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The regulation of PTC containing transcripts of the human NDUFS4 gene of complex I of respiratory chain and the impact of pathological mutations
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Biochimie 90 (2008) 1452e1460 www.elsevier.com/locate/biochi
Research paper
The regulation of PTC containing transcripts of the human NDUFS4 gene of complex I of respiratory chain and the impact of pathological mutations Damiano Panelli b, Vittoria Petruzzella a,b, Rita Vitale a, Domenico De Rasmo a, Arnold Munnich c, Agne`s Ro¨tig c, Sergio Papa a,b,* a
Department of Medical Biochemistry, Biology and Physics, University of Bari, Piazza G. Cesare, Bari 70124, Italy b Institute of Biomembranes and Bioenergetics, Italian Research Council, Bari, Piazza G. Cesare 70124, Italy c De´partement de Ge´ne´tique, Maternite´ and INSERM U393, Hoˆpital Necker-Enfants Malades, 149 Rue de Se`vres, 75015 Paris, France Received 14 January 2008; accepted 25 April 2008 Available online 27 May 2008
Abstract The regulation of alternative transcripts of the NDUFS4 gene of complex I of the respiratory chain has been studied in human cell lines. One of the alternative transcripts (SV1) is subjected to the NMD degradation pathway which involves the hUPF1 and hUPF2 factors. Another transcript (SV3) appears to be controlled in the nuclear fraction and to be enhanced when hUPF1 is depleted, but unaffected by translation inhibitors or when hUPF2 expression is down-regulated. A pathological homozygous nonsense mutation in exon 1, found in a patient affected by mitochondrial disorder, inactivated in the patient’s fibroblasts NMD degradation of SV1 and enhanced the nuclear production of SV3. In another patient with a homozygous splice acceptor site mutation in intron 1, SV3, which was the only transcript of NDUFS4 gene to be produced, accumulated in fibroblasts. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Alternative splicing; Nonsense altered splicing; Nonsense mediated decay; Premature translationetermination codon; Respiratory complex I
1. Introduction Alternative splicing is a fundamental mechanism which increases the coding capacity of a single gene producing proteomic complexity [1]. Mammalian pre-mRNA splicing machinery selects correct pairs of splicing sites among potential but
Abbreviations: PTC, premature termination codon; UPF1, up-frame 1; UPF2, up-frame 2; NMD, nonsense mediated mRNA decay; EJs, exone exon junctions; EJCs, exon junction complexes; NAS, nonsense-associated altered splicing; NDUFS4, Homo sapiens NADH dehydrogenase (ubiquinone) FeeS protein 4; PUR, puromycin; CHX, cycloheximide; siRNA, small interfering RNA; ESE, splicing enhancer element. * Corresponding author. Department of Medical Biochemistry, Biology and Physics, University of Bari, Piazza G. Cesare, Bari 70124, Italy. Tel.: þ390805448541; fax: þ390805448538. E-mail address: [email protected] (S. Papa). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.04.017
inappropriate splice sites [2,3]. Pathological mutations or inefficient intron removal can alter this selectivity generating abnormal alternative transcripts. Transcripts containing premature termination codon (PTC) are deleterious for eukaryotic cells, as they can produce truncated potentially toxic proteins [4e6]. In mammalian cells mRNA quality control is assured by nonsense mediated mRNA decay (NMD). A recognized NMD mechanism is the hUPF1ehUPF2 dependent pathway, which requires for PTC recognition a pioneer translation round of the newly made RNA [4e8]. This process is therefore lessened by conditions that prevent translation termination at nonsense codon [9e 11]. In the pre-mRNA splicing process a set of proteins are deposited at exoneexon junctions (EJs) forming the exon junction complexes (EJCs) [12,13]. In the NMD pathway EJCs are removed from the ribosome during a pioneer round of translation. If a ribosome reaches a PTC it is not able to
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B). The PTC containing isoforms could be bonafide candidates for NMD having a stop codon more than 55 nucleotides upstream of the 30 -most exoneexon junction [30]. In the patient harbouring the 44G / A mutation, NMD inhibition by puromycin, which in control cells resulted in a significant cellular level of SV1 and SV2 transcripts, had no effect on these transcripts, already present at a significant level [29]. This suggested that the 44G / A mutation stabilized the SV1 and SV2 alternative transcripts, generated by the wildtype gene but degraded by NMD. In the normal fibroblasts the cellular level of the third transcript, SV3, despite being up-regulated by the 44G / A nonsense mutation, like SV1 and SV2, was not affected by puromycin treatment [29]. In a different patient with a homozygous splice acceptor site mutation in intron 1 (IVS1nt-1,G / A), accumulation of a transcript skipping exon 2 has been reported [28]. In this paper a study on the regulation of the SV1 and SV3 alternative transcripts, generated by the NDUFS4 gene in HeLa cells as well as in normal and patients’ fibroblasts, is presented. The results show that SV1 and SV3 are controlled by two different mechanisms of RNA surveillance. SV1 is degraded by the hUPF1 and hUPF2 dependent NMD, SV3 is down-regulated in the nuclear fraction through a hUPF1 dependent, hUPF2 independent mechanism. Both mechanisms are impaired in the two different pathological nonsense mutations of the gene.
release EJCs from mRNA. In this case the transcript is targeted to be destroyed by NMD through the interaction of phosphorylated UPF1 RNA helicase with EJC-bound UPF2 protein [14]. Results have been presented showing that in mammalian cells, in addition to the hUPF1ehUPF2 dependent NMD pathway, also a hUPF1 dependent but hUPF2 independent pathway can be utilized [15]. Less characterized nuclear RNA surveillance mechanisms have also been reported [16e21]. The nuclear NDUFS4 gene encodes for one of the 45 subunits of mitochondrial NADH: ubiquinone oxidoreductase, complex I [22]. The five exons of the human NDUFS4 gene result in a canonical mRNA coding a 175 residue protein (Fig. 1, Panels AeB). The NDUFS4 protein is an essential component for the overall architecture of the complex [23] and is a hotspot for mutations in complex I deficiency [24e 28]. In a patient affected by Leigh Syndrome, an autosomical recessive form of fatal infantile neurological disorder, a 44G / A nonsense mutation in the NDUFS4 gene, which introduces a PTC in the first exon, in proximity to the canonical ATG start codon, has been identified [26]. This PTC, rather than eliciting nonsense mediated decay of the canonical mRNA, up-regulated three PTC containing alternative isoforms. The splice variants 1 and 2 (SV1 and SV2) result from the insertion between exon 2 and exon 3 of a cryptoexon which uses two alternative acceptor sites. The splice variant 3 (SV3) derives from exon 2 skipping [29] (Fig. 1, Panel
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Fig. 1. Organization of the human NDUFS4 locus and its alternative splicing pathways (Panel A). Boxes represent exons. Schematic representation of the NDUFS4 alternative transcripts with numbers indicating the premature termination codon (PTC) distance in codons (cds) with respect to the AUG start codon and in nucleotides with respect to the 30 most exoneexon junction, respectively (Panel B). The positions of the primers used to amplify alternative transcripts by real-time PCR are also indicated as black bars. The reverse primer M18K2B designed in exon 3 was the same in all amplifications. For the canonical transcript the forward primer F-Ex2-Ex3Real spanned the specific exon 2/exon 3 boundary; for SV1 the forward primer F840-Real was designed in the first 83 nucleotides of the cryptoexon specific for this isoform; for SV3 the forward primer spanned the specific exon 1/exon 3 boundary.
