Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on July 22, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 18 2341-2348
DOI: 10.1093/hmg/ddg238
© 2003 Oxford University Press

Investigation of a pathogenic mtDNA microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria

Richard J. Temperley¹, Sara H. Seneca², Katarzyna Tonska³, Ewa Bartnik³, Laurence A. Bindoff⁴, Robert N. Lightowlers^1,* and Zofia M.A. Chrzanowska-Lightowlers¹

¹School of Neurology, Neurobiology and Psychiatry, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK, ²Department of Medical Genetics, AZ-VUB, Laarbeeklaan 101, B-1090 Brussels, Belgium, ³Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Genetics, University of Warsaw, Pawinskiego, 5A 02-106, Warsaw, Poland and ⁴Department of Neurology, University of Bergen, Haukeland Sykehus, 5021 Bergen, Norway

Received May 12, 2003; Accepted July 10, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human mtDNA is transcribed from both strands, producing polycistronic RNA species that are immediately processed. Discrete RNA units are matured by the addition of nucleotides at their 3' termini: -CCA trinucleotide is added to mt-tRNAs, whilst mt-rRNAs and mt-mRNAs are oligo- or polyadenylated, respectively. The cis-acting elements, enzymes and indeed the mechanisms involved in these processes are still largely uncharacterized. Further, the function of polyadenylation in promoting stability, translation or decay of human mt-mRNA is unclear. A microdeletion has been identified in a patient presenting with mtDNA disease. Loss of these two residues removes the termination codon for MTATP6 and sets MTCO3 immediately in frame. Accurate processing at this site still occurs, but there is a markedly decreased steady-state level of RNA14, the ATPase 8- and 6-encoding bi-cistronic mRNA unit, establishing that an mtDNA mutation can cause dysregulation of mRNA stability. Analysis of the polyadenylation profile of the processed RNA14 at steady state revealed substantial abnormalities. The majority of mutated RNA14 terminated with short poly (A) extensions and a second, partially truncated population, was also present. Initial maturation of mutated RNA14 was unaffected, but deadenylation occurred rapidly. Inhibition of mitochondrial protein synthesis showed that the deadenylation was dependent on translation. Finally, deadenylation was shown to enhance mRNA decay, explaining the decrease in steady-state RNA14. An hypothesis is presented to describe how an mtDNA mutation that results in the loss of a termination codon causes enhanced mt-mRNA decay by translation-dependent deadenylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human mtDNA, a circular duplex of 16 569 bp, is present in multicopy in all nucleated cells of the body. It encodes 13 polypeptides, all of which are believed to be essential components of the enzymatic complexes that couple cellular respiration to ATP synthesis. In addition, the genome encodes 22 tRNA and 2 rRNA molecules that are required for intramitochondrial protein synthesis. Mitochondrial transfection in mammals remains intractable and in the absence of faithful reconstitution systems many aspects of mitochondrial gene expression are not well understood. It has been known for over 20 years that the genome is almost fully transcribed from both strands (1). These polycistronic RNA units are rapidly processed into discrete RNA species, which are immediately matured to produce mt-rRNAs and mt-mRNAs that are oligo- and polyadenylated, or mt-tRNA species that carry a 3' CCA trinucleotide (1,2). There is good evidence that mitochondrial RNase P is involved in the processing of mt-tRNAs (3,4) and a mitochondrial [CCA] nucleotidyltransferase has been categorized (5). The enzymes responsible for RNA processing events that do not involve mt-tRNAs and for the maturation of all mRNAs have not been identified. Further, the cis-acting elements necessary for accurate RNA processing have also not been defined.

