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, 02-106 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 12-

nucleotide sequence (the dodecamer) 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 . The SUV3 gene was then found to encode a protein from the large

superfamily of ATP-dependent RNA helicases . Margossian et al. demonstrated that the Suv3

protein was a component of the 3’-5’ exoribonucleolytic activity previously isolated and

described by Min et al. 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 , but it soon became

apparent that its role was not limited to processing of transcripts containing any particular

intron 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 .

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 . In the landmark study,

Dziembowski et al. 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 . 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

. 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 .

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 . As the polycistronic primary transcripts in yeast mitochondria undergo extensive

and complex posttranscriptional processing (reviewed in ), 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 .

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 .

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 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 . Interestingly, overexpression of PET127 can partially complement the

phenotype resulting from the degradosome deficiency . The protein encoded by the NUC1

gene, once considered as the candidate for a general mitochondrial ribonuclease is now

known to be a DNA endonuclease involved in mtDNA recombination and in cell cycle

regulation , and plays no discernible role in RNA degradation . For other putative

ribonucleases that localize in the mitochondrion according to proteomic studies and high-

throughput screens ], such as Rex2p and Ngl1p, no evidence of a significant involvement in

general mitochondrial RNA turnover was found (authors’ unpublished data and ). 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 .

The analysis of the enzymatic properties of the yeast mitochondrial degradosome

reconstituted in vitro from Suv3 and Dss1 proteins expressed in E. coli 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 . When the substrate is an RNA/RNA duplex, the helicase activity

precedes the exoribonuclease – the loading strand (with the 3’ single-stranded 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 .

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 .

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 .

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 . 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 than to bacterial RNase R or yeast exosome subunit

Dis3p 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 , 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 , 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 . A similar role was proposed for the

human exosome-associated proteins Ski2p and RHAU . Structural and mechanistic data

obtained for DExH/D helicases NS3 and NPH-II 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 and Rrp44 , 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. . 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 . 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 , 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 . 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 . 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 , 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 . 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 . 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. suggest that the main RNA degradation activity of S. pombe

mitochondria is the 5’-3’ exoribonuclease governed by the ortholog of Pet127p , 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 . 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 . The mRNA for hSuv3p was detected in all tissues but the highest level was

observed in tissues with increased energy requirements . Further research showed that pre-

mRNA of hSuv3p undergoes alternative splicing which results in formation of two messenger

RNA . 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 .

hSuv3p is localized in the mitochondrial matrix, and its N-terminal fragment contains

a mitochondrial localisation signal . The protein was found to be a component of the

mitochondrial nucleoid . Moreover, in some cell types hSuv3p is also localized in the

nucleus .

The hSuv3p helicase has all catalytic domains found in its yeast ortholog .

Biochemical studies of hSuv3p protein purified from heterologous system showed that it has

ATPase activity, which is modestly stimulated by polynucleotides . Hydrolysis of ATP is

necessary for hSuv3p to unwind double-stranded nucleic acids . Although it was claimed

initially that this protein acts in the 5’-3’ direction our studies (unpublished data) and

experiments performed by others 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 . 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 . The

latter feature was surprising because based on previous studies of yeast Suv3p the human

homolog had been expected to prefer RNA substrates .

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 . In

contrast to S. cerevisiae, mammalian mitochondrial mRNAs are polyadenylated . 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 and by

overexpression of a dominant-negative, catalytically inactive mutant version of the protein .

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 and causes stabilization of the ND3 mRNA . At the same

time, long-term expression of the gene encoding the inactive version of hSuv3p results in

decrease of mature mRNA ND3 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . In case of

the yeast mitochondrial degradosome the helicase Suv3p moves along the substrate and

delivers it to the catalytic center of Dss1p . 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 . 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 .

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 . 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 . This is in agreement with previous studies showing

existence of adenylated L-strand trancripts . 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 .

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 . Thus, their

detection confirms that the L-strand transcript, like the policistronic H-strand transcript, is

processed by the tRNA punctuation mechanism . 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 . 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 .

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. 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 . Studies performed by Mukherjee et al. 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 . 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 .

Moreover, in human mitochondria silencing of polynucleotide phosphorylase (PNPase) leads

to increase of the aberrantly proccessed COX1 transcripts. Most importantly, these transcripts

were uridylated .

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 . This pathway of mtRNA degradation seems to

depend on mitochondrial translation . It was also reported that RNase L is capable to degrade

mitochondrial mRNA when mouse cells were treated with the ionophore monensin .

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 .

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 . 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 .

Two forms of PNPase exist in plants, one targeted into mitochondria, the other located

in plastids . 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 . The mitochondrial enzyme takes part in degradation of rRNA and tRNA processing

intermediates, non-coding RNA transcribed from intergenic regions and antisense RNA .

Human PNPase was identifided during a screen for genes upregulated in the process of

terminal cellular differentation and senescence . The protein was shown to localize to

mitochondria and its phosphorolytic activity was confirmed in vitro . 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 . The

situation became even more complicated when polynucleotide phosphorylase was shown to

localize into the mitochondrial intermembrane space (IMS) where it is involved in control of

RNA import into mitochondria .

We showed that PNPase co-purifies with hSuv3p and vice-versa . Moreover, both

proteins form an active complex in vitro . 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 . In addition, Liou et al. 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 AIF-

dependent apoptosis . 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 and the gene expression

profile, which takes place in all the tissues examined . On the basis of these studies hSUV3

gene has been classified to the group of constitutively expressed housekeeping genes .

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 . Moreover, it has been shown that SUV3

gene deletion in mice also causes embryonic lethality , 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 .

Using the yeast two-hybrid system HBXIP was identified as a protein interacting with

the helicase hSuv3p . 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 . This complex inhibits binding of procaspase-9 to the

Apaf1 protein, which is responsible for its activation . Caspase-9 activity is associated with

the intrinsic mitochondrial-dependent apoptotic pathway . Importantly, reduction of the

hSuv3p protein level leads to activation of caspase-9, which indicates that this process

involves the above mentioned pathway .

However, the available data is insufficient to confirm or reject the hypotheses

generated by Minczuk et al. . 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 .

Perhaps hSuv3p can control other interactions of the HBXIP protein. On the other hand,

HBXIP is mainly localized in the cytoplasm , 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 . 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 . hSuv3p presence in the nucleus of HeLa

cells was determined by immunofluorescence staining using anti-hSuv3p antibodies . Our

unpublished data indicate that the EGFP protein synthesized in fusion with the C-terminal

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 . 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 .

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 . 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 . Pereira et al. 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 . 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 . This prompted the suggestion that human

hSuv3p helicase may participate in maintaining nuclear genome stability . Recent studies

showed that the hSuv3p helicase interacts with the nuclease FEN1 and the RPA protein ,

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 .

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 . These changes

correlated with a slightly increased level of the p27 protein, which is known to be an inhibitor

of the G1/S transition . 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 damage-

induced apoptosis . 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 .

On the other hand, studies using 293 cells (human embryonic kidney) showed that in

those cells the hSuv3p helicase is localized only in mitochondria . 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. 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. , 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. observed an

increased number of cells in G2/M phase and polyploid cells, and not in G1 phase, as reported

earlier . This discrepancy is currently hard to explain. Moreover, it was also reported that cells

with reduced hSuv3p levels have features indicating cellular senescence .

4. Concluding remarks

RNA degradation in human mitochondria seems to be mostly dependent on a hSuv3p-

contaning 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 . 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 .

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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