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2. Materials and methods
2.6. Reverse transcription-PCR (RT-PCR)
2.1. Cell culture and RNA isolation
RNA was treated with DNAase RNAase free (Roche) and then (1 mg) reverse transcribed with 0.5 mg of oligo dT18 and 200 U of M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega). RT-PCRs of Fig. 3, Panel B contained cDNA synthesized from 0.2 mg of total RNA, 25 pmol each of the specific plasmid primers T7 (50 -TTAATACGACTCACTATAGGG-30 ) and BGH20N (50 -CTAGAAGGCACAGTCGA GGC-30 ), and 2.0 units of TaqDNA polymerase (Eppendorf). The PCR conditions were 30 sec at 94 C, 30 sec at 60 C, 1 min at 72 C for 30 cycles. The amplified products were analysed by electrophoresis in 1.5% agarose gel. The bands were extracted from gel, purified with a purification kit (Invitrogen) and confirmed by sequencing. RT-PCRs of Fig. 4, Panel A contained cDNA synthesized from 0.1 mg of total RNA, 12.5 pmol each of the specific primers and 1.0 units of TaqDNA polymerase (Eppendorf). The PCR conditions were 30 sec at 94 C, 30 sec at 59 C, 30 sec at 72 C for 30 cycles. The primers’ sequence used in each amplification are indicated in Table S1 (see Supplementary Data).
Primary fibroblast lines from skin biopsies of control subjects and patients were grown as described in Ref. [23]. HeLa cells were grown at 37 C under 5% CO2 in DMEM supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. RNA was isolated using the Trizol reagent (Roche); nuclear and cytoplasmic RNA fractions were obtained using PARIS kit (Ambion) according to the manufacturer’s protocol. Before RNA extraction, nuclear contamination by cytosolic fraction was estimated by measurement of LDH activity [31]. 2.2. Inhibition of protein synthesis HeLa cells were grown to 80% confluence and puromycin (PUR) (100 mg/ml) or cycloheximide (CHX) (50 mg/ml) was added to the culture medium. The medium was removed after 6 h and cells washed twice with PBS solution before RNA extraction.
2.7. Half-life experiments 2.3. Plasmids construction A minigene generating the SV1 transcript (minigene i) was obtained amplifying from a human control genome the NDUFS4 exons and sequences flanking using restriction sitetagged primers and cloning them sequentially in the pCDNA3.1(þ) vector. The minigenes generating canonical and SV3 transcripts (minigenes ii and iii) were made from the minigene i by EcoN I/BstX I double digestion to eliminate the crypto-exon and Not I single digestion to eliminate the exons 2 and crypto, respectively. Plasmid constructs were confirmed by DNA sequencing. 2.4. Cell transfection HeLa cells were seeded in 6-well plates to 80e90% optical confluence. Minigenes constructs were transiently transfected using metafectene according to the manufacturer’s recomendations (Biontex). After 24 h the medium was replaced with new medium, the cells grown for 24 h and RNA was extracted 48 h post-transfection. Treatment with cycloheximide (50 mg/ ml) was performed for 6 h, 42 h after transfection. RNA was extracted at the end of the cycloheximide treatment. 2.5. siRNA experiments HeLA cells were seeded in 12-well plates and transfected with 100 nM of hUPF1 or hUPF2 siRNAs using 4 ml of metafectene (Biontex) according to the manufacturer’s recommendations. Annealed siRNA duplexes were purchased from Ambion (for hUPF1 ID#: 142478; for hUPF2 ID#: 217153); mRNAs targets for hUPF1 and hUPF2 specific knockdown were: 50 -CGGACGTGGAAATACTTCT-30 and 50 -GCCGAC CAGAGGAAAACTT-30 , respectively.
HeLa cells after 24 h of hUPF1 silencing (see siRNA experiments) were treated with 100 mg/ml of 5,6-dichloro-1-b-Dribofuranosylbenzimidazole, RNA was collected after 3, 6, 9 h and the levels of SV1 and SV3 transcripts were monitored by real-time PCR. 2.8. Real-time PCR RNA was treated with DNAase RNAase free (Roche) and then 1 mg of RNA was reverse transcribed with 0.5 mg of oligo dT18 or 0.5 mg of random examers and 200 U of M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega); for minigenes analysis to distinguish between endogenous and exogenous NDUFS4 transcripts, the cDNAs were obtained by the BGH20N and NeoR (50 -AATATCACGGGTAGCCAACG30 ) primers specific for the constructs NDUFS4 splicing isoforms and neomycin transcript, respectively. Quantitative real-time PCR experiments were performed by IQ Sybr Green super mix (Biorad), and 40 ng cDNA were used to measure GAPDH, b-actin and neomycin mRNAs and 400 pg of cDNA to measure 18S rRNA, respectively. The NDUFS4 splice isoforms and the hUPF1 and hUPF2 mRNAs were measured using 40 ng of cDNA. In each amplification, 200 nM of specific primers (Table S1, see Supplementary Data) were used. Relative RNA levels were calculated from CT values according to the DCT method (Biorad) and relative NDUFS4 splice isoforms levels were normalized with the appropriate housekeeping gene. The PCR conditions were 20 sec at 94 C, 30 sec at 59 C, 45 sec at 72 C for 45 cycles, followed by a melt curve cycle. The primers used and the specific melting-temperature for each transcripts are listed in Table S1 (see Supplementary Data). The exponential curves of real-time PCR reaction of the NDUFS4 alternative transcripts are presented in
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Panel A of Fig. S1 (see Supplementary Data). Panel B of Fig. S1 presents the melting-temperatures of the NDUFS4 alternative transcripts.
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2.9. Data analysis Statistical significance was assessed by Student’s t test using a spreadsheet program.
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Fig. 2. Effect of puromycin and cycloheximide on the relative expression of canonical, SV1 and SV3 NDUFS4 alternative transcripts in HeLa cells. The expression level of the transcripts was analysed by real-time PCR. Normalization was made with respect to b-actin. In all the experiments the average values and standard deviations from five real-time PCR analyses are shown. *P < 0.001. For other details see Section 2.
Real-time analysis of the NDUFS4 transcripts shows that in HeLa cells, like in normal human fibroblasts [29], inhibition of cytoplasmic translation by puromycin (PUR) increased the
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Fig. 3. Schematic representation of the NDUFS4 minigenes in the pCDNA3.1(þ) Vector (Panel A). RT-PCR analysis of the production of exogenous transcripts, SV1, CAN and SV3, 48 h after HeLa cells transfection with the NDUFS4 minigenes (Panel B). Effect of cycloheximide on the expression level of the exogenous SV1, canonical and SV3 transcripts in transiently transfected HeLa cells (Panel C). The level of exogenous NDUFS4 isoforms in transfected cells, normalized with respect to the plasmid neomycin resistance transcript, was measured by real-time PCR. In untreated transfected cells the level of exogenous NDUFS4 isoforms was set as 1. Average values and standard deviations in three real-time PCR analyses are shown. *P < 0.005. For other details see Section 2.