Defects of the mitochondrial genome are known to cause a spectrum of disorders commonly known as mitochondrial cytopathies (6). One such patient harboured a micro deletion at bp 9204/5 (or 9205/6, referred to here as µ9205) that removes the termination codon for RNA14, the bi-cistronic RNA unit encoding ATPase 8 and 6 (Fig. 1A) (7,8). Further, µ9205 brings a termination codon-less MTATP6 gene into direct apposition with MTCO3, generating a predicted ATPase 6/COX3 fusion protein, if processing is defective. The patient presented with seizures and several bouts of lactic acidaemia, symptoms consistent with mitochondrial dysfunction. µ9205 was reported to be present in homoplasmic form in the patient and we have confirmed that the maximal level of wild-type mtDNA is <2% in cultured fibroblasts (unpublished data). Here, we report that µ9205 has only a minimal effect on processing, but causes a decrease in the steady-state level of the mutated RNA14. Further investigation of the patient-derived fibroblast cell lines showed that the enhanced turnover of RNA14 was due to a translation-dependent deadenylation decay mechanism.

View larger version (33K): [in this window] [in a new window]   Figure 1. Steady state level of RNA14 is decreased in a patient-derived cell line carrying an mtDNA microdeletion at the RNA14/15 processing site. (A) Schematic showing processing and maturation of mitochondrial RNA14 and RNA15. (i) Initially, a tri-cistronic RNA species is formed which includes the partially overlapping open reading frames encoding ATPase 6 and 8 (RNA14) and cytochrome c oxidase subunit 3 (RNA15). (ii) Cleavage occurs in the control, resulting in a 3' terminal UA dinucleotide in RNA14 and a 5' AUG translation initiation codon in RNA15. Only after 3' polyadenylation of RNA14 is a UAA translation termination codon generated. (iii) If correct processing of the tri-cistronic RNA carrying the microdeletion still occurs, RNA15 will retain an intact initiation codon identical to the control. However, µRNA14 will have lost a natural termination codon, irrespective of polyadenylation. (B) Northern analysis shows that processing of RNA14/15 does occur in the presence of the microdeletion [µ], but the steady-state level of µRNA14 is substantially lower than RNA14 in three non-disease control primary fibroblast lines (controls A, B and C). Levels of RNA14 are expressed as a percentage of total RNA14+RNA14/15. Probes are shown to the left of each panel. MTND1 (complex I, subunit 1) was used as a control for mitochondrial transcript levels. (C) To determine the 3' sequence of fully matured RNA14 in patient and control, polyadenylated RNA14 was amplified, cloned and individual clones subjected to DNA sequencing. Approximately 86% (32 of 37) of the µRNA14 species carried 3' terminal sequences that would be expected following accurate processing 5' proximal to the AUG of RNA15, as described in (A).

 

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The steady-state level of RNA14 is selectively reduced in the patient-derived cell line
The pathogenic µ9205 mutation lies at the junction between the two genes MTATP6 and MTCO3. The micro-deletion removes an A : T and T : A base pair, which could correspond either to residues 9205/6 or 9204/5 (Fig. 1A). If processing at this site were unaffected, the micro-deletion would be predicted to remove the 3' terminal U of RNA14 that, after polyadenylation of the transcript, would normally form a termination codon. First, to determine whether the mutation affected RNA processing between RNA14 and 15 (RNA transcribed from MTCO3), northern analysis of RNA isolated from the patient-derived and three control fibroblast cell lines was employed. As shown in Figure 1B, the unprocessed intermediate, RNA14/15, is present in both patient-derived cell line and controls at approximately equal levels. The steady-state level of processed RNA14, however, was markedly lower in the patient (µRNA14). In contrast, there was little difference in RNA15 or of ND1, another mitochondrial transcript. The µ9205, therefore, does not prevent cleavage between µRNA14 and 15, so the decrease in steady-state level of µRNA14 when compared with controls cannot simply be explained by a partial processing deficiency.

The micro-deletion between RNA14 and 15 does not affect the accuracy of processing but alters the polyadenylation profile of RNA14
Northern analysis confirmed that RNA14/15 is processed, however as µ9205 removes two residues that are very close to the natural processing site, it is possible that the accuracy of processing was affected. To determine whether processing and maturation were still precise, the 3' termini of polyadenylated RNA14 were analysed from patient and control fibroblasts as detailed in materials and methods. Nearly all species were processed immediately upstream of the 5' initiation codon of RNA15 (Fig. 1C). The majority of the 3' termini of RNA14 were consistent with polyadenylation of species that had been generated by accurate processing. All polyadenylated µRNA14 species were, as predicted from the genome sequence, devoid of a translation termination codon.

