- Research article
- Open Access
Sequence features involved in the mechanism of 3' splice junction wobbling
© Tsai et al; licensee BioMed Central Ltd. 2010
- Received: 5 November 2009
- Accepted: 7 May 2010
- Published: 7 May 2010
Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms.
By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site. Although a lower frequency of alternative splicing occurs within the intronic region (alternative splicing at the proximal AG) than in the exonic region (alternative splicing at the distal AG), alternative AG sites located within the intronic region show stronger potential as the acceptor. These observations revealed that the choice of 3' splice sites during 3' splicing junction wobbling could depend on the distance between the duplicated AG and the branch point site (BPS). Further mutagenesis experiments demonstrated that the distance of AG-to-AG and BPS-to-AG can greatly influence 3' splice site selection. Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices.
Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function. In this study, we showed that occurrence of 3' wobble splicing occurs in a distance-dependent manner and that most of this wobble splicing is probably caused by steric hindrance from a factor bound at the neighboring tandem motif sequence.
- Alternative Splice
- Splice Site
- Splice Site Selection
- Branch Point Sequence
- Splice Site Recognition
Alternative splicing is an important mechanism of gene regulation in the human genome, occurring in around 40-60% of human genes . The 5' and 3' alternative splicing events contribute 25% of all alternative splicing instances and such events often result in frameshift mutations or insertion/deletion of amino acids in the expressed proteins . Recent studies indicated that certain alternative 5'/3' tandem splice site selections are only a few nucleotides apart, and that such short nucleotide length variations can still lead to subtle changes in protein structure through the modification of coding amino acids [3–5]. Interestingly, this phenomenon occurs throughout the genome, which could result in many protein isoforms with one amino acid insertion or deletion [6–15]. Splicing at junctions that contain GTNGT at the 5' splice site or NAGNAG at the 3' splice site would generate long transcripts (3 bp included) or short transcripts (3 bp excluded) during the wobble splicing process [7, 9, 16]. Although tandem motifs are common in human genes, only a small fraction of them can produce wobble splicing isoforms (GTNGT: 2%; NAGNAG: 16%) [7, 9–11]. Therefore, a complicated mechanism could exist for regulating tandem splice site choice during the wobble splicing process. Previous studies have shown that the high fidelity of splice site recognition involves specific networks of RNA-protein, protein-protein and RNA-RNA interactions [17–19]. The mechanism of acceptor site choice requires more complicated control than for donor sites, because the splicing factors interact flexibly with cis-elements such as branch point sequences (BPS), polypyrimidine tracts and the acceptor AG site. In our previous studies, we found that selection of acceptor sites in 3' wobble splicing could be affected by (i) a tandem splice site (NAGNAG) and (ii) component sequences occurring between the BPS and the NAGNAG [including BPS and polypyrimidine tracts (PPTs)] . Interestingly, we also found that mutations or single nucleotide polymorphisms (SNPs) at the BPS can disturb 3' wobble splicing selections by creating an aberrant branch point . This would affect the BPS-to-AG nucleotide distance and hence alter the wobble splicing selection pattern. However, the detailed mechanisms for such wobble splicing choices are currently unclear and remain to be studied. In this study, we utilized a minigene approach to demonstrate that the distance between two tandem splice sites or between BPS and AG plays an important role in 3' alternative splicing choice at nearby tandem splice sites, and that this phenomenon is indiscriminate.