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Fig. 4. Transient silencing of the hUPF1 and hUPF2 genes in HeLa cells. RT-PCR analysis of hUPF1 and hUPF2 mRNAs in control HeLa cells (1) and HeLa cells treated with siRNA (2) against hUPF1 and hUPF2 genes, respectively (Panel A). PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. Loading of the same cell amounts was assessed by analyzing b-actin mRNA levels. The same data were obtained by real-time PCR analysis. Relative hUPF1 and hUPF2 mRNA levels were determined and normalized to b-actin mRNA levels. The level of hUPF1 and hUPF2 mRNAs in untreated cells was set to 1. In all experiments the average values and standard deviations in three real-time PCR analyses are shown. Only samples with a down-regulation >60% were selected and used for subsequent investigations. *P < 0.001. Relative expression of SV1, canonical and SV3 NDUFS4 transcripts in hUPF1 and hUPF2 depleted HeLa cells (Panel B). The level of the NDUFS4 transcripts in the untreated samples as well as treated samples was calculated with respect to the b-actin. In all experiments the average values and standard deviations in five real-time PCR analyses are shown. *P < 0.001; **P < 0.005. Effect of hUPF1 silencing on the decay rate of SV1 and SV3 isoforms in HeLa cells (Panel C). Seventy-two hours after treatment with siRNA against hUPF1 gene, transcription in control and treated cells was inhibited with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole. The level of SV1 and SV3 transcripts were determined by real-time PCR in total RNA extracts collected at 0, 3, 6, 9 h after the addition of the inhibitor. The level of each transcript at time zero was arbitrarily set to 100 and the other values were expressed as percentage of this value using GAPDH to normalize real-time PCR data. The values, representative of three different experiments with similar results, are represented as semi-log plots.
level of the SV1 alternative transcript, but had no effect on SV3 and canonical transcripts. A similar pattern was obtained with cycloheximide (CHX) (Fig. 2). To exclude the possibility that the different response to puromycin and cycloheximide of SV1 and SV3 isoforms was due to their different levels of expression, three NDUFS4 minigenes were generated (Fig. 3, Panel A). Forty-eight hours
following HeLa cells transfection with constructs their splice products were analysed by RT-PCR using two plasmid primers encompassing all possible splice isoforms. Agarose gel electrophoresis of the RT-PCR products and their sequencing showed that the major transcription products of minigenes i, ii, iii, corresponded to SV1, canonical and SV3 transcripts, respectively (Fig. 3, Panel B).
D. Panelli et al. / Biochimie 90 (2008) 1452e1460
Forty-eight hours after transfection of HeLa cells with siRNAs against hUPF1 and hUPF2 genes the level of hUPF1 and hUPF2 transcripts were decreased by approximately 60e70% (Fig. 4, Panel A) by their respective siRNAs. This resulted in abrogation of NMD as shown by the accumulation of several PTC containing alternative spliced mRNAs, bonafide NMD substrates, generated by different complex I genes (paragraph A.2.1 of Supplementary Data). The impact of hUPF1 and hUPF2 silencing on the level of SV1 and SV3 NDUFS4 transcripts was then analysed by real-time PCR. The results show that hUPF1 silencing induced up-regulation of SV1 and SV3 transcripts. The level of the canonical transcript, used as control, was unaffected by hUPF1 (Fig. 4, Panel B). Silencing of hUPF2 resulted in significant up-regulation of the SV1 isoform only (Fig. 4, Panel B). The real-time analysis of the levels of SV1 and SV3, after transcription inhibition, shows that hUPF1 silencing stabilized the level of SV1 but had no effect on SV3 (Fig. 4, Panel C).
A Relative Expression
3.2. Effects of hUPF1 and hUPF2 silencing on the level and stability of NDUFS4 transcripts
transcripts (Fig. 6, Panel B; see also Ref. [29]). In a patient with a homozygous splice acceptor site mutation in intron 1 (IVSnt-1, G / A) of the NDUFS4 gene (Fig. 6, Panel A) only an mRNA transcript, in which exon 2 was skipped, was detected [28]. Amplification of the abnormal transcript and nucleotide sequencing showed that it corresponded exactly to the
3.4. NDUFS4 exonic or intronic pathological mutations abrogate down-regulation of the SV1 and SV3 transcripts The 44G / A homozygous pathological mutation in exon 1 which introduced a PTC at codon 15 (Fig. 6, Panel A) abrogated the NMD dependent and NMD independent control of SV1 and SV3 transcripts, respectively, up-regulating both
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Real-time PCR analysis of endogenous SV1, SV3 and canonical NDUFS4 transcripts was performed on nuclear and cytoplasmic RNA fractions extracted from HeLa cells. Measurement of lactate dehydrogenase activity showed that contamination of the nuclear fraction by cytoplasm was less than 5%. In addition denaturing agarose gel-electrophoretic analysis showed that high molecular weight rRNA precursors were only detectable in the nuclear fraction. The real-time PCR results (Fig. 5, Panel A) show that whilst the SV1 level in the nuclear fraction was significantly higher with respect to that in the cytoplasmic fraction, the SV3 level was similar in both fractions. Down-regulation by RNA interference of hUPF1 increased the SV1 and SV3 cytoplasmic levels, but in the nuclear fraction only the level of the SV3 transcript was increased. In both fractions the canonical transcript was, as expected, unaffected by hUPF1 silencing (Fig. 5, Panel BeC).
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Forty-two hours following minigene transfection, protein synthesis was inhibited with CHX treatment. Total RNA samples were prepared 6 h after CHX addition. The levels of the exogenous canonical and SV3 transcripts were unaffected by CHX treatment. The level of the exogenous SV1 isoform was, on the other hand, up-regulated by the drug (Fig. 3, Panel C).
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Fig. 5. Relative expression of SV1, canonical and SV3 NDUFS4 transcripts in the nuclear fraction with respect to the cytoplasmic fraction (Panel A). The expression level of the transcripts was analysed by real-time PCR. Normalization was performed with respect to the ribosomal 18S transcript. In the cytoplasmic fraction the level of the NDUFS4 transcripts was set as 1. Relative amount of the SV1, canonical and SV3 transcripts in the nuclear fraction after silencing of the hUPF1 gene (Panel B). Relative amount of the SV1, canonical and SV3 transcripts in the cytoplasmic fraction after silencing of the hUPF1 gene (Panel C). Normalization was made with respect to the b-actin transcript and the level of the NDUFS4 transcripts in the untreated samples was set as 1. In all the experiments the average values and standard deviations in five real-time PCR analyses are shown. *P < 0.001.
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Fig. 6. Impact of exonic and intronic mutations on the levels of NDUFS4 transcripts in patients fibroblasts. Schematic representation of the exonic 44G / A and intronic IVS1nt-1,G / A NDUFS4 mutations (Panel A). Real-time PCR analysis of NDUFS4 SV1, canonical and SV3 transcript levels in control and patients fibroblasts (Panel B). The level of the NDUFS4 transcripts in the normal and patients fibroblasts was normalized with respect to the GAPDH transcript. In all experiments the average values and standard deviations in three real-time PCR analyses are shown.