These data only show that accurately processed µRNA14 is polyadenylated. It is possible that processing is inexact, but only the correctly cleaved species are polyadenylated. To deliver a snapshot of the termini at steady state, a newly modified assay was employed (9). The Mitochondrial Poly(A)Tail-length assay (MPAT), is described in the materials and methods and relies on the ligation of a primer to all free 3' hydroxyl termini. To establish the efficacy of the assay, the polyadenylation profile of RNA15 was examined in various fibroblast cell lines. As illustrated in Figure 2A, the processing and polyadenylation of RNA15 is similar in the patient and control RNA, with the majority of species carrying between 51 and 63 adenyl additions. This is consistent with an early report of a generic mitochondrial poly(A) tail length in HeLa cells of approximately 56 nucleotides (10). RNA14 from the control also shows a discrete population of poly(A) tail lengths (45–56) with a slightly shorter median. In marked contrast, however, µRNA14 demonstrates a very different profile. There are two populations of extended poly(A) tail length, one similar to the control (A_45–56) and one slightly shorter (A_26–37). There is also a third, severely truncated but prevalent population with between -1 and 11 residue extensions at the 3' terminus (A_-1–11).

View larger version (42K): [in this window] [in a new window]   Figure 2. µ9205 results in deadenylation of µRNA14. (A) The polyadenylation profiles of RNA14 and 15 were assessed in patient [µ] and control [cont] by MPAT. Profiles were similar for RNA15. For RNA14, in addition to a subpopulation with similar length extensions to the control, µRNA14 carried two shorter populations. The most apparent species contained very limited extensions, representing either short poly(A) additions or incorrectly processed species. (B) Prior to the final step in the MPAT assay, the short PCR products bridging the µRNA14 processing site were cloned and a subset sequenced. Sequences from all 11 clones are shown and each contained adenyl additions to accurately processed 3' termini. The number of clones with identical inserts are indicated. (C) To determine whether µRNA14(-1–11) was generated by aberrant maturation or by degradation of the full-length poly(A) tail, mitochondrial transcription was inhibited by the addition of ethidium bromide for 2 days and MPAT performed on RNA isolated at the indicated time points after release from transcription inhibition. Full extension is visible prior to the formation of µRNA14(-1–11) confirming that these shorter species are degradation products.

 
The latter species could represent limited adenylation, or, as argued above, could indicate inaccurate processing around the cleavage site. To establish whether these short extensions were indeed due to polyadenylation, cDNA copies were cloned and subjected to DNA sequence analysis (Fig. 2B). Clones revealed consistent processing, followed exclusively with short poly(A) tails. Accurate cleavage of µRNA14/15 was also confirmed by analysing the 5' termini of RNA15, with the great majority of species carrying a 5' terminal AUG (unpublished data).

Maturation of µRNA14 is unaffected
Processing of µRNA14 is efficient and relatively accurate. The polyadenylation profile of µRNA14 at steady state is, however, markedly affected. Although MPAT is only semi-quantitative, the profiles show that the majority of processed µRNA14 at steady state carry substantially truncated poly(A) tails. This could either reflect a defect in the initial maturation, whereby the full complement of adenyl residues is not added, or a rapid deadenylation. To address this, cells were subjected to inhibition of mtDNA transcription by the addition of ethidium bromide for 2 days, washed to release the cells from this inhibition and RNA prepared from aliquots of cells at the indicated time points following release. MPAT was then performed to determine the pre-steady-state polyadenylation profile. As shown in Figure 2C, de novo polyadenylation of the control and patient RNA14 are very similar, confirming that the truncated µRNA14(A_-1–11) population present at steady state is formed by decay, rather than incomplete maturation.