Occurrence of wobble splicing at tandem splice sites separated by a short distance
Slu7 did not affect 3' wobble splicing at tandem motif sites
Tandem splice site-based wobble splicing depends on the AG-to-AG and BPS-to-AG distance
Previous studies have shown that the high fidelity of splice site recognition involves specific networks of RNA-protein, protein-protein and RNA-RNA interactions [17–19]. However, the detailed mechanisms for alternative splicing at tandem motifs are currently unclear. Previous studies indicated that such tandem motifs at splicing junctions are common in human genes, but only a small fraction of them can generate wobble in splicing selection [7, 9–11]. Our data confirmed that a high frequency of 5' wobble splicing events is located at four nucleotides from the dominant donor site and this is hypothesized to be associated with the binding affinity of U1 snRNA [9, 25]. Based on this, 5' wobble splicing could occur when one donor site is effectively competing with the other donor site for U1 binding. Alternatively, 3' alternative wobble splicing seems to occur more frequently at closely associated tandem acceptor sites (< 6 nucleotides). Thus, the choice between 3' acceptor sites appears to be more complicated than that of 5' donor sites. It is possible that splicing factors flexibly interact with cis-elements, such as BPS, PPTs and AG splice sites, during 3' splicing. According to the proposed linear scanning mechanism model [21–24], the spliceosome recognizes the branch point and scans downstream for the first AG. However, in this study we also observed many instances in which use of the distal AG was preferred, which could not be explained by the scanning model alone.
RNA surveillance, also known as nonsense-mediated mRNA decay (NMD), is an mRNA quality-control mechanism that degrades abnormal mRNAs such as misspliced mRNA transcripts . By recognizing mRNAs containing a premature termination codon, NMD eliminates the production of the truncated protein encoded by the misspliced transcripts that could function to the detriment of cells . Putative splicing sites located close to the dominant splice site may cause wobble splicing, resulting in small insertion/deletion changes in transcripts [9, 12, 25, 30]. Our data also showed that NMD is involved in distribution of short-distance alternative splicing. Figure 1 and 2 show that while most of the wrongly spliced mRNA transcripts were degraded by NMD, those wobble spliced at tandem motifs, AG(N)nAG or GT(N)nGT (n = 1, 4, 7, 10, 13 or 16), might have a better chance to escape from the NMD surveillance because of the occurrence of an in-frame insertion/deletion of one or two amino acids in these mRNAs without generating a premature stop codon. Frame-shifting a tandem splice site (n = 0, 2 or 3) has severe consequences for protein function because of the creation of altered protein residues or loss of mRNA transcripts by the wobble splicing process. One high frequency 5' wobble splicing is located four nucleotides from the dominant donor site, and most of the frame-shifting transcripts would be disrupted by NMD. In contrast to 5' alternative splicing, a high frequency of 3' alternative splicing occurs at ± three nucleotides (635 cases), which can increase protein diversity by altering 1-2 amino acids. Although this only subtly changes the protein sequence, it might influence protein function, for example in NR3C1, DRPLA, PAX3, PAX7, IGF1R and ING4 [10, 31–34]. Previous studies indicated that four wobble-splicing isoforms of ING4 differ in several functional aspects including protein localization, protein degradation, protein-protein interactions, transcriptional activity and cell spreading and migration [13, 35, 36]. Moreover, alternative splicing at a 5' or 3' tandem splice site may play an important role in the progression of disease, because reported cases include human genes WT1 and ABCA4[37, 38].
Traditionally, alternative splicing is expressed in a tissue type or developmental stage-dependent manner through regulating certain splicing factors. However, most NAGNAG- or GTNGT-based wobble splicing events did not show differential expression patterns of spliced isoforms in various tissues. Only a small fraction of these were reported to be tissue specific in genes including ITGAM, SMARCA4 and BTNL2. In this study, hSlu7 failed to alter short-distance wobble splicing, which may be due to the close distance (< 6 nucleotides) between proximal and distal AG sites. Therefore, the trans-splicing factor, hSlu7, might not be involved in the recognition of proximal and distance splice sites in NAGNAG-based wobble splicing. According features of the neighboring nucleotides around tandem splice sites and G/C content of PPT, Shina et al. successfully developed a method to accurately predict of NAGNAG-based wobble splicing pattern. In this study, we revealed that distance of AG-to-AG and BPS-to-AG both influenced choice of tandem acceptor sites during 3'-short-distance wobble splicing. Based on these observations, we believe that most of this wobble splicing is most likely caused by steric hindrance from a factor bound at the surrounding tandem motif sequence. Based on this hypothesis, wobble splicing could be predicted according to cis-element sequence features as reported [39, 40].
