RNA turnover in human mitochondria: More questions than answers?

Biochimica et Biophysica Acta 1797 (2010) 1066–1070



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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o


Review

RNA turnover in human mitochondria: More questions than answers?
Lukasz S. Borowski a, Roman J. Szczesny a,b, Lien K. Brzezniak a, Piotr P. Stepien a,b,⁎
a
Institute of Genetics and Biotechnology, Faculty of Biology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw, Poland
b
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland




a r t i c l e i n f o a b s t r a c t

Article history: Protein complexes responsible for RNA degradation play important role in three key aspects of RNA
Received 2 November 2009 metabolism: they control stability of physiologically functional transcripts, remove the unnecessary RNA
Received in revised form 19 January 2010 processing intermediates and destroy aberrantly formed RNAs. In mitochondria the post-transcriptional
Accepted 25 January 2010
events seem to play a major role in regulation of gene expression, therefore RNA turnover is of particular
Available online 2 February 2010
importance. Despite many years of research, the details of this process are still a challenge. This review
Keywords:
summarizes emerging landscape of interplay between the Suv3p helicase (SUPV3L1, Suv3), poly(A)
Mitochondrion polymerase and polynucleotide phosphorylase in controlling RNA degradation in human mitochondria.
RNA degradation © 2010 Elsevier B.V. All rights reserved.
RNA surveillance
Polynucleotide phosphorylase (PNPase)
Poly(A) polymerase (PAP)
Suv3 helicase (SUPV3L1, hSuv3p)




1. Introduction sary to translate the encoded subunits of the oxidative phosphory-
lation complexes [3]. Proper expression of mitochondrial genes is vital
Today's mitochondrial genomes are an evolutionary remnant of for functioning of mitochondria, but since approximately 1500 other
the endosymbiont which colonized pre-eukaryotic cells about mitochondrial proteins are nuclear-encoded, a precisely tuned
2 billion years ago [1]. Subsequently most of its genes were either regulation of gene expression of both genomes is necessary.
lost or transferred to the nuclear genome. Despite the monophyletic Disturbances in proper functioning of mitochondrial gene expres-
origin of mitochondria organization of mitochondrial genomes, RNA sion have been implicated in many human conditions including aging,
processing and RNA decay pathways are diverse in cells of different cancer and neurodegenerative diseases [4–7]. Thus understanding the
eukaryotic organisms. Plant mitochondrial genomes are relatively mechanisms of mtDNA expression is of importance, but despite many
large, encode 50–60 genes, some of which contain introns. The genes years of investigations, many aspects of mammalian gene expression
have multiple promoters driving expression of monocistronic or are still unknown or are the subject of controversy.
polycistronic transcripts. The decay pathway of plant mtRNA is Human mitochondrial transcripts are synthesized as polycistronic
dependent on post-transcriptional addition of poly(A) tails. The yeast molecules complementary to the H-strand and L-strand of the
Saccharomyces cerevisiae mitochondrial genes are transcribed from 13 mtDNA. Subsequently the long primary transcripts are quickly
promoters, some genes contain multiple introns, but mRNAs are not processed into mRNAs, tRNAs and rRNAs by endonucleases acting in
polyadenylated and RNA stability is achieved by the encoded AU-rich most cases specifically at tRNA sequences flanking mRNAs and rRNAs
sequence at the 3′ ends [2]. [8]. In principle this tRNA-punctuation processing should result in
The miniature mammalian mitochondrial genomes are organized stoichiometric amounts of all mtRNAs, but the final concentration of a
in an extremely compact way; they are about 16 kbp long and encode given RNA also depends on premature termination of transcription [9]
only 13 proteins plus two ribosomal RNAs and a set of tRNAs neces- and on RNA degradation.
In all biological systems RNA decay plays three important roles:
it determines the half-life of a given RNA species, it destroys the
Abbreviations: mtDNA, mitochondrial DNA; mtRNA, mitochondrial RNA; IMS, aberrantly formed RNA molecules which might interfere with the
intermembrane space; PNPase, polynucleotide phosphorylase; mtPAP, mitochondrial translation machinery, and it degrades the processing intermediates.
poly(A) polymerase; hSuv3p, human Suv3 helicase This is particularly important for human mitochondria, as the large
⁎ Corresponding author. Institute of Genetics and Biotechnology, Faculty of Biology,
Warsaw University, Pawinskiego 5a, 02-106 Warsaw, Poland. Tel.: +48 225922240;
polycistronic RNA transcribed from the L-strand contains only one
fax: +48 226584176. polypeptide (ND6) and 8 tRNAs, while the remaining vast intergenic
E-mail address: (P.P. Stepien). regions do not seem to play any physiological role.

