yeast Dss1 exoribonuclease is lacking and PNPase is localized in
mitochondria, several research groups considered PNPase as the mitochondrial degradosome
RNase. However, experiments with depletion of PNPase led to conflicting results [80, 98, 99].
The situation became even more complicated when polynucleotide phosphorylase was shown
to localize into the mitochondrial intermembrane space (IMS) [98] where it is involved in
control of RNA import into mitochondria [100].
We showed that PNPase co-purifies with hSuv3p and vice-versa [73]. Moreover, both
proteins form an active complex in vitro [66]. Therefore it is possible that a small fraction of
PNPase, difficult to detect during fractionation studies, resides in the mitochondrial matrix
and takes part in mtRNA decay as a component of mitochondrial degradosome together with
hSuv3p. This would be similar to Escherichia coli where most of the PNPase pool seems to
not participate in degradosome formation [101]. In addition, Liou et al. [102] reported that
PNPase with RhlB helicase can form a complex without RNAse E. So the question of PNPase
involvement in mammalian mitochondrial RNA degradation still remains open.
3.4. Non-mitochondrial protein partners of the hSuv3p helicase
Lowering the level of hSuv3p protein in HeLa cells induces caspase- and AIFdependent apoptosis [63]. The fact that depriving a cell of the function performed by the
hSuv3p helicase leads to the cell death indicates that the hSuv3p protein plays a fundamental
role. This is consistent with the result of hSUV3 gene promoter analysis [59] and the gene
expression profile, which takes place in all the tissues examined [58]. On the basis of these
studies hSUV3 gene has been classified to the group of constitutively expressed housekeeping
genes [59].
The importance of the function performed by the hSuv3p protein at the organism level
highlights the fact that reducing hSUV3 orthologue expression in Caenorhabditis elegans is
lethal in early stages of embryonic development [103]. Moreover, it has been shown that
SUV3 gene deletion in mice also causes embryonic lethality [104], and the use of conditional
knock-out revealed that the absence of the SUV3 gene product leads to shortening mice’s life
span and changes characteristic for premature aging [105].
Using the yeast two-hybrid system HBXIP was identified as a protein interacting with
the helicase hSuv3p [106]. The HBXIP protein is a cofactor of the anti-apoptotic survivin
protein and it has been suggested that the hSuv3p protein may participate in the control of
apoptosis via the HBXIP-survivin complex [106]. This complex inhibits binding of
procaspase-9 to the Apaf1 protein, which is responsible for its activation [107]. Caspase-9
activity is associated with the intrinsic mitochondrial-dependent apoptotic pathway [108].
Importantly, reduction of the hSuv3p protein level leads to activation of caspase-9, which
indicates that this process involves the above mentioned pathway [63].
However, the available data is insufficient to confirm or reject the hypotheses
generated by Minczuk et al. [106]. Using purified proteins it was shown that the interaction of
HBXIP and survivin is on its own sufficient to efficiently inhibit the caspase-9 activation
[107]. Perhaps hSuv3p can control other interactions of the HBXIP protein. On the other
hand, HBXIP is mainly localized in the cytoplasm [106, 107], while the hSuv3p helicase is
not observed in this cell compartment. So far there is no data on whether silencing of the
hSuv3p helicase influences the levels of HBXIP or its intracellular localization.
Taken together, it seems more likely that the cell death observed after silencing of
hSUV3 gene expression is due to hSuv3p function being indispensable for survival, but not
directly linked to the control of apoptosis.
In silico analysis suggested that hSuv3p has a fragment directing this protein to the
nucleus in the C-terminal region [63]. Double, nuclear-mitochondrial hSuv3p localization was
likely as such intracellular distribution was also observed for other proteins, for exemple the
helicase MDDX28 or the endo/exonuclease FEN1 [109, 110]. hSuv3p presence in the nucleus
of HeLa cells was determined by immunofluorescence staining using anti-hSuv3p antibodies
[63]. Our unpublished data indicate that the EGFP protein synthesized in fusion with the Cterminal part of hSuv3p is located in the nucleus (Szczesny, unpublished data). Interestingly,
this C-terminal fragment of hSuv3p protein includes the region responsible for interaction
with HBXIP [106]. This suggests that the HBXIP protein may influence the distribution of
hSuv3p in the cell. Such a role of the HBXIP-hSuv3p interaction has been already proposed
[106].