PTC containing SV3 isoform described in this work. The amount of the SV3 mRNA generated from the NDUFS4 (IVS1nt-1,G / A) mutant in the patients’ fibroblasts was 50 times higher than in normal fibroblast cells (Fig. 6, Panel B). 4. Discussion The present results reveal features of surveillance mechanisms regulating the levels of two alternative transcripts (SV1 and SV3) of the NDUFS4 gene of complex I, found to accumulate in patients with pathological mutations in this gene [28,29]. Whilst one of these transcripts, SV1, is found to be degraded by NMD which is translation dependent and involves both hUPF1 and hUPF2 factors, the other, SV3, appeared to be down-regulated in the nucleus by a translation and hUPF2 independent pathway. The pathological 44G / A mutation in exon 1 abrogated down-regulation of both SV1 and SV3 transcripts. The pathological IVSnt-1, G / A intron mutation resulted in high level of SV3 as single transcript of the gene, which escaped NMD. Real-time PCR analysis of the canonical mRNA, SV1 and SV3 NDUFS4 transcripts in HeLa cells showed that, like in fibroblasts, inhibition of protein synthesis by puromycin or cycloheximide, stabilized SV1 but had no effect on SV3. The lack of SV3 stabilization by translation inhibitors was not due to the low expression level of this transcript. The same differential effect of translation inhibition on SV1 and SV3 was also observed when these transcripts were over-expressed in HeLa cells transfected with minigene constructs. The insensitivity of SV3 to translation inhibition indicated that this transcript, although containing a PTC in a position which would candidate it for NMD, could be down-regulated by a different surveillance process.
There is evidence that some gene transcripts are scanned in the nucleus for ORF integrity [19e21,32e34]. Quantitative analysis of the NDUFS4 transcripts in subcellular fractions showed relative abundance of SV1 in the nuclear fraction with respect to the cytosol at difference with SV3 and canonical transcripts whose levels were practically the same in the two fractions. hUPF1 inactivation by siRNA induced significant up-regulation of SV1 and SV3 in total cell extracts, but only of SV3 in the nuclear fraction. hUPF2 silencing upregulated, on the other hand, SV1 but not SV3 in total HeLa cell preparation. The SV1 transcript exhibited prolonged half-life in hUPF1-depleted HeLa cells, whereas the SV3 transcript stability was unaffected. Thus the SV1 transcript undergoes the hUPF1ehUPF2 dependent NMD mechanism following its export from the nucleus into the cytoplasm. The SV3 transcript is, on the other hand, depressed in the nucleus through a translation independent but hUPF1 dependent process. Also this process is abrogated in the patient with the 44G / A mutation. The possibility that SV3 is an alternatively spliced transcript, produced at low levels in normal condition, but overproduced in the 44G / A pathological mutant since of altered exonic splicing enhancer element (ESE) [17,18], should also be considered. Use of three algorithms (RescueESE, ESEfinder and PESX) rules out, however, the possibility that the 44G / A mutation could have an effect on consensus ESE. In addition previous work, using an in vitro splicing system in pSPL3 vector, showed that the 44G / A mutation did not activate per se aberrant splicing [29]. A likely explanation for the escape of SV3 to NMD, although it is apparently candidate for this process, is that the stop codon is too close to the start codon (37 codons) to prevent downstream translation reinitiation and hence the ribosome continues translation in a reading frame that is not prematurely
D. Panelli et al. / Biochimie 90 (2008) 1452e1460
terminated. In silico analysis reveals the presence in SV3 of two translation competent AUGs (Fig. S3, Panel A of Supplementary Data). In vitro translation experiments showed that the canonical cDNA construct produced, in addition to the expected band of z20 kDa, also a small amount of a lower mW (z10 kDa) protein. The SV3 cDNA produced, on the other hand, only, and in high amount, the smaller protein recognized, only, by the anti-C NDUFS4 antibody (Fig. S3, Panel B of Supplementary Data). Thus the PTC introduced in the SV3 transcript could not activate NMD because AUGs, downstream the PTC, were still able to initiation translation. The results presented on the insensibility of the SV3 level to translation inhibition (Figs. 2 and 3, Panel C), the halflife of endogenous SV3 transcript in HeLa cells (Fig. 4, Panel C), the relative levels of the SV3 in the nuclear and cytoplasmic fractions (Fig. 5), the translation competence of SV3 (Fig. S3, Panel B of Supplementary Data), the expression level of the SV3 minigenes constructs (Fig. S4 of Supplementary Data), all provide converging evidence showing that it is the nuclear production and not the degradation of SV3 to be influenced by PTCs inserted in exon 1 or 3. Acknowledgments Supported by National Project on Bioenergetics, ‘‘Molecular Mechanisms, Physiology and Pathology of Membrane Bioenergetics System’’, 2005-Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR), Italy; and the Research Foundation Cassa di Risparmio di Puglia, 2005.
Appendix A. Supplementary information Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biochi.2008.04. 017. References [1] S. Stamm, S. Ben-Ari, I. Rafalska, Y. Tang, Z. Zhang, D. Toiber, T.A. Thanaraj, H. Soreq, Function of alternative splicing, Gene 344 (2005) 1e20. [2] J.P. Staley, C. Guthrie, Mechanical devices of the spliceosome: motors, clocks, springs, and things, Cell 92 (1998) 315e326 Review. [3] R. Reed, L. Palandjian, Spliceosome assembly, in: A.R. Krainer (Ed.), Eukaryotic mRNA Processing, IRL Press, Oxford, 1997, pp. 103e129. [4] S. Li, M.F. Wilkinson, Nonsense surveillance in lymphocytes? Immunity 8 (1998) 135e141 Review. [5] P.A. Frischmeyer, H.C. Dietz, Nonsense-mediated mRNA decay in health and disease, Hum. Mol. Genet. 8 (1999) 1893e1900 Review. [6] L.E. Maquat, Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics, Nat. Rev. Mol. Cell Biol. 5 (2004) 89e99. [7] A.B. Maderazo, J.P. Belk, F. He, A. Jacobson, Nonsense-containing mRNAs that accumulate in the absence of a functional nonsense-mediated mRNA decay pathway are destabilized rapidly upon its restitution, Mol. Cell. Biol. 23 (2003) 842e851. [8] Y. Ishigaki, X. Li, G. Serin, L.E. Maquat, Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20, Cell 106 (2001) 607e617.