In summary, this study supplies further evidence of the involvement of acceptor site selection in wobble splicing at close tandem splice sites. Overall, our data reveal that the mechanism of short-distance 3' wobble splicing is stochastic and depends on the BPS-to-AG and AG-to-AG distance.
Frequency of 3' and 5' alternative splicing
In this study, all investigations were based on the third release of human 36.35i from the ASD http://www.ebi.ac.uk/asd/. For the analysis of the human 5' and 3' alternative splicing, the splicing events data file (AltSplice-rel3.events.txt) and gene sequence file (AltSplice-rel3.genes.txt) were downloaded from the ASD. The interesting intron splicing events were extracted from the isoform, and thus were classified according to their location 5' or 3' to introns. A total of 19,874 intron isoform events were reported in the ASD, which comprised 8,772 and 9,491 distinct alternative sites in 5' and 3', respectively. In each of the instances of splicing, the splice site used to produce the major transcripts was defined as the dominant splice site. An alternative site with equal EST support was identified as an ambiguous case. An alternative site with a majority of disagreement among all corresponding II events was treated as a conflicting case. After filtering all ambiguous and conflicting cases, 7,400 and 8,223 explicit alternative sites remained in 5' and 3', respectively. For each of these events, the number of ESTs that occurred was recorded in relation to the distance from the dominant splice site.
The genomic DNA of NM_014226-Exon 6-7 and NM_015179-Exon 32-33 was amplified by PCR using primer pair NM_014226-F/R and NM_015179-F/R from genomic DNA of the AZ-521 cell line. The amplified fragments were cloned into pGEM-T easy vector (Promega). After determining their sequence by an autosequencer, a minigene construct was generated by subcloning the genomic DNA of NM_014226-Exon 6-7 and NM_015179-Exon 32-33 into the EcoRI site of the pEGFP-C1 vector (Clontech). The minigenes containing various AG-to-AG or BPS-to-AG distances were generated by overlapping PCR as follows. The plasmid human minigene was used as template for a first PCR with a variant AG-to-AG distance primer set or BPS-to-AG distance primer set and the PCR products were used as a megaprimer. A second PCR was performed using the original minigene as template and the product subcloned into pGEM-T easy vector. After confirming the sequence, we subcloned the amplified product into pEGFP-C1 expression vector. The specific PCR primer pairs are listed in Additional file 2, Table S1. The 11AG/23AG plasmid construct is provided by Dr. R. Reed and subcloned into pRGFP-C1 expression vector.
Splicing analysis in vivo
The minigene plasmids were introduced into HeLa cells by Lipofectamine 2000 (Invitrogen) according to the methods provided by the manufacturer. At 48 h posttransfection, total RNA was extracted, and reverse transcription was carried out using 2.5 μg of poly(A)(+) RNA, oligo-(dT)15 and SuperScript II reverse transcriptase (Invitrogen). The splicing products were analyzed by capillary electrophoresis using the FAM-labeled primer set described in Additional file 2, Table S1.
Western blotting assay
HeLa cells were transfected with either of the RNAi oligonucleotides (5'-UUCAGAUCCCUUGUCAUAGGCUUCC-3') directed against hSlu7, and random sequence siRNA oligonucleotides (Invitrogen) were used as a negative control. Forty-eight hours after transfection, whole-cell extracts were obtained, subjected to SDS-PAGE and immunoblotted using hSlu7 (sc-10828, Santa Cruz Biotechnology) and anti-actin antibody (sc-1616, Santa Cruz Biotechnology).