Deadenylation of µRNA14 is translation-dependent
The marked decrease in poly(A) tail length was due to rapid deadenylation of µRNA14. How could this be explained, as the control and patient-derived RNA14 differ by only two residues? One striking difference is that all µRNA14 species have lost a translation termination codon. Natural termination codons are recognized by release factors in a wide variety of species and there have been reports of mitochondrial release factors in man (11–13). Without these factors it is highly likely that the mitoribosome will either process through the poly(A) tail, elongating ATPase 6 by the addition of polylysine, or will be inhibited from procession by proteins bound to the poly(A) tail. Each of these possibilities differs from the natural mechanism and could provide targets for a mitochondrial mRNA surveillance system. Interestingly, a system for the decay of transcripts devoid of natural termination codons has been recently reported in the cytosol of yeast and man (14,15). Degradation of these ‘non-stop’ transcripts requires translation. No evidence of an mRNA surveillance acting in mammalian mitochondria has ever been reported. A similar system, however, could potentially be conserved in mitochondria, whereby transcripts without translation termination codons would be recognised as aberrant and rapidly degraded. To determine the role of translation on deadenylation of µRNA14, cells were subjected to thiamphenicol (TAP) treatment for 2 days to inhibit mitochondrial protein synthesis and MPAT assays performed on RNA isolated from cells during this treatment (Fig. 3). TAP had little effect on the polyadenylation profile of RNA14 in control cells, or on the longer poly(A) tails of µRNA14. TAP, however, clearly inhibited the formation of the severely truncated µRNA14 (A_-1–11) (Fig. 3 cf. lanes 6 and 9), illustrating that the rapid deadenylation of µRNA14 is translation-dependent.

View larger version (42K): [in this window] [in a new window]   Figure 3. Deadenylation of µRNA14 is translation-dependent. Mitochondrial protein synthesis was inhibited in patient-derived (µ) and control cells by the addition of thiamphenicol (TAP) for the indicated times prior to RNA isolation. RNA14 was then subjected to MPAT. Relative quantification of the polyadenylated species was assessed (ImageQuant) and is shown for the control (lanes 1 and 4) and patient (lanes 6 and 9), prior to (lanes 1 and 6) and following (lanes 4 and 9) 48 h in TAP. No difference is apparent for the control, whilst TAP treatment resulted in the loss of µRNA14(-1–11), showing that deadenylation is translation dependent.

 
Deadenylation of µRNA14 leads to enhanced mRNA decay
It is currently unknown whether polyadenylation of mitochondrial transcripts protects them from decay. Therefore, although the absence of a translation termination codon promotes rapid deadenylation, it is not known whether this would necessarily lead to an instability of the transcript. Initially, to determine the relative decay rate of RNA14, patient and control cells were treated with the transcriptional inhibitor ethidium bromide, RNA isolated post inhibition and the relative stability of transcripts carrying the short or long poly(A) extensions was assessed using MPAT. As can be seen in Figure 4A, µRNA14 (A_-1–11) is clearly lost before the fully adenylated species, consistent with decreased stability, but it can also be seen that this treatment promotes de novo extension of the poly(A) tail. The mechanism for this is unknown, but a similar observation was reported almost 30 years ago (10). We do not know whether this de novo extension could, itself, promote increased transcript stability. Therefore, it was necessary to reassess the steady-state levels of RNA14 in the TAP-treated cells. Inhibition of translation prevents the rapid deadenylation of µRNA14. Consequently, if µRNA14 (A_-1–11) is more rapidly degraded, there should be a substantial difference between the steady-state levels of µRNA14 in the TAP-treated and untreated cells. If deadenylation does not modulate the turnover of µRNA14, TAP will have no effect. TAP addition for 2 days had only a minimal effect on the steady-state level of RNA14 in controls. However, Figure 4B shows that the amounts are greatly increased in the patient cells after TAP treatment, with µRNA14 recovering to levels almost identical to the control. This confirms that µRNA14(A_-1–11) is more rapidly degraded than the extended species.