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[9] M.S. Carter, J. Doskow, P. Morris, S. Li, R.P. Nhim, S. Sandstedt, M.F. Wilkinson, A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro, J. Biol. Chem. 270 (1995) 28995e29003. [10] E.N. Noensie, H.C. Dietz, A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition, Nat. Biotechnol. 19 (2001) 434e439. [11] P. Belgrader, J. Cheng, L.E. Maquat, Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 482e486. [12] H. Le Hir, E. Izaurralde, L.E. Maquat, M.J. Moore, The spliceosome deposits multiple proteins 20e24 nucleotides upstream of mRNA exonexon junctions, EMBO J. 19 (2000) 6860e6869. [13] H. Le Hir, M.J. Moore, L.E. Maquat, Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions, Genes Dev. 14 (2000) 1098e1108. [14] X. Sun, H.A. Perlick, H.C. Dietz, L.E. Maquat, A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 10009e10014. [15] N.H. Gehring, J.B. Kunz, G. Neu-Yilik, S. Breit, M.H. Viegas, M.W. Hentze, A.E. Kulozik, Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements, Mol. Cell 20 (2005) 65e75. [16] M.W. Hentze, A.E. Kulozik, A perfect message: RNA surveillance and nonsense-mediated decay, Cell 96 (1999) 307e310. [17] M. Caputi Jr., R.J. Kendzior, K.L. Beemon, A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer, Genes Dev. 16 (2002) 1754e1759. [18] L. Cartegni, S.L. Chew, A.R. Krainer, Listening to silence and understanding nonsense: exonic mutations that affect splicing, Nat. Rev. Genet. 3 (2002) 285e298. [19] S. Aoufouchi, J. Yelamos, C. Milstein, Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis, Cell 85 (1996) 415e422. [20] J. Wang, Y.F. Chang, J.I. Hamilton, M.F. Wilkinson, Nonsense-associated altered splicing: a frame-dependent response distinct from nonsensemediated decay, Mol. Cell 10 (2002) 951e957. [21] C. Wachtel, B. Li, J. Sperling, R. Sperling, Stop codon-mediated suppression of splicing is a novel nuclear scanning mechanism not affected by elements of protein synthesis and NMD, RNA 10 (2004) 1740e1750. [22] J. Carroll, I.M. Fearnley, J.M. Skehel, R.J. Shannon, J. Hirst, J.E. Walker, Bovine complex I is a complex of 45 different subunits, J. Biol. Chem. 281 (2006) 32724e32727. [23] S. Scacco, V. Petruzzella, S. Budde, R. Vergari, R. Tamborra, D. Panelli, L.P. van den Heuvel, J.A. Smeitink, S. Papa, Pathological mutations of the human NDUFS4 gene of the 18 kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex, J. Biol. Chem. 278 (2003) 44161e44167. [24] L. van den Heuvel, W. Ruitenbeek, R. Smeets, Z. Gelman-Kohan, O. Elpeleg, J. Loeffen, F. Trijbels, E. Mariman, D. de Bruijn, J. Smeitink, Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18kD (AQDQ) subunit, Am. J. Hum. Genet. 62 (1998) 262e268. [25] S.M. Budde, L.P. van den Heuvel, A.J. Janssen, R.J. Smeets, C.A. Buskens, L. DeMeirleir, R. Van Coster, M. Baethmann, T. Voit, J.M. Trijbels, J.A. Smeitink, Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene, Biochem. Biophys. Res. Commun. 18 (2000) 63e68. [26] V. Petruzzella, R. Vergari, I. Puzziferri, D. Boffoli, E. Lamantea, M. Zeviani, S. Papa, A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome, Hum. Mol. Genet. 10 (2001) 529e535. [27] V. Petruzzella, S. Papa, Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene, Gene 286 (2002) 149e154 Review.
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[28] P. Benit, J. Steffann, S. Lebon, D. Chretien, N. Kadhom, P. de Lonlay, A. Goldenberg, Y. Dumez, M. Dommergues, P. Rustin, A. Munnich, A. Rotig, Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt-1) in the NDUFS4 gene in Leigh syndrome, Hum. Genet. 112 (2003) 563e566. [29] V. Petruzzella, D. Panelli, A. Torraco, A. Stella, S. Papa, Mutations in the NDUFS4 gene of mitochondrial complex I alter stability of the splice variants, FEBS Lett. 579 (2005) 3770e3776. [30] E. Nagy, L.E. Maquat, A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance, Trends Biochem. Sci. 23 (1998) 198e199.
[31] H.U. Bergmeyer, E. Bernt, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press Inc, New York, 1963, p. 324. [32] H.C. Dietz Jr., R.J. Kendzior, Maintenance of an open reading frame as an additional level of scrutiny during splice site selection, Nat. Genet. 8 (1994) 183e188. [33] S. Li, D. Leonard, M.F. Wilkinson, T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism, J. Exp. Med. 185 (1997) 985e992. [34] Y.F. Chang, W.K. Chan, J.S. Imam, M.F. Wilkinson, Alternatively spliced T-cell receptor transcripts are up-regulated in response to disruption of either splicing elements or reading frame, J. Biol. Chem. 282 (2007) 29738e29747.
Biochimie 90 (2008) 1452e1460 www.elsevier.com/locate/biochi
Research paper
The regulation of PTC containing transcripts of the human NDUFS4 gene of complex I of respiratory chain and the impact of pathological mutations Damiano Panelli b, Vittoria Petruzzella a,b, Rita Vitale a, Domenico De Rasmo a, Arnold Munnich c, Agne`s Ro¨tig c, Sergio Papa a,b,* a
Department of Medical Biochemistry, Biology and Physics, University of Bari, Piazza G. Cesare, Bari 70124, Italy b Institute of Biomembranes and Bioenergetics, Italian Research Council, Bari, Piazza G. Cesare 70124, Italy c De´partement de Ge´ne´tique, Maternite´ and INSERM U393, Hoˆpital Necker-Enfants Malades, 149 Rue de Se`vres, 75015 Paris, France Received 14 January 2008; accepted 25 April 2008 Available online 27 May 2008
Abstract The regulation of alternative transcripts of the NDUFS4 gene of complex I of the respiratory chain has been studied in human cell lines. One of the alternative transcripts (SV1) is subjected to the NMD degradation pathway which involves the hUPF1 and hUPF2 factors. Another transcript (SV3) appears to be controlled in the nuclear fraction and to be enhanced when hUPF1 is depleted, but unaffected by translation inhibitors or when hUPF2 expression is down-regulated. A pathological homozygous nonsense mutation in exon 1, found in a patient affected by mitochondrial disorder, inactivated in the patient’s fibroblasts NMD degradation of SV1 and enhanced the nuclear production of SV3. In another patient with a homozygous splice acceptor site mutation in intron 1, SV3, which was the only transcript of NDUFS4 gene to be produced, accumulated in fibroblasts. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Alternative splicing; Nonsense altered splicing; Nonsense mediated decay; Premature translationetermination codon; Respiratory complex I
1. Introduction Alternative splicing is a fundamental mechanism which increases the coding capacity of a single gene producing proteomic complexity [1]. Mammalian pre-mRNA splicing machinery selects correct pairs of splicing sites among potential but
Abbreviations: PTC, premature termination codon; UPF1, up-frame 1; UPF2, up-frame 2; NMD, nonsense mediated mRNA decay; EJs, exone exon junctions; EJCs, exon junction complexes; NAS, nonsense-associated altered splicing; NDUFS4, Homo sapiens NADH dehydrogenase (ubiquinone) FeeS protein 4; PUR, puromycin; CHX, cycloheximide; siRNA, small interfering RNA; ESE, splicing enhancer element. * Corresponding author. Department of Medical Biochemistry, Biology and Physics, University of Bari, Piazza G. Cesare, Bari 70124, Italy. Tel.: þ390805448541; fax: þ390805448538. E-mail address: [email protected] (S. Papa). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.04.017
inappropriate splice sites [2,3]. Pathological mutations or inefficient intron removal can alter this selectivity generating abnormal alternative transcripts. Transcripts containing premature termination codon (PTC) are deleterious for eukaryotic cells, as they can produce truncated potentially toxic proteins [4e6]. In mammalian cells mRNA quality control is assured by nonsense mediated mRNA decay (NMD). A recognized NMD mechanism is the hUPF1ehUPF2 dependent pathway, which requires for PTC recognition a pioneer translation round of the newly made RNA [4e8]. This process is therefore lessened by conditions that prevent translation termination at nonsense codon [9e 11]. In the pre-mRNA splicing process a set of proteins are deposited at exoneexon junctions (EJs) forming the exon junction complexes (EJCs) [12,13]. In the NMD pathway EJCs are removed from the ribosome during a pioneer round of translation. If a ribosome reaches a PTC it is not able to
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B). The PTC containing isoforms could be bonafide candidates for NMD having a stop codon more than 55 nucleotides upstream of the 30 -most exoneexon junction [30]. In the patient harbouring the 44G / A mutation, NMD inhibition by puromycin, which in control cells resulted in a significant cellular level of SV1 and SV2 transcripts, had no effect on these transcripts, already present at a significant level [29]. This suggested that the 44G / A mutation stabilized the SV1 and SV2 alternative transcripts, generated by the wildtype gene but degraded by NMD. In the normal fibroblasts the cellular level of the third transcript, SV3, despite being up-regulated by the 44G / A nonsense mutation, like SV1 and SV2, was not affected by puromycin treatment [29]. In a different patient with a homozygous splice acceptor site mutation in intron 1 (IVS1nt-1,G / A), accumulation of a transcript skipping exon 2 has been reported [28]. In this paper a study on the regulation of the SV1 and SV3 alternative transcripts, generated by the NDUFS4 gene in HeLa cells as well as in normal and patients’ fibroblasts, is presented. The results show that SV1 and SV3 are controlled by two different mechanisms of RNA surveillance. SV1 is degraded by the hUPF1 and hUPF2 dependent NMD, SV3 is down-regulated in the nuclear fraction through a hUPF1 dependent, hUPF2 independent mechanism. Both mechanisms are impaired in the two different pathological nonsense mutations of the gene.
