Tissue-specific isoforms of the single C. elegans Ryanodine receptor gene unc-68 control specific functions


Autoři: Filipe Marques aff001;  Saurabh Thapliyal aff001;  Avelino Javer aff002;  Priyanka Shrestha aff002;  André E. X. Brown aff002;  Dominique A. Glauser aff001
Působiště autorů: Department of Biology, University of Fribourg, Fribourg, Switzerland aff001;  MRC London Institute of Medical Sciences, London, United Kingdom aff002;  Institute of Clinical Sciences, Imperial College London, London, United Kingdom aff003
Vyšlo v časopise: Tissue-specific isoforms of the single C. elegans Ryanodine receptor gene unc-68 control specific functions. PLoS Genet 16(10): e1009102. doi:10.1371/journal.pgen.1009102
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009102

Souhrn

Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.

Klíčová slova:

Alternative splicing – Caenorhabditis elegans – Calcium signaling – Invertebrate genomics – Neurons – Pharyngeal muscles – Phenotypes – Swimming


Zdroje

1. Clapham DE. Calcium signaling. Cell. 2007;131(6):1047–58. Epub 2007/12/18. doi: 10.1016/j.cell.2007.11.028 18083096.

2. Rizzuto R, Pozzan T. When calcium goes wrong: genetic alterations of a ubiquitous signaling route. Nat Genet. 2003;34(2):135–41. doi: 10.1038/ng0603-135 12776115.

3. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 2000;1(1):11–21. doi: 10.1038/35036035 11413485.

4. Coronado R, Morrissette J, Sukhareva M, Vaughan DM. Structure and function of ryanodine receptors. Am J Physiol. 1994;266(6 Pt 1):C1485–504. doi: 10.1152/ajpcell.1994.266.6.C1485 8023884.

5. Verkhratsky A, Shmigol A. Calcium-induced calcium release in neurones. Cell Calcium. 1996;19(1):1–14. Epub 1996/01/01. doi: 10.1016/s0143-4160(96)90009-3 8653752.

6. Zalk R, Lehnart SE, Marks AR. Modulation of the ryanodine receptor and intracellular calcium. Annu Rev Biochem. 2007;76:367–85. doi: 10.1146/annurev.biochem.76.053105.094237 17506640.

7. Van Petegem F. Ryanodine receptors: structure and function. J Biol Chem. 2012;287(38):31624–32. doi: 10.1074/jbc.R112.349068 22822064; PubMed Central PMCID: PMC3442496.

8. Hakamata Y, Nakai J, Takeshima H, Imoto K. Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain. FEBS Lett. 1992;312(2–3):229–35. doi: 10.1016/0014-5793(92)80941-9 1330694.

9. Hwang JH, Zorzato F, Clarke NF, Treves S. Mapping domains and mutations on the skeletal muscle ryanodine receptor channel. Trends Mol Med. 2012;18(11):644–57. doi: 10.1016/j.molmed.2012.09.006 23069638.

10. Brini M. Ryanodine receptor defects in muscle genetic diseases. Biochemical and biophysical research communications. 2004;322(4):1245–55. Epub 2004/09/01. doi: 10.1016/j.bbrc.2004.08.029 15336972.

11. Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, et al. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation. 2002;106(1):69–74. Epub 2002/07/03. doi: 10.1161/01.cir.0000020013.73106.d8 12093772.

12. Yano M, Yamamoto T, Kobayashi S, Matsuzaki M. Role of ryanodine receptor as a Ca(2)(+) regulatory center in normal and failing hearts. Journal of cardiology. 2009;53(1):1–7. Epub 2009/01/27. doi: 10.1016/j.jjcc.2008.10.008 19167631.

13. Supnet C, Noonan C, Richard K, Bradley J, Mayne M. Up-regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer's disease. J Neurochem. 2010;112(2):356–65. Epub 2009/11/12. doi: 10.1111/j.1471-4159.2009.06487.x 19903243.

14. Kushnir A, Betzenhauser MJ, Marks AR. Ryanodine receptor studies using genetically engineered mice. FEBS Lett. 2010;584(10):1956–65. Epub 2010/03/11. doi: 10.1016/j.febslet.2010.03.005 20214899; PubMed Central PMCID: PMC3690514.

15. De Mandal S, Shakeel M, Prabhakaran VS, Karthi S, Xu X, Jin F. Alternative splicing and insect ryanodine receptor. Archives of Insect Biochemistry and Physiology. 2019;102(3):e21590. doi: 10.1002/arch.21590 31218747

16. Maryon EB, Coronado R, Anderson P. unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction. J Cell Biol. 1996;134(4):885–93. Epub 1996/08/01. doi: 10.1083/jcb.134.4.885 8769414; PubMed Central PMCID: PMC2120954.

17. Maryon EB, Saari B, Anderson P. Muscle-specific functions of ryanodine receptor channels in Caenorhabditis elegans. J Cell Sci. 1998;111 (Pt 19):2885–95. Epub 1998/09/10. 9730981.

18. Hamada T, Sakube Y, Ahnn J, Kim DH, Kagawa H. Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans. J Mol Biol. 2002;324(1):123–35. Epub 2002/11/08. doi: 10.1016/s0022-2836(02)01032-x 12421563.

19. Liu Q, Chen B, Yankova M, Morest DK, Maryon E, Hand AR, et al. Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction. J Neurosci. 2005;25(29):6745–54. Epub 2005/07/22. doi: 10.1523/JNEUROSCI.1730-05.2005 16033884.

