mRNA turnover in human mitochondria, one of the key mechanisms governing mitochondrial gene expression, still presents an unsolved puzzle. The present article summarizes the current research on the mechanisms and enzymes that may be involved in that process.

Mitochondrial DNA (mtDNA), transcription and RNA processing

Human mtDNA (hmtDNA) is a small (16569 nt) double-stranded circular molecule containing no introns, with the main regulatory elements involved in replication and transcription initiation positioned within a single major non-coding region (the D-loop). This remarkably compact genome encodes two ribosomal RNAs: 12S and 16S, 22 tRNAs and 13 polypeptides. The latter are all components of four of the multiprotein complexes that couple oxidative phosphorylation.

Transcription initiates from two main sites in mtDNA: the HSP (heavy-strand promoter) and the LSP (light strand promoter). This gives rise to polycistronic RNA precursors with tRNA molecules interspersed between the protein and rRNA-coding sequences. The tRNA structures form recognition sites for nascent transcript-processing enzymes, generating mature mRNAs and rRNAs [1]. The endonucleolytic cleavage at the 5′-end of tRNA by RNase P has been suggested to initiate the process [2]; however the 3′-tRNA-cleavage activity has not yet been unequivocally identified. Addition of CCA by a tRNA-nucleotidyl transferase completes maturation of the tRNA 3′-termini [3]. All other RNAs are constitutively adenylated, with one to ten adenine residues and 50–60 nt long poly(A) tails added to the 3′-termini of rRNAs and mRNAs respectively. Processed human mt-mRNAs (mitochondrial mRNAs) display a striking heterogeneity, the lack of 5′-cap structure being one of the few traits shared by all transcripts (Table 1).

Table 1
Human mitochondrially encoded transcripts

UTR, untranslated region.

Gene 5′-UTR (nt) Open reading frame (nt) 3′-UTR (nt) Initiation codon Stop codon Poly(A) required to complete stop codon (nt) 
ND1 955 AUA UAA 
ND2 1041 AUU UAA 
ND3 345 AUA UAA 
ND4L/4 0/296 296/1377 1371/0 AUG/AUG UAA/UAA 0/2 
ND5 1811 568 AUA UAA 
ND6 524 500–550* AUG AGG 
CO1 1541 72 AUG AGA 
CO2 708 24† AUG UAG 
CO3 783 AUG UAA 
Cytb 1140 AUG UAA 
ATP8/6 1/161 206/679 634/0 AUG/AUG UAG/UAA 0/1 
Gene 5′-UTR (nt) Open reading frame (nt) 3′-UTR (nt) Initiation codon Stop codon Poly(A) required to complete stop codon (nt) 
ND1 955 AUA UAA 
ND2 1041 AUU UAA 
ND3 345 AUA UAA 
ND4L/4 0/296 296/1377 1371/0 AUG/AUG UAA/UAA 0/2 
ND5 1811 568 AUA UAA 
ND6 524 500–550* AUG AGG 
CO1 1541 72 AUG AGA 
CO2 708 24† AUG UAG 
CO3 783 AUG UAA 
Cytb 1140 AUG UAA 
ATP8/6 1/161 206/679 634/0 AUG/AUG UAG/UAA 0/1 
*

The precise 3′-end of MTND6 (mtDNA-encoded NADH dehydrogenase 6) has not been unequivocally determined; see [13].

Can be polymorphic, but this is correct for HeLa cells.

Mammalian mRNA turnover: an unsolved puzzle

Polyadenylation plays a critical role in controlling mRNA stability in many systems (for a review, see [4]). In the eukaryotic cytosol, in co-operation with a protective poly(A)-binding protein, the poly(A) tail acts as a stabilizing factor, whereas, in bacteria and chloroplasts, polyadenylation promotes transcript degradation. The monophyletic origin of mitochondria might suggest the existence of an uniform bacterial-like regulation in these organelles; interestingly, however, although polyadenylation in plant mitochondria does lead to transcript degradation, the situation is surprisingly different in protist and yeast mitochondria. The stabilizing role of poly(A) tails of edited protist mRNAs compared with rapid decay of unedited polyadenylated transcripts has been demonstrated (reviewed in [4]). Yeast mt-mRNAs lack polyadenylation; instead, an encoded dodecamer sequence is implicated in controlling transcript stability.