Capillary electrophoresis analysis
PCR reactions were performed in a 20 μl final volume, including 10× PCR buffer, FAM-labeled primer pairs, dNTPs and Takara Taq DNA polymerase (Takara Shuzo Company, Shiga, Japan). PCR conditions were as follows: 94°C for 5 min; 26 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min; 72°C for 10 min and cooling at 4°C. One microliter of the PCR mix was diluted to 10 μl with formamide (Applied Biosystems, Foster City, CA, USA), containing 1 μl ROX 350 fluorescent size standards (Applied Biosystems), denatured at 95°C for 5 min and cooled at 4°C. Amplified PCR products were separated by an ABI 3100-Avant DNA analyzer using Polymer 3100 POP4, then quantified with GeneScan 3.7 software. The ratio of the wobble splicing isoforms was determined by dividing the peak area of the individual forms by the total area.
We are grateful to Dr R. Reed for the 11AG/23AG construct. This work was supported by research grants from Academia Sinica.
- Black DL: Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003, 72: 291-336. 10.1146/annurev.biochem.72.121801.161720View ArticlePubMedGoogle Scholar
- Sugnet CW, Kent WJ, Ares M, Haussler D: Transcriptome and genome conservation of alternative splicing events in humans and mice. Pacific Symposium on Biocomputing. 2004, 66-77.Google Scholar
- Stetefeld J, Ruegg MA: Structural and functional diversity generated by alternative mRNA splicing. Trends Biochem Sci. 2005, 30 (9): 515-521. 10.1016/j.tibs.2005.07.001View ArticlePubMedGoogle Scholar
- Zavolan M, Kondo S, Schonbach C, Adachi J, Hume DA, Hayashizaki Y, Gaasterland T: Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome. Genome Res. 2003, 13 (6B): 1290-1300. 10.1101/gr.1017303View ArticlePubMedPubMed CentralGoogle Scholar
- Wen F, Li F, Xia H, Lu X, Zhang X, Li Y: The impact of very short alternative splicing on protein structures and functions in the human genome. Trends Genet. 2004, 20 (5): 232-236. 10.1016/j.tig.2004.03.005View ArticlePubMedGoogle Scholar
- Akerman M, Mandel-Gutfreund Y: Alternative splicing regulation at tandem 3' splice sites. Nucleic Acids Res. 2006, 34 (1): 23-31. 10.1093/nar/gkj408View ArticlePubMedPubMed CentralGoogle Scholar
- Hiller M, Huse K, Szafranski K, Jahn N, Hampe J, Schreiber S, Backofen R, Platzer M: Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity. Nat Genet. 2004, 36 (12): 1255-1257. 10.1038/ng1469View ArticlePubMedGoogle Scholar
- Hiller M, Huse K, Szafranski K, Jahn N, Hampe J, Schreiber S, Backofen R, Platzer M: Single-Nucleotide Polymorphisms in NAGNAG Acceptors Are Highly Predictive for Variations of Alternative Splicing. Am J Hum Genet. 2006, 78 (2): 291-302. 10.1086/500151View ArticlePubMedPubMed CentralGoogle Scholar
- Hiller M, Huse K, Szafranskzi K, Rosenstiel P, Schreiber S, Backofen R, Platzer M: Phylogenetically widespread alternative splicing at unusual GYNGYN donors. Genome Biol. 2006, 7 (7): R65- 10.1186/gb-2006-7-7-r65View ArticlePubMedPubMed CentralGoogle Scholar
- Tadokoro K, Yamazaki-Inoue M, Tachibana M, Fujishiro M, Nagao K, Toyoda M, Ozaki M, Ono M, Miki N, Miyashita T: Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products. J Hum Genet. 2005, 50 (8): 382-394. 10.1007/s10038-005-0261-9View ArticlePubMedGoogle Scholar