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2010.01.028

, L.S. Borowski et al. / Biochimica et Biophysica Acta 1797 (2010) 1066–1070 1067


2. RNA turnover in yeast mitochondria trimming of the poly(A) tails would regulate the stability of a given
transcript.
So far the best characterized mitochondrial RNA decay system has In addition to its mitochondrial functions, the hSuv3p helicase is
been described for the yeast S. cerevisiae, because both genetical and present in the nuclei, where it interacts with WRN and BLM helicases
biochemical analysis was available. Yeast RNA turnover is accom- [26], both implicated in ensuring chromatin stability and DNA repair.
plished by the two-subunit protein complex called mtEXO or The hSuv3p protein was also found to form a complex with HBXIP
mitochondrial degradosome. It consists of an RNA helicase encoded protein, which is a necessary partner of survivin in suppression of
by the nuclear gene SUV3 [10] and of an exoribonuclease encoded by apoptosis [27]. So far it is not clear how this multiple subcellular
the nuclear gene DSS1 [11]. Both proteins form a complex where the localization is achieved, are mitochondria a reservoir of hSuv3p or
helicase feeds the RNA substrate at the expense of ATP hydrolysis separate pools of the protein co-exist in the cell.
into the active center of Dss1 ribonuclease, and the degradation
reaction proceeds from the 3′ end to 5′ end yielding nucleoside mono- 4. The diverse functions of polyadenylation
phosphates [12]. Mutations inactivating either of the two enzyme
subunits result in a plethora of phenotypes: yeast cells stop respiring, Polyadenylation is a template-independent process where aden-
mitochondrial translation is abolished, mtDNA is unstable and various osine residues are covalently attached to the 3′ end of RNA molecules,
abnormal RNAs accumulate, including unprocessed precursors, forming a poly(A) tail which can be as long as several hundred
introns, and transcripts with abnormal 5′ or 3′ termini [13–15]. nucleotides in the case of nuclear-encoded eukaryotic mRNAs, but only
The yeast mitochondrial degradosome functions as a regulator of about 50 nt for mammalian mitochondrial mRNAs. Polyadenylation is
mature RNA half-life and as a surveillance system, degrading unneces- common to all living organisms, but, interestingly, it plays opposite
sary or aberrant RNA molecules [14]. Interestingly, most of the SUV3 roles in various taxa: in eukaryotes nuclear transcripts generated by
or DSS1 mutant phenotypes can be suppressed when a secondary polymerase II are stabilized by polyadenylation which occurs con-
missense mutation in either of the two subunits of the mtRNA poly- comitantly with transcription termination and maturation events [28].
merase is introduced [16]. This phenomenon shows that a reduction The newly synthesized poly(A) tails bind specific poly(A)-binding
in transcription efficiency can rescue the effects of perturbations in proteins which control the length of the tail, ensure protection from
RNA decay: thus the proper balance between synthesis and decay of degradation, and enable the release of mRNA from its template, its
mtRNA is essential for functioning of mitochondria. It is important to export to the cytoplasm and subsequent interaction with the 5′end of
note that there is no polyadenylation in yeast mitochondria, instead the mRNA which enables translation [29].
an A-rich twelve nucleotide sequence called dodecamer is encoded at In contrast to the above, polyadenylation is a degradation signal
the 3′ end of each of the mRNAs [17]. for bacteria, several Archaea, chloroplasts and plant mitochondria, so
Orthologous two-subunit complex has been also found in the poly(A) tail is synthesized as the “kiss of death” and is a necessary
mitochondria of Trypanosoma [18], where it was shown that subunit step in recruiting exonucleases to the 3′ end [30,31]. Interestingly,
of this complex (TbDSS-1 exoribonuclease) participates mainly in polyadenylation-dependent RNA degradation was recently discovered
RNA surveillance [19,20]. In mitochondria of Schizosaccharomyces in the nucleus of S. cerevisiae, Drosophila melanogaster and humans