The presence of stable poly(A) extensions at the 3′-termini of mitochondrial transcripts remains a puzzling feature. Oligoadenylation at the 3′-termini has been suggested to precede the addition of long poly(A) tails, but the source of oligo(A) extensions remains unclear [5]. The enzymes capable of polyadenylation, hmtPAP [human mitochondrial poly(A)-specific polymerase], or deadenylation, hmtPNPase (human mitochondrial polynucleotide phosphorylase), a homologue of bacterial and plant PNPases (polynucleotide phosphorylases), have been identified in human mitochondria. In an siRNA (small interfering RNA) hmtPAP-knockdown experiment, the three mt-mRNAs analysed seemed to be relatively stable, despite the shortening of poly(A) tails, suggesting that long poly(A) tails may not be required for mt-mRNA stability [5,6]. In a similar study, the role of the hmtPAP and hmtPNPase in transcript polyadenylation and poly(A) shortening respectively were demonstrated by siRNA knockdown, with mtPAP depletion resulting in reduced steady-state levels of several mt-mRNAs and their translation products, causing mitochondrial dysfunction. This presented some discrepancies with the previous observations as to the role of poly(A) tails [7]. Nevertheless, consensus candidates for the components of the mt-mRNA turnover system seemed to be at hand, at least until recently (see below). The RNA-degrading machinery (degradosome) has been described in yeast and Escherichia coli [8,9]. Although the subunit composition is not conserved, among the core components of both systems are an RNA helicase and exoribonuclease activities: Suv3p and Dss1p in yeast, and RhlB and PNPase in E. coli. Apart from the homologue of bacterial PNPase, the orthologue of yeast SUV3 RNA helicase gene has been found in human mitochondria; however, the human Suv3p (hSuv3p) has 104-fold greater activity in unwinding dsDNA (double-stranded DNA) than dsRNA (double-stranded RNA), therefore its role in RNA degradation is unclear. Surprisingly, PNPase has been shown recently to reside in the mitochondrial intermembrane space, hence physically separated from the matrix-localized mitochondrial transcripts, clouding its role, if any, in mitochondrial mRNA metabolism. Subsequently, a potential role in many different processes maintaining mitochondrial homoeostasis has been attributed to that enzyme [10].

Why is the human mitochondrial mRNA polyadenylated?

One undeniable reason for polyadenylation of certain mt-mRNAs is the necessity of completing the translation stop codons [1] (Table 1). The other roles are still uncertain. A recent report described a patient harbouring a microdeletion in mtDNA, resulting in the loss of the termination codon for MTATP6 (mtDNA-encoded ATPase 6). This mutation did not prevent the proper processing of the 3′-end of the transcript (RNA14) or the synthesis of functional polypeptides from the non-stop template [11,12]. However the translation-dependent shortening of RNA14 poly(A) tail and decreased steady-state level of that transcript were observed. Whether this could represent the natural deadenylation-dependent mRNA degradation or rather an alternative surveillance pathway remains to be seen. Interestingly, a multistep RNA-amplification method revealed a low abundance of internally polyadenylated mRNAs. Poly(A) tails were proposed to target these truncated or fragmented transcripts for degradation, suggesting a coexistence of stable and unstable polyadenylation in human mitochondria [13]. A stabilizing role of polyadenylation of the natural 3′-termini, however, has not been confirmed, and the existence of a counterpart of the cytosolic protective poly(A)-binding protein in human mitochondria is uncertain. Discovery of the intermembrane space localization of hmtPNPase and differences in biochemical characteristics between hmtPNPase and the bacterial and plant enzymes [14] support the suggestion of its role in maintaining mitochondrial homoeostasis, with mRNA metabolism being one of the processes influenced by PNPase activity in an indirect fashion. Constitutive knockdown of PNPase in human cell lines demonstrated an effect on mt-mRNA processing and polyadenylation, but to different degrees for various transcripts [15]. These effects, which included abnormal 5′- and 3′-end processing and fluctuations in the length of the poly(A) tails, did not seem to influence mt-mRNA steady-state levels nor the polypeptide-synthesis rate and accumulation.

Although it has been studied extensively, the role of polyadenylation remains unclear. Further analysis is ongoing with respect to both mt-mRNA stability and also the possible roles in translation or co-translational insertion of nascent polypeptides (all highly hydrophobic intermembrane proteins) into the inner mitochondrial membrane.

RNA UK 2008: Independent Meeting held at The Burnside Hotel, Bowness on Windermere, Cumbria, U.K., 18–20 January 2008. Organized and Edited by David Elliot (Newcastle, U.K.), Sarah Newbury (Sussex, U.K.) and Alison Tyson-Capper (Newcastle, U.K.).

Abbreviations

     
  • hmtPAP

    human mitochondrial poly(A) polymerase

  •  
  • mtDNA

    mitochondrial DNA

  •  
  • hmtDNA

    human mtDNA

  •  
  • mt-mRNA

    mitochondrial mRNA

  •  
  • PNPase

    polynucleotide phosphorylase

  •  
  • hmtPNPase

    human mitochondrial PNPase

  •  
  • siRNA

    small interfering RNA

A.J.B. is a Ph.D. student sponsored by EU (European Union)-MCEST (Marie Curie Early Stage Training) grant (contract MEST-CT-FP6-503684).

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