release EJCs from mRNA. In this case the transcript is targeted to be destroyed by NMD through the interaction of phosphorylated UPF1 RNA helicase with EJC-bound UPF2 protein [14]. Results have been presented showing that in mammalian cells, in addition to the hUPF1ehUPF2 dependent NMD pathway, also a hUPF1 dependent but hUPF2 independent pathway can be utilized [15]. Less characterized nuclear RNA surveillance mechanisms have also been reported [16e21]. The nuclear NDUFS4 gene encodes for one of the 45 subunits of mitochondrial NADH: ubiquinone oxidoreductase, complex I [22]. The five exons of the human NDUFS4 gene result in a canonical mRNA coding a 175 residue protein (Fig. 1, Panels AeB). The NDUFS4 protein is an essential component for the overall architecture of the complex [23] and is a hotspot for mutations in complex I deficiency [24e 28]. In a patient affected by Leigh Syndrome, an autosomical recessive form of fatal infantile neurological disorder, a 44G / A nonsense mutation in the NDUFS4 gene, which introduces a PTC in the first exon, in proximity to the canonical ATG start codon, has been identified [26]. This PTC, rather than eliciting nonsense mediated decay of the canonical mRNA, up-regulated three PTC containing alternative isoforms. The splice variants 1 and 2 (SV1 and SV2) result from the insertion between exon 2 and exon 3 of a cryptoexon which uses two alternative acceptor sites. The splice variant 3 (SV3) derives from exon 2 skipping [29] (Fig. 1, Panel
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Fig. 1. Organization of the human NDUFS4 locus and its alternative splicing pathways (Panel A). Boxes represent exons. Schematic representation of the NDUFS4 alternative transcripts with numbers indicating the premature termination codon (PTC) distance in codons (cds) with respect to the AUG start codon and in nucleotides with respect to the 30 most exoneexon junction, respectively (Panel B). The positions of the primers used to amplify alternative transcripts by real-time PCR are also indicated as black bars. The reverse primer M18K2B designed in exon 3 was the same in all amplifications. For the canonical transcript the forward primer F-Ex2-Ex3Real spanned the specific exon 2/exon 3 boundary; for SV1 the forward primer F840-Real was designed in the first 83 nucleotides of the cryptoexon specific for this isoform; for SV3 the forward primer spanned the specific exon 1/exon 3 boundary.
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2. Materials and methods
2.6. Reverse transcription-PCR (RT-PCR)
2.1. Cell culture and RNA isolation
RNA was treated with DNAase RNAase free (Roche) and then (1 mg) reverse transcribed with 0.5 mg of oligo dT18 and 200 U of M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega). RT-PCRs of Fig. 3, Panel B contained cDNA synthesized from 0.2 mg of total RNA, 25 pmol each of the specific plasmid primers T7 (50 -TTAATACGACTCACTATAGGG-30 ) and BGH20N (50 -CTAGAAGGCACAGTCGA GGC-30 ), and 2.0 units of TaqDNA polymerase (Eppendorf). The PCR conditions were 30 sec at 94 C, 30 sec at 60 C, 1 min at 72 C for 30 cycles. The amplified products were analysed by electrophoresis in 1.5% agarose gel. The bands were extracted from gel, purified with a purification kit (Invitrogen) and confirmed by sequencing. RT-PCRs of Fig. 4, Panel A contained cDNA synthesized from 0.1 mg of total RNA, 12.5 pmol each of the specific primers and 1.0 units of TaqDNA polymerase (Eppendorf). The PCR conditions were 30 sec at 94 C, 30 sec at 59 C, 30 sec at 72 C for 30 cycles. The primers’ sequence used in each amplification are indicated in Table S1 (see Supplementary Data).
Primary fibroblast lines from skin biopsies of control subjects and patients were grown as described in Ref. [23]. HeLa cells were grown at 37 C under 5% CO2 in DMEM supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. RNA was isolated using the Trizol reagent (Roche); nuclear and cytoplasmic RNA fractions were obtained using PARIS kit (Ambion) according to the manufacturer’s protocol. Before RNA extraction, nuclear contamination by cytosolic fraction was estimated by measurement of LDH activity [31]. 2.2. Inhibition of protein synthesis HeLa cells were grown to 80% confluence and puromycin (PUR) (100 mg/ml) or cycloheximide (CHX) (50 mg/ml) was added to the culture medium. The medium was removed after 6 h and cells washed twice with PBS solution before RNA extraction.
2.7. Half-life experiments 2.3. Plasmids construction A minigene generating the SV1 transcript (minigene i) was obtained amplifying from a human control genome the NDUFS4 exons and sequences flanking using restriction sitetagged primers and cloning them sequentially in the pCDNA3.1(þ) vector. The minigenes generating canonical and SV3 transcripts (minigenes ii and iii) were made from the minigene i by EcoN I/BstX I double digestion to eliminate the crypto-exon and Not I single digestion to eliminate the exons 2 and crypto, respectively. Plasmid constructs were confirmed by DNA sequencing. 2.4. Cell transfection HeLa cells were seeded in 6-well plates to 80e90% optical confluence. Minigenes constructs were transiently transfected using metafectene according to the manufacturer’s recomendations (Biontex). After 24 h the medium was replaced with new medium, the cells grown for 24 h and RNA was extracted 48 h post-transfection. Treatment with cycloheximide (50 mg/ ml) was performed for 6 h, 42 h after transfection. RNA was extracted at the end of the cycloheximide treatment. 2.5. siRNA experiments HeLA cells were seeded in 12-well plates and transfected with 100 nM of hUPF1 or hUPF2 siRNAs using 4 ml of metafectene (Biontex) according to the manufacturer’s recommendations. Annealed siRNA duplexes were purchased from Ambion (for hUPF1 ID#: 142478; for hUPF2 ID#: 217153); mRNAs targets for hUPF1 and hUPF2 specific knockdown were: 50 -CGGACGTGGAAATACTTCT-30 and 50 -GCCGAC CAGAGGAAAACTT-30 , respectively.