View larger version (65K): [in this window] [in a new window]   Figure 4. The rapid decay of µRNA14 is translation-dependent. (A) To determine the stability of RNA14 in patient-derived (µ) and control cells, transcription was inhibited, RNA isolated at the indicated times post-inhibition and RNA14 subjected to MPAT. µRNA14(-1–11) clearly disappears more rapidly than the longer species. Although ethidium bromide inhibits de novo synthesis of RNA14, there is evidence of transcription-independent polyadenylation. (B) To show unequivocally that µRNA14(-1–11) is rapidly degraded, RNA was isolated from untreated cells (0) or after 2 days in thiamphenicol (48) and subjected to northern analysis. As shown in Figure 3, TAP treatment prevents the formation of µRNA14(-1–11). TAP has a minor affect on the steady-state levels of RNA14 in the control; however, the amount of µRNA14 increases dramatically, confirming that µRNA14(-1–11) decays rapidly. (C) Suggested mechanism describing how loss of a translation termination codon in mt-mRNAs leads to decreased transcript stability. Polyadenylation promotes transcript stability by binding of protein factors to the poly(A) tail that prevent the approach of various ribonucleases. When the translocating mitoribosome reaches a termination codon, release factors (RF) and ribosomal recycling factors (RRF) are recruited, leading to release of the nascent polypeptide and mitoribosome disassembly. In the absence of a termination codon (µRNA14) the mitoribosome continues translocating into the poly(A) tail, displacing the poly(A) binding protein. This reveals a region of the tail accessible for endonucleolytic degradation, resulting in species with the shortened tail (µRNA14(-1–11)), which are swiftly degraded.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
There is a paucity of knowledge concerning post-transcriptional events in mammalian mitochondria. Although there is an increasing number of mtDNA mutations recorded that affect the processing, maturation and stability of mt-tRNAs (16–18), this report describes how an mtDNA mutation can influence the turnover of a human mitochondrial messenger RNA. The enzyme(s) responsible for polyadenylation of mammalian mt-mRNAs and -rRNAs have yet to be identified and searches of the human genome database do not yield any candidate. In the absence of a poly(A) polymerase, it is formally possible that the [CCA]-nucleotidyltransferase may play this essential role, consistent with the observation that 3' termini of messenger RNAs are occasionally extended with cytosine rather than adenosine (Fig. 1C). Analysis of the polyadenylation profile of µRNA14 showed that the major subset carried severely truncated tails (µRNA14_(-1–11)). Production of these short tails was translation-dependent, as evidenced by their disappearance in cells treated with the inhibitor, thiamphenicol. So what are the consequences of non-stop translation? In the absence of faithful translational termination, the nascent protein would be predicted to carry a polylysine extension and it is difficult to see how this new polypeptide would be liberated from its association with tRNA. Further, the absence of a termination codon might be predicted to prevent mitoribosome disassembly. It is not the aim of this report to focus on the defect in the respiratory chain biochemistry of this patient-derived cell line. However, it has previously been reported to result in a partial biochemical defect (8) and we have noted a minor fault in the assembly of complex V (Z.M. Chrzanowska-Lightowlers, unpublished data). Irrespective of these abnormalities, the absence of conventional translation termination of RNA14 can be tolerated, at least in fibroblasts and lymphocytes.

How does translation of the defective µRNA14 cause truncation of the poly(A) tail? It is possible that the translocating mitoribosome removes endogenous poly(A) binding proteins, revealing sites prone to nuclease digestion (Fig. 4C). This may reflect a mitochondrial mRNA surveillance mechanism capable of detecting mRNAs that do not contain termination codons. As mentioned earlier, this may have some similarity to a recently characterized mRNA decay process (14,15). The absence of a termination codon was shown in yeast and mammalian cells to induce a translation-dependent rapid degradation of the transcript, mediated by Ski7p, which recognizes the empty A site on the ribosome and recruits the exosome, initiating a 3'–5' exonucleolytic degradation. Although such a mitochondrial mRNA surveillance system is credible, our data may not be consistent with a 3'–5' exonucleolytic activity, as the profile of the truncated species does not change markedly, immediately after either translational or transcriptional inhibition and there is suggestion of a protein ‘footprint’ in the polyadenylation profiles. This could be generated by the bound mitoribosome, stalled at the 3' terminus (Fig. 4C).