pombe, interestingly, the latter was found to affect RNA processing [32–34]. The yeast S. cerevisiae complex called TRAMP is responsible
exclusively [21]. for polyadenylation of aberrant nuclear rRNAs, snoRNAs and cryptic
unstable transcripts, which subsequently are degraded by the nuclear
3. RNA surveillance and decay in human mitochondria: exosome complex [35]. This shows that the poly(A) tails can have
the involvement of hSuv3p helicase opposite functions within the same cells.
Polyadenylation of mammalian mitochondrial mRNAs was discov-
The search for enzymes responsible for human mtRNA turnover ered a long time ago [36,37], and one of its functions was to ensure
and surveillance has proven to be difficult. The human nuclear proper stop codons, because some of the mtDNA-encoded sequences
genome encodes an orthologue of the yeast Suv3 helicase (SUPV3L1; comprise of U or UA and lack the remaining A's which have to
hSuv3p; Suv3), but no orthologues of the Dss1 exoribonuclease were be added post-transcriptionally. Other functions of mitochondrial
found. The hSuv3p helicase localizes predominantly to the mitochon- polyadenylation still remain unresolved. Two groups reported the
drial matrix, but small amounts could be detected in the cell nucleus identification of human mitochondrial poly(A) polymerase (hmtPAP)
[22]. In vitro studies showed that the enzyme is an ATP-dependent [38,39]. Silencing of human mtPAP resulted in shortening of poly(A)
multisubstrate helicase, able to unwind dsDNA, dsRNA and RNA–DNA tails from about 50 nt to about 8 nt, so the short oligo(A) tails were
heteroduplexes [23,24]. Experiments using a dominant-negative still present. The short tails were found to be precursors of the long
hSuv3p mutant and siRNA knockdown revealed that the helicase is ones [40] but the question remains whether they are the product of
indeed necessary component of the RNA turnover system in human the hmtPAP activity or is there another, yet unidentified oligoadeny-
mitochondria: it regulates the half-life of correctly formed mature lating enzyme in mammalian mitochondria? The second question is
mRNAs, the decay of aberrantly formed transcripts and the rapid what is the function of short and long mitochondrial poly(A) tails? Are
degradation of processing intermediates, which are derived predom- they stability factors or do they play a role in translation? Conflicting
inantly from the L-strand transcripts [25]. Perturbation of the hSuv3p results have been obtained by studying the effects of knocking down
activity significantly inhibits RNA degradation and therefore reveals the human mtPAP: one report suggested no effects on steady state
RNA species that are impossible to detect in normal cells due to their levels of mitochondrial mRNAs [39], while another group reported a
extremely short half-life. One of such class of RNA is so-called mirror decrease of some, but not all mitochondrial mRNAs [38].
RNAs: i.e. antisense RNA transcribed and processed from the opposite Lower level of some (but not all) oligoadenylated mtRNAs [38]
strand of mitochondrial genome and subsequently polyadenylated suggests a protective role for human mitochondrial poly(A) tails.
and degraded [25]. It remains to be seen if mirror RNAs are only an On the other hand, G. Schuster et al. [41] suggested the existence
unnecessary processing by-product or if they play a regulatory role in of polyadenylation-dependent degradation of human mtRNAs. Such
translation or mtDNA replication. a decay mechanism would operate in parallel with protective
Interestingly, perturbations in the hSuv3p helicase activity polyadenylation. This hypothesis was based on the observation of
resulted in lengthening of the poly(A) tails of several mitochondrial so-called internal polyadenylation of human mtRNAs: the analysis of
transcripts [25]. This may indicate a functional coupling between truncated mRNAs revealed that they are polyadenylated. We think
polyadenylation and degradation: the balance between synthesis and it is possible that polyadenylation of aberrant RNAs or processing