HeLa cells after 24 h of hUPF1 silencing (see siRNA experiments) were treated with 100 mg/ml of 5,6-dichloro-1-b-Dribofuranosylbenzimidazole, RNA was collected after 3, 6, 9 h and the levels of SV1 and SV3 transcripts were monitored by real-time PCR. 2.8. Real-time PCR RNA was treated with DNAase RNAase free (Roche) and then 1 mg of RNA was reverse transcribed with 0.5 mg of oligo dT18 or 0.5 mg of random examers and 200 U of M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega); for minigenes analysis to distinguish between endogenous and exogenous NDUFS4 transcripts, the cDNAs were obtained by the BGH20N and NeoR (50 -AATATCACGGGTAGCCAACG30 ) primers specific for the constructs NDUFS4 splicing isoforms and neomycin transcript, respectively. Quantitative real-time PCR experiments were performed by IQ Sybr Green super mix (Biorad), and 40 ng cDNA were used to measure GAPDH, b-actin and neomycin mRNAs and 400 pg of cDNA to measure 18S rRNA, respectively. The NDUFS4 splice isoforms and the hUPF1 and hUPF2 mRNAs were measured using 40 ng of cDNA. In each amplification, 200 nM of specific primers (Table S1, see Supplementary Data) were used. Relative RNA levels were calculated from CT values according to the DCT method (Biorad) and relative NDUFS4 splice isoforms levels were normalized with the appropriate housekeeping gene. The PCR conditions were 20 sec at 94 C, 30 sec at 59 C, 45 sec at 72 C for 45 cycles, followed by a melt curve cycle. The primers used and the specific melting-temperature for each transcripts are listed in Table S1 (see Supplementary Data). The exponential curves of real-time PCR reaction of the NDUFS4 alternative transcripts are presented in
D. Panelli et al. / Biochimie 90 (2008) 1452e1460
Panel A of Fig. S1 (see Supplementary Data). Panel B of Fig. S1 presents the melting-temperatures of the NDUFS4 alternative transcripts.
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Fig. 2. Effect of puromycin and cycloheximide on the relative expression of canonical, SV1 and SV3 NDUFS4 alternative transcripts in HeLa cells. The expression level of the transcripts was analysed by real-time PCR. Normalization was made with respect to b-actin. In all the experiments the average values and standard deviations from five real-time PCR analyses are shown. *P < 0.001. For other details see Section 2.
Real-time analysis of the NDUFS4 transcripts shows that in HeLa cells, like in normal human fibroblasts [29], inhibition of cytoplasmic translation by puromycin (PUR) increased the
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Fig. 3. Schematic representation of the NDUFS4 minigenes in the pCDNA3.1(þ) Vector (Panel A). RT-PCR analysis of the production of exogenous transcripts, SV1, CAN and SV3, 48 h after HeLa cells transfection with the NDUFS4 minigenes (Panel B). Effect of cycloheximide on the expression level of the exogenous SV1, canonical and SV3 transcripts in transiently transfected HeLa cells (Panel C). The level of exogenous NDUFS4 isoforms in transfected cells, normalized with respect to the plasmid neomycin resistance transcript, was measured by real-time PCR. In untreated transfected cells the level of exogenous NDUFS4 isoforms was set as 1. Average values and standard deviations in three real-time PCR analyses are shown. *P < 0.005. For other details see Section 2.
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Fig. 4. Transient silencing of the hUPF1 and hUPF2 genes in HeLa cells. RT-PCR analysis of hUPF1 and hUPF2 mRNAs in control HeLa cells (1) and HeLa cells treated with siRNA (2) against hUPF1 and hUPF2 genes, respectively (Panel A). PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. Loading of the same cell amounts was assessed by analyzing b-actin mRNA levels. The same data were obtained by real-time PCR analysis. Relative hUPF1 and hUPF2 mRNA levels were determined and normalized to b-actin mRNA levels. The level of hUPF1 and hUPF2 mRNAs in untreated cells was set to 1. In all experiments the average values and standard deviations in three real-time PCR analyses are shown. Only samples with a down-regulation >60% were selected and used for subsequent investigations. *P < 0.001. Relative expression of SV1, canonical and SV3 NDUFS4 transcripts in hUPF1 and hUPF2 depleted HeLa cells (Panel B). The level of the NDUFS4 transcripts in the untreated samples as well as treated samples was calculated with respect to the b-actin. In all experiments the average values and standard deviations in five real-time PCR analyses are shown. *P < 0.001; **P < 0.005. Effect of hUPF1 silencing on the decay rate of SV1 and SV3 isoforms in HeLa cells (Panel C). Seventy-two hours after treatment with siRNA against hUPF1 gene, transcription in control and treated cells was inhibited with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole. The level of SV1 and SV3 transcripts were determined by real-time PCR in total RNA extracts collected at 0, 3, 6, 9 h after the addition of the inhibitor. The level of each transcript at time zero was arbitrarily set to 100 and the other values were expressed as percentage of this value using GAPDH to normalize real-time PCR data. The values, representative of three different experiments with similar results, are represented as semi-log plots.
level of the SV1 alternative transcript, but had no effect on SV3 and canonical transcripts. A similar pattern was obtained with cycloheximide (CHX) (Fig. 2). To exclude the possibility that the different response to puromycin and cycloheximide of SV1 and SV3 isoforms was due to their different levels of expression, three NDUFS4 minigenes were generated (Fig. 3, Panel A). Forty-eight hours
following HeLa cells transfection with constructs their splice products were analysed by RT-PCR using two plasmid primers encompassing all possible splice isoforms. Agarose gel electrophoresis of the RT-PCR products and their sequencing showed that the major transcription products of minigenes i, ii, iii, corresponded to SV1, canonical and SV3 transcripts, respectively (Fig. 3, Panel B).
D. Panelli et al. / Biochimie 90 (2008) 1452e1460
Forty-eight hours after transfection of HeLa cells with siRNAs against hUPF1 and hUPF2 genes the level of hUPF1 and hUPF2 transcripts were decreased by approximately 60e70% (Fig. 4, Panel A) by their respective siRNAs. This resulted in abrogation of NMD as shown by the accumulation of several PTC containing alternative spliced mRNAs, bonafide NMD substrates, generated by different complex I genes (paragraph A.2.1 of Supplementary Data). The impact of hUPF1 and hUPF2 silencing on the level of SV1 and SV3 NDUFS4 transcripts was then analysed by real-time PCR. The results show that hUPF1 silencing induced up-regulation of SV1 and SV3 transcripts. The level of the canonical transcript, used as control, was unaffected by hUPF1 (Fig. 4, Panel B). Silencing of hUPF2 resulted in significant up-regulation of the SV1 isoform only (Fig. 4, Panel B). The real-time analysis of the levels of SV1 and SV3, after transcription inhibition, shows that hUPF1 silencing stabilized the level of SV1 but had no effect on SV3 (Fig. 4, Panel C).
A Relative Expression
3.2. Effects of hUPF1 and hUPF2 silencing on the level and stability of NDUFS4 transcripts
transcripts (Fig. 6, Panel B; see also Ref. [29]). In a patient with a homozygous splice acceptor site mutation in intron 1 (IVSnt-1, G / A) of the NDUFS4 gene (Fig. 6, Panel A) only an mRNA transcript, in which exon 2 was skipped, was detected [28]. Amplification of the abnormal transcript and nucleotide sequencing showed that it corresponded exactly to the
3.4. NDUFS4 exonic or intronic pathological mutations abrogate down-regulation of the SV1 and SV3 transcripts The 44G / A homozygous pathological mutation in exon 1 which introduced a PTC at codon 15 (Fig. 6, Panel A) abrogated the NMD dependent and NMD independent control of SV1 and SV3 transcripts, respectively, up-regulating both
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Real-time PCR analysis of endogenous SV1, SV3 and canonical NDUFS4 transcripts was performed on nuclear and cytoplasmic RNA fractions extracted from HeLa cells. Measurement of lactate dehydrogenase activity showed that contamination of the nuclear fraction by cytoplasm was less than 5%. In addition denaturing agarose gel-electrophoretic analysis showed that high molecular weight rRNA precursors were only detectable in the nuclear fraction. The real-time PCR results (Fig. 5, Panel A) show that whilst the SV1 level in the nuclear fraction was significantly higher with respect to that in the cytoplasmic fraction, the SV3 level was similar in both fractions. Down-regulation by RNA interference of hUPF1 increased the SV1 and SV3 cytoplasmic levels, but in the nuclear fraction only the level of the SV3 transcript was increased. In both fractions the canonical transcript was, as expected, unaffected by hUPF1 silencing (Fig. 5, Panel BeC).