Other evidence supporting nuclear function of hSuv3p comes from yeast studies as
high throughput identification of protein complexes suggests that yeast Suv3p interacts with
nuclear proteins involved in replication, repair and recombination of DNA [111]. The Sgs1
helicase, which is a homologue of human WRN helicase and BLM helicase, was identified
among these partners. These two proteins belong to the RecQ helicase family, members of
which are involved in maintaining the integrity of the nuclear genome [112]. Pereira et al.
[104] showed that the hSuv3p protein interacts with both the BLM and WRN helicases in
vitro, however, higher affinity was observed in case of the hSuv3p-BLM interaction. It was
found that lowering the hSuv3p level increases the number of sister chromatid exchange
events [104]. The same effect is observed in cells with impaired function of the BLM
helicase, but not for the other four human helicases of the RecQ family [104, 113]. This
prompted the suggestion that human hSuv3p helicase may participate in maintaining nuclear
genome stability [104]. Recent studies showed that the hSuv3p helicase interacts with the
nuclease FEN1 and the RPA protein [64], which play a key role in DNA replication and
repair. They interact in vitro and co-purify from the nuclear fraction of HeLa cells, which
implies that their interactions take place in the nucleus. In addition, interactions of these
proteins modulate their activities: RPA inhibits helicase activity of hSuv3p, whereas the latter
stimulates the endonucleolytic activity of FEN1 [64].
Lowering the level of hSuv3p protein raises levels of p53 and causes cell cycle
disturbance, in particular it increases the number of cells in G1 phase [63]. These changes
correlated with a slightly increased level of the p27 protein, which is known to be an inhibitor
of the G1/S transition [114]. These results are another indication of hSuv3p taking part in the
maintenance of nuclear DNA. p53 plays a key role in cell cycle regulation and DNA damageinduced apoptosis [115]. It therefore seems possible that the decrease in the level of hSuv3p
protein induces nuclear DNA damage, which in turn can lead to increased p53 levels,
inhibition of the cell cycle and if the damage is too strong to be repaired, the induction of
apoptosis. Interestingly, the reduction in the HBXIP protein level also leads to an increased
number of cells in G1 and increased p27 protein levels [116].
On the other hand, studies using 293 cells (human embryonic kidney) showed that in
those cells the hSuv3p helicase is localized only in mitochondria [73]. The reason for the
observed differences in hSuv3p localization in different cell lines is currently unknown. It
could be an inherent difference between different types of cells, or a result of changes in the
cell lines’ biology that occurred during their many years of maintenance in vitro. The
examination of hSuv3p protein localization in different cell types, both transformed and
normal, including primary cells, should provide information allowing more precise
conclusions.
The main cell compartment, in which the hSuv3p helicase is located is the
mitochondrial network. Research by Khidr et al. [72] provides comprehensive data on the
significance of hSuv3p for functioning of mitochondria. It was found that hSuv3p helicase is
essential for maintaining mitochondrial homeostasis. hSuv3p silencing by shRNA leads to
decreased activity of oxidative phosphorylation complexes, reduction of the inner
mitochondrial membrane potential and consequently lowering the ATP level in the cell. As in
Szczesny et al. [63], it was shown that silencing of hSuv3p results in cell death and cell cycle
changes as well as increased levels of p53 protein. However, Khidr et al. [72] observed an
increased number of cells in G2/M phase and polyploid cells, and not in G1 phase, as reported
earlier [63]. This discrepancy is currently hard to explain. Moreover, it was also reported that
cells with reduced hSuv3p levels have features indicating cellular senescence [72].
4. Concluding remarks
RNA degradation in human mitochondria seems to be mostly dependent on a hSuv3pcontaning protein complex. However, other systems controlling RNA abundance might play a
role as well. The question of possible endonucleolytic cleavage is still open. More research is
needed on the role of polyuridylation in RNA degradation in mitochondria. The discovery of
a deadenylating enzyme in human mitochondria calls for more research on oligo- and
polyadenylation of mitochondrial transcripts [117]. Finally, recent data point to the presence
of both nuclear- and mitochondrially encoded small RNA in human mitochondria, raising
possibilities of a small RNA-dependent RNA decay [118, 119].
All these data indicate that the landscape of RNA degradation in mitochondria is still a
fascinating subject for research.
Acknowledgements
The authors thank Prof Ewa Bartnik and Aleksander Chlebowski for their help with the
manuscript. This work was supported by grant N N303 550639 and N N302 663940 from the
Ministry of Science and Higher Education of Poland and by the Polish National Centre for
Research and Development (NR13004704). RJS is the recipient of the Stipend for Young
Researchers from the Foundation for Polish Science.
Figure legend
Figure 1. Functional interplay between the activities of the yeast mitochondrial degradosome.
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