Does polyadenylation promote transcript stability or translation in the mitochondrion? In plant organelles and bacteria poly(A) addition promotes rapid decay, in the cytosol of eukaryotes it increases stability and stimulates translation (19–22). The µRNA14(A_-1–11) species are more rapidly degraded than those with more extended poly(A) tails. This is clearly shown by the comparison of µRNA14 steady-state levels in TAP-treated and untreated patient-derived cells (Fig. 4B). Although it is tempting to conclude that polyadenylation does indeed lead to increased stability, it must be borne in mind that the truncated species are generated following translation run on. As shown in Frischmeyer et al. (14) and van Hoof et al. (15), at least for cytosolic RNA, non-stop transcripts are destabilized by a process of RNA decay that is unlike the natural decay pathway. This may also be the case with mitochondrial RNA species that do not carry a translation termination codon. The natural mechanism of RNA decay in mammalian mitochondria is not known. Recent evidence suggests that at least in yeast a mitochondrial degradosome exists that contains two or more proteins, one an RNA helicase and one a nuclease (23). Until the endogenous mechanisms of mt-mRNA decay are known in mammals, it cannot be excluded that the rapid decay noted for µRNA14(A_-1–11) is due to a novel decay pathway that has been retained for non-stop transcripts or as part of a general mitochondrial mRNA surveillance mechanism.

Finally, it is important to note the surprisingly minimal effect the microdeletion has on processing of RNA14/15. Although the mutation occurs at this natural processing site, enzymes responsible for this cleavage are still capable of recognizing it. We therefore remain ignorant of the cis-acting element that is crucial for maintaining correct processing at this site.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tissue culture
Passage matched patient-derived and control primary fibroblasts were cultured in a humidified atmosphere of 5% CO₂ at 37°C in EMEM with 10% fetal bovine serum, 2 mM L-glutamine, 1xnon-essential amino acids, 1 mM sodium pyruvate and 50 µg/ml uridine. Where indicated, the medium was supplemented with ethidium bromide (final concentration 250 ng/ml), or thiamphenicol (50 µg/ml). For the ethidium bromide release experiment, all cells were treated with ethidium bromide for 48 h, rinsed twice with PBS and ethidium bromide-free medium added. All reagents were from Sigma and plasticware from Corning-Costar.

RNA isolation and northern blot procedure
RNA was prepared by TRIZOL (Invitrogen) extraction following manufacturer's recommendations. Northerns were performed as described (24). Briefly, aliquots (5 µg) of RNA were electrophoresed through 1.2% agarose under denaturing conditions and transferred to Genescreen membrane (NEN duPont) following the manufacturer's recommendations. Probes were generated from PCR products. Blots were probed with random hexamer labelled DNA fragments corresponding to internal regions of MTATP6 (nt 8563–8989), MTCO3 (nt 9440–9841) and MTND1 (nt 3384–4250) (7).

MPAT assay
The assay is based on the method described by Couttet (9). For the initial RNA ligation step, 2.5 µg RNA were annealed to 20 pmol of our universal linker DNA oligonucleotide 5'-phospho-ATG TGA GAT CAT GCA CAG TCA TA-3'-NH₂. After chilling on ice, PEG 1000 (final 12.5% v : v) was added and ligation performed in the recommended buffer by 20 Units T4 RNA Ligase (New England Biolabs, 37°C for 3 h.) The reaction was subjected to phenol/chloroform extraction and sodium acetate/ethanol precipitation. Half of the reaction product was reverse transcribed using the Superscript enzyme (GIBCO BRL) primed with 10 pmol of 5'-GAC TGT GCA TGA TCT CAC-3' (ANTI-LIGN). An aliquot (25%) of this cDNA was used to programme a standard PCR reaction (0.2 mM dNTPs, 1.5 mM MgCl₂, 3.5 Units Taq polymerase), with 1 µM ANTI-LIGN in combination with 1 µM of an upstream primer specific to the gene of interest: MTATP6 (nt 8863–8880) 5'-GTG ATT ATA GGC TTT CGC-3', MTCO3 (nt 9856–9875) 5'-TCT GCT TCA TCC GCC AA CTA-3'. Primers and free nucleotides were removed after 35 cycles using a QIAquick PCR purification column (Qiagen).

