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• W2012054423 abstract "TO THE EDITOR The role of lysyl hydroxylase 2 (LH2) as an essential enzyme in the collagen biosynthesis pathway contributes significantly to the maintenance of skin architecture. This enzyme is required for the hydroxylation of specific collagen lysines prior to the formation of cross-links that stabilize the collagen fibrils (Takaluoma et al., 2007Takaluoma K. Lantto J. Myllyharju J. Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.Matrix Biol. 2007; 26: 396-403Crossref PubMed Scopus (54) Google Scholar). There are two alternately spliced LH2 isoforms encoded by two distinct mRNA transcripts that either include or exclude the 63 nucleotide exon 13A (LH2(long) and LH2(short); Yeowell and Walker, 1999Yeowell H.N. Walker L.C. Tissue specificity of a new splice form of the human lysyl hydroxylase 2 gene.Matrix Biol. 1999; 18: 179-187Crossref PubMed Scopus (68) Google Scholar). LH2(long) mRNA is the major LH2 splice form in skin and has been reported to be markedly increased in scleroderma and other fibrotic conditions by several laboratories (van der Slot et al., 2003van der Slot A.J. Zuurmond A.M. Bardoel A.F. et al.Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis.J Biol Chem. 2003; 278: 40967-40972Crossref PubMed Scopus (270) Google Scholar; Walker et al., 2005Walker L.C. Overstreet M.A. Yeowell H.N. Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.Matrix Biol. 2005; 23: 515-523Crossref PubMed Scopus (27) Google Scholar; Wu et al., 2006Wu J. Reinhardt D.P. Batmunkh C. et al.Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts.Exp Cell Res. 2006; 312: 3485-3494Crossref PubMed Scopus (26) Google Scholar). This is accompanied by an overaccumulation of cross-linked collagen that contributes to the fibrosis (van der Slot et al., 2005van der Slot A.J. van Dura E.A. de Wit E.C. et al.Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels.Biochim Biophys Acta. 2005; 1741: 95-102Crossref PubMed Scopus (78) Google Scholar). We have examined the mechanism of LH2(long) splicing regulation by identification of the cis-elements and trans-acting protein factors involved. Recently, we have reported that RNA-binding proteins (TIA and Fox-2) act on intronic sequences flanking exon 13A (E13A) to enhance expression of fibrosis-associated LH2(long) (Yeowell et al., 2009Yeowell H.N. Walker L.C. Mauger D.M. et al.TIA nuclear proteins regulate the alternate splicing of lysyl hydroxylase 2.J Investig Dermatol. 2009; 129: 1402-1411Crossref PubMed Scopus (8) Google Scholar; Seth and Yeowell, 2010Seth P. Yeowell H.N. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.Arthritis Rheumat. 2010; 62: 1167-1175Crossref PubMed Scopus (11) Google Scholar). We have shown that suppression of these trans-acting factors by siRNAs decreased the levels of LH2(long). This indicates the important functions that TIA, Fox-2, and potentially other regulatory splicing factors may have in the pathobiology of scleroderma by their ability to increase the levels of LH2(long). To our knowledge, this study is the first to identify cis-elements within E13A itself, which can either upregulate or downregulate the expression of LH2(long) mRNA. The majority of human transcripts are alternately spliced and this accounts for the vast diversity of the human proteome (Blencowe, 2006Blencowe B.J. Alternative splicing: new insights from global analyses.Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). Depending on their exonic/intronic location, motifs on pre-mRNA that affect alternative splicing can function either as enhancers or silencers of exonic splicing (ESE or ESS) or intronic splicing (ISE or ISS) (Wang and Burge, 2008Wang Z. Burge C.B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.RNA. 2008; 14: 802-813Crossref PubMed Scopus (635) Google Scholar). These motifs serve as sequence-specific docking sites for trans-acting protein factors that can either activate or repress the use of proximal splice sites by various mechanisms. To date, the cis-elements and trans-factors identified in the regulation of the alternate splicing of LH2 have been located in the introns flanking the alternately spliced E13A. To examine E13A for possible exonic regulatory cis-elements, in this study we probed the 63 nucleotide alternately spliced exon by sequential substitutions. This enabled us to identify one major ESE and two ESS within E13A that affected its splicing. Using a wild-type LH2 minigene (Seth and Yeowell, 2010Seth P. Yeowell H.N. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.Arthritis Rheumat. 2010; 62: 1167-1175Crossref PubMed Scopus (11) Google Scholar), we initially mutated the entire alternate E13A, with the exception of six nucleotides (Figure 1). As with all alternately spliced and constitutively spliced exons, two nucleotides at the beginning and end of each exon are essential for its splicing. To ensure that we were not interfering with these essential exonic nucleotides, we retained the wild-type sequence for the first three and last three nucleotides of this exon for each of the substitutions. Moreover, the strategy of using substitutions ensured that the size of the exon, which can influence the splicing pattern, was unchanged (Baek and Green, 2005Baek D. Green P. Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing.Proc Natl Acad Sci USA. 2005; 102: 12813-12818Crossref PubMed Scopus (105) Google Scholar). The accuracy of the mutations was confirmed by sequence analysis and the mutated minigene constructs were stably transfected in HEK293 cells as previously described (Seth and Yeowell, 2010Seth P. Yeowell H.N. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.Arthritis Rheumat. 2010; 62: 1167-1175Crossref PubMed Scopus (11) Google Scholar). As shown in Figure 1, a 57 nucleotide substitution of E13A in our prevalidated LH2 minigene (Seth and Yeowell, 2010Seth P. Yeowell H.N. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.Arthritis Rheumat. 2010; 62: 1167-1175Crossref PubMed Scopus (11) Google Scholar) from nucleotides (+4 to +60) resulted in a dramatic decrease in the level of LH2(long) mRNA. The substitution sequence was derived from intron 8 of the fibroblast growth factor receptor 2 (FGFR2) gene, a sequence that has previously been shown to be inert and devoid of any splicing motifs (Baraniak et al., 2003Baraniak A.P. Lasda E.L. Wagner E.J. et al.A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.Mol Cell Biol. 2003; 23: 9327-9337Crossref PubMed Scopus (71) Google Scholar). To address the concern that the observed decrease in LH2(long) mRNA levels on substitution of its internal nucleotides was independent of the sequence used for substitutions, we replaced the same 57 nucleotides of LH2 E13A with two different 57 nucleotide sequences. One sequence was derived from a different part of the FGFR2 intron 8, whereas the other was derived from the completely unrelated β-globin gene. Both these sequences have been confirmed as splicing inert and have no identifiable splicing cis-elements (Baraniak et al., 2003Baraniak A.P. Lasda E.L. Wagner E.J. et al.A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.Mol Cell Biol. 2003; 23: 9327-9337Crossref PubMed Scopus (71) Google Scholar; Seth et al., 2008Seth P. Miller H.B. Lasda E.L. et al.Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc.J Biol Chem. 2008; 283: 10058-10067Crossref PubMed Scopus (13) Google Scholar). As shown in Figure 1b and quantitated in Figure 1c, a significant decrease in LH2(long) mRNA levels was observed in all minigene substitutions irrespective of the origin of the substitution sequence. These results suggested the presence of at least one cis-enhancer element within E13A of LH2. To identify the location and number of these regulatory cis-elements affecting the alternate splicing of E13A, we examined the effect of sequential, smaller substitutions. To accomplish this, seven nucleotides were substituted in the minigene constructs 7.1 to 7.7 and eight nucleotides were substituted in 7.8 (Figure 2a). All these substitutions were transversions, and the substituted sequence was analyzed using bioinformatics for the absence of any putative splicing motifs. These minigene constructs were then stably transfected in HEK293 cells as described. As shown in Figure 2b and c, the results suggest the presence of multiple cis-elements within E13A. In comparison to the wild-type, the mutation of the 7.2 sequence virtually abolished the levels of LH2(long) mRNA, whereas the substitution of the nucleotides in minigenes 7.4, 7.7, and 7.8 produced the opposite effect of increasing the levels of LH2(long) mRNA by over 5-fold. The other exonic substitutions only slightly increased LH2(long) mRNA levels. Overall, these substitutions strongly suggest the presence of an ESE within the nucleotides that were mutated in construct 7.2 and, in addition, the presence of two ESS in the exonic regions mutated in the constructs 7.4, 7.7, and 7.8. As three separate exonic regions are represented by constructs 7.2, 7.3–7.4, and 7.7–7.8, our results reveal the presence of multiple cis-elements within E13A that affect its own alternative splicing. However, it should be noted that when both the ESE and ESS sequences are removed by substitution of the entire E13A (Figure 1), the overall effect is virtual elimination of LH2(long). This suggests that the ESE in construct 7.2 has a major role in the regulation of LH2 alternate splicing and offers a target for decreasing the expression of the fibrosis-associated LH2(long) mRNA. Potentially this could be achieved by trans-splicing approaches that are currently being tested in case of the cystic fibrosis trans-membrane receptor gene or by using antisense oligonucleotides against the identified LH2 cis-elements to alter the pattern of LH2 alternate splicing and directly modulate the levels of LH2(long) mRNA (Song et al., 2009Song Y. Lou H.H. Boyer J.L. et al.Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.Hum Gene Ther. 2009; 20: 267-281Crossref PubMed Scopus (41) Google Scholar; Perez et al., 2010Perez B. Rodriguez-Pascau L. Vilageliu L. et al.Present and future of antisense therapy for splicing modulation in inherited metabolic disease.J Inherit Metab Dis. 2010; 33: 397-403Crossref PubMed Scopus (29) Google Scholar). This approach, and the potential knockdown of the currently unknown trans-acting protein factor(s) that may bind to the identified splicing motifs, could offer fresh perspectives to therapeutic strategies directed against scleroderma. This work was supported by NIH grant RO1 AG10215 from the National Institute on Aging (to HNY) and by the Scleroderma Foundation (to HNY). The authors thank Robert D. Streilein of Duke Dermatology for help with the figures." @default.
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• W2012054423 title "Identification of Exonic Cis-Elements Regulating the Alternative Splicing of Scleroderma-Associated Lysyl Hydroxylase 2 mRNA" @default.
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