Intronic gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
Splicing defects constitute a major cause of human disease. Mutations affecting conserved splicing sequences at exon-intron junctions are easily recognized as possibly pathogenic, whereas variants in exonic or intronic regions are difficult to classify without functional evidence provided by transcript analysis or in vitro analysis using minigenes. In this work, we sought out to study the pathogenicity of two novel intronic PAH variants identified in phenylketonuria patients. Both mutations resulted in exon skipping in minigenes. We demonstrate that U1snRNP70 binds to the intronic region and that this binding is abolished in the mutant sequences. Correction of the splicing defect was achieved using modified U1 snRNA perfectly complementary to each of the mutant sequences. The results extend the repertoire of natural U1 snRNP cellular functions by including its role as splicing enhancer via binding downstream of the natural 5’ splice site. In addition, our results correlate with the described therapeutic effect of modified U1snRNP for splicing mutations in different genes, thus having a significant impact in the development of specific therapies for splicing defects.
Vyšlo v časopise:
Intronic gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site. PLoS Genet 14(4): e32767. doi:10.1371/journal.pgen.1007360
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1007360
Souhrn
Splicing defects constitute a major cause of human disease. Mutations affecting conserved splicing sequences at exon-intron junctions are easily recognized as possibly pathogenic, whereas variants in exonic or intronic regions are difficult to classify without functional evidence provided by transcript analysis or in vitro analysis using minigenes. In this work, we sought out to study the pathogenicity of two novel intronic PAH variants identified in phenylketonuria patients. Both mutations resulted in exon skipping in minigenes. We demonstrate that U1snRNP70 binds to the intronic region and that this binding is abolished in the mutant sequences. Correction of the splicing defect was achieved using modified U1 snRNA perfectly complementary to each of the mutant sequences. The results extend the repertoire of natural U1 snRNP cellular functions by including its role as splicing enhancer via binding downstream of the natural 5’ splice site. In addition, our results correlate with the described therapeutic effect of modified U1snRNP for splicing mutations in different genes, thus having a significant impact in the development of specific therapies for splicing defects.
Zdroje
1. Scotti MM, Swanson MS. RNA mis-splicing in disease. Nat Rev Genet. 2016;17(1):19–32. doi: 10.1038/nrg.2015.3 26593421
2. Manning KS, Cooper TA. The roles of RNA processing in translating genotype to phenotype. Nat Rev Mol Cell Biol. 2017;18(2):102–14. doi: 10.1038/nrm.2016.139 27847391
3. Baralle D, Buratti E. RNA splicing in human disease and in the clinic. Clin Sci (Lond). 2017;131(5):355–68.
4. Andresen BS, Krainer AR. When the genetic code is not enough-how sequence variations can alter pre-mRNA splicing and cause (complex) disease. In: Almasy L, Al-Chalabi A, editors. Genetics of Human Complex Diseases. New York: Cold Spring Harbor Laboratory Press; 2009. p. 165–82.
5. Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, et al. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat. 2007;28(2):150–8. doi: 10.1002/humu.20400 17001642
6. Egloff S, O'Reilly D, Murphy S. Expression of human snRNA genes from beginning to end. Biochem Soc Trans. 2008;36(Pt 4):590–4. doi: 10.1042/BST0360590 18631122
7. Carmel I, Tal S, Vig I, Ast G. Comparative analysis detects dependencies among the 5' splice-site positions. Rna. 2004;10(5):828–40. doi: 10.1261/rna.5196404 15100438
8. Roca X, Olson AJ, Rao AR, Enerly E, Kristensen VN, Borresen-Dale AL, et al. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics. Genome Res. 2008;18(1):77–87. doi: 10.1101/gr.6859308 18032726
9. Madsen PP, Kibaek M, Roca X, Sachidanandam R, Krainer AR, Christensen E, et al. Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping. Hum Genet. 2006;118(6):680–90. doi: 10.1007/s00439-005-0070-4 16317551
10. Merkhofer EC, Johnson TL. U1 snRNA rewrites the "script". Cell. 2012;150(1):9–11. doi: 10.1016/j.cell.2012.06.017 22770211
11. Xiong HY, Alipanahi B, Lee LJ, Bretschneider H, Merico D, Yuen RK, et al. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease. Science. 2015;347(6218):1254806. doi: 10.1126/science.1254806 25525159
12. Desviat LR, Perez B, Ugarte M. Minigenes to confirm exon skipping mutations. Methods Mol Biol. 2012;867:37–47. doi: 10.1007/978-1-61779-767-5_3 22454053
13. Havens MA, Hastings ML. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016;44(14):6549–63. doi: 10.1093/nar/gkw533 27288447