- Tsai KW, Lin WC: Quantitative analysis of wobble splicing indicates that it is not tissue specific. Genomics. 2006, 88 (6): 855-864. 10.1016/j.ygeno.2006.07.004View ArticlePubMedGoogle Scholar
- Tsai KW, Tarn WY, Lin WC: Wobble splicing reveals the role of the branch point sequence-to-NAGNAG region in 3' tandem splice site selection. Mol Cell Biol. 2007, 27 (16): 5835-5848. 10.1128/MCB.00363-07View ArticlePubMedPubMed CentralGoogle Scholar
- Tsai KW, Tseng HC, Lin WC: Two wobble-splicing events affect ING4 protein subnuclear localization and degradation. Experimental cell research. 2008, 314 (17): 3130-3141. 10.1016/j.yexcr.2008.08.002View ArticlePubMedGoogle Scholar
- Atkinson TP, Dai Y: Activation-induced changes in alternate splice acceptor site usage. Biochem Biophys Res Commun. 2007, 358 (2): 590-595. 10.1016/j.bbrc.2007.04.158View ArticlePubMedGoogle Scholar
- Schindler S, Szafranski K, Hiller M, Ali GS, Palusa SG, Backofen R, Platzer M, Reddy AS: Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes. BMC genomics. 2008, 9: 159- 10.1186/1471-2164-9-159View ArticlePubMedPubMed CentralGoogle Scholar
- Lai CH, Hu LY, Lin WC: Single amino-acid InDel variants generated by alternative tandem splice-donor and -acceptor selection. Biochem Biophys Res Commun. 2006, 342 (1): 197-205. 10.1016/j.bbrc.2006.01.101View ArticlePubMedGoogle Scholar
- Staley JP, Guthrie C: Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998, 92 (3): 315-326. 10.1016/S0092-8674(00)80925-3View ArticlePubMedGoogle Scholar
- Reed R: Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev. 1996, 6 (2): 215-220. 10.1016/S0959-437X(96)80053-0View ArticlePubMedGoogle Scholar
- Reed R: Mechanisms of fidelity in pre-mRNA splicing. Curr Opin Cell Biol. 2000, 12 (3): 340-345. 10.1016/S0955-0674(00)00097-1View ArticlePubMedGoogle Scholar
- Thanaraj TA, Stamm S, Clark F, Riethoven JJ, Le Texier V, Muilu J: ASD: the Alternative Splicing Database. Nucleic Acids Res. 2004, D64-69. 32 DatabaseGoogle Scholar
- Smith CW, Chu TT, Nadal-Ginard B: Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol Cell Biol. 1993, 13 (8): 4939-4952.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen S, Anderson K, Moore MJ: Evidence for a linear search in bimolecular 3' splice site AG selection. Proc Natl Acad Sci USA. 2000, 97 (2): 593-598. 10.1073/pnas.97.2.593View ArticlePubMedPubMed CentralGoogle Scholar
- Smith CW, Porro EB, Patton JG, Nadal-Ginard B: Scanning from an independently specified branch point defines the 3' splice site of mammalian introns. Nature. 1989, 342 (6247): 243-247. 10.1038/342243a0View ArticlePubMedGoogle Scholar
- Chua K, Reed R: An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing. Mol Cell Biol. 2001, 21 (5): 1509-1514. 10.1128/MCB.21.5.1509-1514.2001View ArticlePubMedPubMed CentralGoogle Scholar
- Dou Y, Fox-Walsh KL, Baldi PF, Hertel KJ: Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site. Rna. 2006, 12 (12): 2047-2056. 10.1261/rna.151106View ArticlePubMedPubMed CentralGoogle Scholar
- Liang XH, Haritan A, Uliel S, Michaeli S: trans and cis splicing in trypanosomatids: mechanism, factors, and regulation. Eukaryot Cell. 2003, 2 (5): 830-840. 10.1128/EC.2.5.830-840.2003View ArticlePubMedPubMed CentralGoogle Scholar
- Chua K, Reed R: The RNA splicing factor hSlu7 is required for correct 3' splice-site choice. Nature. 1999, 402 (6758): 207-210. 10.1038/46086View ArticlePubMedGoogle Scholar