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0
Relative Expression
Forty-two hours following minigene transfection, protein synthesis was inhibited with CHX treatment. Total RNA samples were prepared 6 h after CHX addition. The levels of the exogenous canonical and SV3 transcripts were unaffected by CHX treatment. The level of the exogenous SV1 isoform was, on the other hand, up-regulated by the drug (Fig. 3, Panel C).
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Fig. 5. Relative expression of SV1, canonical and SV3 NDUFS4 transcripts in the nuclear fraction with respect to the cytoplasmic fraction (Panel A). The expression level of the transcripts was analysed by real-time PCR. Normalization was performed with respect to the ribosomal 18S transcript. In the cytoplasmic fraction the level of the NDUFS4 transcripts was set as 1. Relative amount of the SV1, canonical and SV3 transcripts in the nuclear fraction after silencing of the hUPF1 gene (Panel B). Relative amount of the SV1, canonical and SV3 transcripts in the cytoplasmic fraction after silencing of the hUPF1 gene (Panel C). Normalization was made with respect to the b-actin transcript and the level of the NDUFS4 transcripts in the untreated samples was set as 1. In all the experiments the average values and standard deviations in five real-time PCR analyses are shown. *P < 0.001.
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IVS1nt-1,G → A th AUG PTC (15 codon)
1 44G →
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B
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Fig. 6. Impact of exonic and intronic mutations on the levels of NDUFS4 transcripts in patients fibroblasts. Schematic representation of the exonic 44G / A and intronic IVS1nt-1,G / A NDUFS4 mutations (Panel A). Real-time PCR analysis of NDUFS4 SV1, canonical and SV3 transcript levels in control and patients fibroblasts (Panel B). The level of the NDUFS4 transcripts in the normal and patients fibroblasts was normalized with respect to the GAPDH transcript. In all experiments the average values and standard deviations in three real-time PCR analyses are shown.
PTC containing SV3 isoform described in this work. The amount of the SV3 mRNA generated from the NDUFS4 (IVS1nt-1,G / A) mutant in the patients’ fibroblasts was 50 times higher than in normal fibroblast cells (Fig. 6, Panel B). 4. Discussion The present results reveal features of surveillance mechanisms regulating the levels of two alternative transcripts (SV1 and SV3) of the NDUFS4 gene of complex I, found to accumulate in patients with pathological mutations in this gene [28,29]. Whilst one of these transcripts, SV1, is found to be degraded by NMD which is translation dependent and involves both hUPF1 and hUPF2 factors, the other, SV3, appeared to be down-regulated in the nucleus by a translation and hUPF2 independent pathway. The pathological 44G / A mutation in exon 1 abrogated down-regulation of both SV1 and SV3 transcripts. The pathological IVSnt-1, G / A intron mutation resulted in high level of SV3 as single transcript of the gene, which escaped NMD. Real-time PCR analysis of the canonical mRNA, SV1 and SV3 NDUFS4 transcripts in HeLa cells showed that, like in fibroblasts, inhibition of protein synthesis by puromycin or cycloheximide, stabilized SV1 but had no effect on SV3. The lack of SV3 stabilization by translation inhibitors was not due to the low expression level of this transcript. The same differential effect of translation inhibition on SV1 and SV3 was also observed when these transcripts were over-expressed in HeLa cells transfected with minigene constructs. The insensitivity of SV3 to translation inhibition indicated that this transcript, although containing a PTC in a position which would candidate it for NMD, could be down-regulated by a different surveillance process.
There is evidence that some gene transcripts are scanned in the nucleus for ORF integrity [19e21,32e34]. Quantitative analysis of the NDUFS4 transcripts in subcellular fractions showed relative abundance of SV1 in the nuclear fraction with respect to the cytosol at difference with SV3 and canonical transcripts whose levels were practically the same in the two fractions. hUPF1 inactivation by siRNA induced significant up-regulation of SV1 and SV3 in total cell extracts, but only of SV3 in the nuclear fraction. hUPF2 silencing upregulated, on the other hand, SV1 but not SV3 in total HeLa cell preparation. The SV1 transcript exhibited prolonged half-life in hUPF1-depleted HeLa cells, whereas the SV3 transcript stability was unaffected. Thus the SV1 transcript undergoes the hUPF1ehUPF2 dependent NMD mechanism following its export from the nucleus into the cytoplasm. The SV3 transcript is, on the other hand, depressed in the nucleus through a translation independent but hUPF1 dependent process. Also this process is abrogated in the patient with the 44G / A mutation. The possibility that SV3 is an alternatively spliced transcript, produced at low levels in normal condition, but overproduced in the 44G / A pathological mutant since of altered exonic splicing enhancer element (ESE) [17,18], should also be considered. Use of three algorithms (RescueESE, ESEfinder and PESX) rules out, however, the possibility that the 44G / A mutation could have an effect on consensus ESE. In addition previous work, using an in vitro splicing system in pSPL3 vector, showed that the 44G / A mutation did not activate per se aberrant splicing [29]. A likely explanation for the escape of SV3 to NMD, although it is apparently candidate for this process, is that the stop codon is too close to the start codon (37 codons) to prevent downstream translation reinitiation and hence the ribosome continues translation in a reading frame that is not prematurely
D. Panelli et al. / Biochimie 90 (2008) 1452e1460
terminated. In silico analysis reveals the presence in SV3 of two translation competent AUGs (Fig. S3, Panel A of Supplementary Data). In vitro translation experiments showed that the canonical cDNA construct produced, in addition to the expected band of z20 kDa, also a small amount of a lower mW (z10 kDa) protein. The SV3 cDNA produced, on the other hand, only, and in high amount, the smaller protein recognized, only, by the anti-C NDUFS4 antibody (Fig. S3, Panel B of Supplementary Data). Thus the PTC introduced in the SV3 transcript could not activate NMD because AUGs, downstream the PTC, were still able to initiation translation. The results presented on the insensibility of the SV3 level to translation inhibition (Figs. 2 and 3, Panel C), the halflife of endogenous SV3 transcript in HeLa cells (Fig. 4, Panel C), the relative levels of the SV3 in the nuclear and cytoplasmic fractions (Fig. 5), the translation competence of SV3 (Fig. S3, Panel B of Supplementary Data), the expression level of the SV3 minigenes constructs (Fig. S4 of Supplementary Data), all provide converging evidence showing that it is the nuclear production and not the degradation of SV3 to be influenced by PTCs inserted in exon 1 or 3. Acknowledgments Supported by National Project on Bioenergetics, ‘‘Molecular Mechanisms, Physiology and Pathology of Membrane Bioenergetics System’’, 2005-Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR), Italy; and the Research Foundation Cassa di Risparmio di Puglia, 2005.
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