14. Tanner G, Glaus E, Barthelmes D, Ader M, Fleischhauer J, Pagani F, et al. Therapeutic strategy to rescue mutation-induced exon skipping in rhodopsin by adaptation of U1 snRNA. Hum Mutat. 2009;30(2):255–63. doi: 10.1002/humu.20861 18837008
15. Pinotti M, Rizzotto L, Balestra D, Lewandowska MA, Cavallari N, Marchetti G, et al. U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency. Blood. 2008;111(5):2681–4. doi: 10.1182/blood-2007-10-117440 18156490
16. Aartsma-Rus A. New Momentum for the Field of Oligonucleotide Therapeutics. Mol Ther. 2016;24(2):193–4. doi: 10.1038/mt.2016.14 26906610
17. Fernandez Alanis E, Pinotti M, Dal Mas A, Balestra D, Cavallari N, Rogalska ME, et al. An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Hum Mol Genet. 2012;21(11):2389–98. doi: 10.1093/hmg/dds045 22362925
18. Rogalska ME, Tajnik M, Licastro D, Bussani E, Camparini L, Mattioli C, et al. Therapeutic activity of modified U1 core spliceosomal particles. Nature communications. 2016;7:11168. doi: 10.1038/ncomms11168 27041075
19. Chao HK, Hsiao KJ, Su TS. A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria. Hum Genet. 2001;108(1):14–9. 11214902
20. Ellingsen S, Knappskog PM, Eiken HG. Phenylketonuria splice mutation (EXON6nt-96A—>g) masquerading as missense mutation (Y204C). Hum Mutat. 1997;9(1):88–90. doi: 10.1002/(SICI)1098-1004(1997)9:1<88::AID-HUMU21>3.0.CO;2-K 8990021
21. Dobrowolski SF, Andersen HS, Doktor TK, Andresen BS. The phenylalanine hydroxylase c.30C>G synonymous variation (p.G10G) creates a common exonic splicing silencer. Mol Genet Metab. 2010;100(4):316–23. doi: 10.1016/j.ymgme.2010.04.002 20457534
22. Heintz C, Dobrowolski SF, Andersen HS, Demirkol M, Blau N, Andresen BS. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11. Mol Genet Metab. 2012;106(4):403–11. doi: 10.1016/j.ymgme.2012.05.013 22698810
23. Acosta AX, Silva WA Jr., Carvalho TM, Zago MA. Ten novel mutations in the phenylalanine hydroxylase gene (PAH) observed in Brazilian patients with phenylketonuria. Hum Mutat. 2001;17(1):77.
24. Guldberg P, Levy HL, Hanley WB, Koch R, Matalon R, Rous BM, et al. Phenylalanine Hydroxylase Gene Mutations in the United States: Report from the Maternal PKU Collaborative Study. Am J Hum Genet. 1996;59(1):84–94. 8659548
25. Gallego-Villar L, Viecelli HM, Perez B, Harding CO, Ugarte M, Thony B, et al. A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver. Mol Ther Nucleic Acids. 2014;3:e193. doi: 10.1038/mtna.2014.44 25226162
26. Hwang DY, Cohen JB. Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron. Mol Cell Biol. 1996;16(6):3012–22. 8649413
27. Hwang DY, Cohen JB. U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells. Genes Dev. 1996;10(3):338–50. 8595884
28. Engreitz JM, Sirokman K, McDonel P, Shishkin AA, Surka C, Russell P, et al. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites. Cell. 2014;159(1):188–99. doi: 10.1016/j.cell.2014.08.018 25259926
29. Eperon IC, Ireland DC, Smith RA, Mayeda A, Krainer AR. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993;12(9):3607–17. 8253084
30. Eperon IC, Makarova OV, Mayeda A, Munroe SH, Caceres JF, Hayward DG, et al. Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1. Mol Cell Biol. 2000;20(22):8303–18. 11046128
31. Cho S, Hoang A, Sinha R, Zhong XY, Fu XD, Krainer AR, et al. Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly. Proc Natl Acad Sci U S A. 2011;108(20):8233–8. doi: 10.1073/pnas.1017700108 21536904
32. Dal Mas A, Rogalska ME, Bussani E, Pagani F. Improvement of SMN2 pre-mRNA processing mediated by exon-specific U1 small nuclear RNA. Am J Hum Genet. 2015;96(1):93–103. doi: 10.1016/j.ajhg.2014.12.009 25557785
33. Tajnik M, Rogalska ME, Bussani E, Barbon E, Balestra D, Pinotti M, et al. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function. PLoS Genet. 2016;12(5):e1006082. doi: 10.1371/journal.pgen.1006082 27227676
34. Pagani F, Buratti E, Stuani C, Bendix R, Dork T, Baralle FE. A new type of mutation causes a splicing defect in ATM. Nat Genet. 2002;30(4):426–9. doi: 10.1038/ng858 11889466
35. Lund E, Dahlberg JE. True genes for human U1 small nuclear RNA. Copy number, polymorphism, and methylation. J Biol Chem. 1984;259(3):2013–21. 6198328
36. Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37(9):e67. doi: 10.1093/nar/gkp215 19339519
37. Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol. 2004;11(2–3):377–94. doi: 10.1089/1066527041410418 15285897
38. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol. 1997;4(3):311–23. doi: 10.1089/cmb.1997.4.311 9278062
39. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res. 2003;31(13):3568–71. 12824367
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2018 Číslo 4
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Intronic gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
- When genes move, genomes collide
- House dust mites use a plant-like siRNA pathway to silence transposable elements