- Maquat LE: Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol. 2004, 5 (2): 89-99. 10.1038/nrm1310View ArticlePubMedGoogle Scholar
- Lareau LF, Green RE, Bhatnagar RS, Brenner SE: The evolving roles of alternative splicing. Curr Opin Struct Biol. 2004, 14 (3): 273-282. 10.1016/j.sbi.2004.05.002View ArticlePubMedGoogle Scholar
- Hiller M, Szafranski K, Sinha R, Huse K, Nikolajewa S, Rosenstiel P, Schreiber S, Backofen R, Platzer M: Assessing the fraction of short-distance tandem splice sites under purifying selection. Rna. 2008, 14 (4): 616-629. 10.1261/rna.883908View ArticlePubMedPubMed CentralGoogle Scholar
- Vogan KJ, Underhill DA, Gros P: An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity. Mol Cell Biol. 1996, 16 (12): 6677-6686.View ArticlePubMedPubMed CentralGoogle Scholar
- Kay PH, Ziman MR: Alternate Pax7 paired box transcripts which include a trinucleotide or a hexanucleotide are generated by use of alternate 3' intronic splice sites which are not utilized in the ancestral homologue. Gene. 1999, 230 (1): 55-60. 10.1016/S0378-1119(99)00049-9View ArticlePubMedGoogle Scholar
- Condorelli G, Bueno R, Smith RJ: Two alternatively spliced forms of the human insulin-like growth factor I receptor have distinct biological activities and internalization kinetics. J Biol Chem. 1994, 269 (11): 8510-8516.PubMedGoogle Scholar
- Rivers C, Levy A, Hancock J, Lightman S, Norman M: Insertion of an amino acid in the DNA-binding domain of the glucocorticoid receptor as a result of alternative splicing. The Journal of clinical endocrinology and metabolism. 1999, 84 (11): 4283-4286. 10.1210/jc.84.11.4283View ArticlePubMedGoogle Scholar
- Unoki M, Shen JC, Zheng ZM, Harris CC: Novel Splice Variants of ING4 and Their Possible Roles in the Regulation of Cell Growth and Motility. J Biol Chem. 2006, 281 (45): 34677-34686. 10.1074/jbc.M606296200View ArticlePubMedGoogle Scholar
- Shen JC, Unoki M, Ythier D, Duperray A, Varticovski L, Kumamoto K, Pedeux R, Harris CC: Inhibitor of growth 4 suppresses cell spreading and cell migration by interacting with a novel binding partner, liprin alpha1. Cancer Res. 2007, 67 (6): 2552-2558. 10.1158/0008-5472.CAN-06-3870View ArticlePubMedPubMed CentralGoogle Scholar
- Maugeri A, van Driel MA, Pol van de DJ, Klevering BJ, van Haren FJ, Tijmes N, Bergen AA, Rohrschneider K, Blankenagel A, Pinckers AJ: The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am J Hum Genet. 1999, 64 (4): 1024-1035. 10.1086/302323View ArticlePubMedPubMed CentralGoogle Scholar
- Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA: Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci USA. 1995, 92 (26): 11960-11964. 10.1073/pnas.92.26.11960View ArticlePubMedPubMed CentralGoogle Scholar
- Chern TM, van Nimwegen E, Kai C, Kawai J, Carninci P, Hayashizaki Y, Zavolan M: A simple physical model predicts small exon length variations. PLoS Genet. 2006, 2 (4): e45- 10.1371/journal.pgen.0020045View ArticlePubMedPubMed CentralGoogle Scholar
- Sinha R, Nikolajewa S, Szafranski K, Hiller M, Jahn N, Huse K, Platzer M, Backofen R: Accurate prediction of NAGNAG alternative splicing. Nucleic Acids Res. 2009, 37 (11): 3569-3579. 10.1093/nar/gkp220View ArticlePubMedPubMed CentralGoogle Scholar
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