Forward primers were designed, internal to the PCR products generated above, so that in combination with ANTI-LIGN a product of exactly 50 bp would be formed assuming correct cleavage and without polyadenylation of the transcript under investigation. These primers, 5'-AGT AAG CCT CTA CCT GCA CG-3' for RNA14 (nt 9177–9196) and 5'-TGT ATG TCT CCA TCT ATT GAT G-3' for RNA15 (nt 9961–9982) were 5' end-labelled by T4 polynucleotide kinase with [ ³²P] dATP (3000 Ci/mmol, Amersham UK) in 50 mM Tris–HCl pH 7.5, 10 mM MgCl₂ and purified prior to use. A five cycle PCR, was performed as above, programmed with the PCR product and 30 nM of both ANTI-LIGN and radiolabelled primer. An aliquot of the product was separated on a 10% polyacrylamide (19 : 1) 8.2 M urea denaturing gel. Products were visualized by PhosphorImage and quantified where necessary using ImageQuant software. Size ranges of the dominant populations were determined by comparison to a 75 nt oligonucleotide, end-labelled as described above. This oligonucleotide migrates to a position corresponding to a poly(A) addition of 25 nt. Using the ladder formed by products in the PCR lanes, it was possible to determine exact product sizes.

Sequencing of polyadenylated RNA14
Reverse transcription reactions were programmed with 5 µg of RNA from either a control or the patient cell line and 1 µM of a 5' tagged oligo(dT) primer 5'-CTA CCA ACT CGA GAG ATC T₃₀-3'. Products were subjected to standard PCR with forward 5'-CAT CAG CCT ACT CAT TCA ACC-3' (MTATP6 nt 8964–8984) and reverse primers 5'- CTA CCA ACT CGA GAG ATC-3' (tag of reverse transcription primer). PCR products were ligated into pGEM T-easy (Promega), and transfected into bacterial strain DH5. DNA was PCR amplified directly from colonies lysed with Triton X-100, using primers designed to regions spanning the plasmid cloning site. These products were purified using QIAquick gel purification column (Qiagen) prior to DNA sequencing using BigDye terminator cycle sequencing chemistry (PE Biosystems) on an ABI 377 automated DNA sequencer.

Sequencing of RNA15 5'ends
cDNA was generated from RNA extracts using the Superscript kit (GIBCO BRL) and a 12–18mer oligo dT primer. The RNA strand was digested with RNase H to leave the single-stranded DNA strand. The procedures described for the MPAT assay were used to tag the 3' end (corresponding to the 5' end of the digested mRNA) and produce double-stranded DNA. MTCO3 was specifically amplified by PCR with primers ANTI-LIGN and 5'-AGG CCT AGT ATG AGG AG-3' (nt 9346–9330). Products were ligated into pGEM T-easy and, following transfection, plasmid DNA was sequenced as described above.

Sequencing of µRNA14_(-1–11) species
The MPAT assay was carried out for RNA14, but using an unlabelled primer in the final PCR. Products were separated on a 4% NuSieve GTG (FMC Bioproducts) agarose gel. Products migrating to the appropriate size were excised and ligated in-gel into pGEM T-easy. Sequences were obtained from the resultant plasmid clones as described above.

	ACKNOWLEDGEMENTS

 
ZMACL and RNL thank the Wellcome Trust and the European Commission (QLG1-CT-2001-00966) for supporting this research and Geoffrey Taylor for expert assistance with the DNA sequencing.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +44 1912228028; Fax: +44 1912228553; Email: r.n.lightowlers{at}ncl.ac.uk

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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