20. Sun L, Shay J, McLoed M, Roodhouse K, Chung SH, Clark CM, et al. Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation. J Neurosci. 2014;34(48):15947–56. Epub 2014/11/28. doi: 10.1523/JNEUROSCI.4238-13.2014 25429136; PubMed Central PMCID: PMC4244466.

21. Nicoll Baines K, Ferreira C, Hopkins PM, Shaw MA, Hope IA. Aging Effects of Caenorhabditis elegans Ryanodine Receptor Variants Corresponding to Human Myopathic Mutations. G3 (Bethesda). 2017;7(5):1451–61. Epub 2017/03/23. doi: 10.1534/g3.117.040535 28325813; PubMed Central PMCID: PMC5427508.

22. Fischer E, Gottschalk A, Schuler C. An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations. Sci Rep. 2017;7(1):17514. Epub 2017/12/14. doi: 10.1038/s41598-017-17819-8 29235522; PubMed Central PMCID: PMC5727474.

23. Sakube Y, Ando H, Kagawa H. An abnormal ketamine response in mutants defective in the ryanodine receptor gene ryr-1 (unc-68) of Caenorhabditis elegans. J Mol Biol. 1997;267(4):849–64. Epub 1997/04/11. doi: 10.1006/jmbi.1997.0910 9135117.

24. Ma X, Zhan G, Sleumer MC, Chen S, Liu W, Zhang MQ, et al. Analysis of C. elegans muscle transcriptome using trans-splicing-based RNA tagging (SRT). Nucleic acids research. 2016;44(21):e156–e. Epub 2016/08/23. doi: 10.1093/nar/gkw734 27557708.

25. Kanagawa T. Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng. 2003;96(4):317–23. Epub 2005/10/20. doi: 10.1016/S1389-1723(03)90130-7 16233530.

26. Kuroyanagi H, Ohno G, Sakane H, Maruoka H, Hagiwara M. Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans. Nat Protoc. 2010;5(9):1495–517. Epub 2010/08/21. doi: 10.1038/nprot.2010.107 20725066.

27. Orengo JP, Bundman D, Cooper TA. A bichromatic fluorescent reporter for cell-based screens of alternative splicing. Nucleic Acids Res. 2006;34(22):e148. Epub 2006/12/05. doi: 10.1093/nar/gkl967 17142220; PubMed Central PMCID: PMC1669726.

28. Trojanowski NF, Raizen DM, Fang-Yen C. Pharyngeal pumping in Caenorhabditis elegans depends on tonic and phasic signaling from the nervous system. Sci Rep. 2016;6:22940. Epub 2016/03/16. doi: 10.1038/srep22940 26976078; PubMed Central PMCID: PMC4791602.

29. Javer A, Ripoll-Sanchez L, Brown AEX. Powerful and interpretable behavioural features for quantitative phenotyping of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 2018;373(1758). Epub 2018/09/12. doi: 10.1098/rstb.2017.0375 30201839; PubMed Central PMCID: PMC6158219.

30. Javer A, Currie M, Lee CW, Hokanson J, Li K, Martineau CN, et al. An open-source platform for analyzing and sharing worm-behavior data. Nat Methods. 2018;15(9):645–6. Epub 2018/09/02. doi: 10.1038/s41592-018-0112-1 30171234; PubMed Central PMCID: PMC6284784.

31. Perez CF, Mukherjee S, Allen PD. Amino acids 1–1,680 of ryanodine receptor type 1 hold critical determinants of skeletal type for excitation-contraction coupling. Role of divergence domain D2. J Biol Chem. 2003;278(41):39644–52. Epub 2003/08/06. doi: 10.1074/jbc.M305160200 12900411.

32. Liu Z, Zhang J, Wang R, Wayne Chen SR, Wagenknecht T. Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel. Journal of molecular biology. 2004;338(3):533–45. doi: 10.1016/j.jmb.2004.03.011 15081811.

33. Yuchi Z, Van Petegem F. Ryanodine receptors under the magnifying lens: Insights and limitations of cryo-electron microscopy and X-ray crystallography studies. Cell Calcium. 2016;59(5):209–27. doi: 10.1016/j.ceca.2016.04.003 27103405

34. Hostettler L, Grundy L, Kaser-Pebernard S, Wicky C, Schafer WR, Glauser DA. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo. G3 (Bethesda). 2017;7(2):607–15. Epub 2017/01/22. doi: 10.1534/g3.116.038133 28108553; PubMed Central PMCID: PMC5295605.

35. El Mouridi S, Lecroisey C, Tardy P, Mercier M, Leclercq-Blondel A, Zariohi N, et al. Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype. G3 (Bethesda). 2017;7(5):1429–37. Epub 2017/03/11. doi: 10.1534/g3.117.040824 28280211; PubMed Central PMCID: PMC5427500.

36. Redemann S, Schloissnig S, Ernst S, Pozniakowsky A, Ayloo S, Hyman AA, et al. Codon adaptation-based control of protein expression in C. elegans. Nat Methods. 2011;8(3):250–2. Epub 2011/02/01. doi: 10.1038/nmeth.1565 21278743.

37. Chai Y, Li W, Feng G, Yang Y, Wang X, Ou G. Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development. Nature protocols. 2012;7(12):2090–102. Epub 2012/11/10. doi: 10.1038/nprot.2012.128 23138350.

38. Schild LC, Glauser DA. Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans. Genetics. 2015;200(4):1029–34. Epub 2015/05/30. doi: 10.1534/genetics.115.177956 26022242; PubMed Central PMCID: PMC4574232.

39. Wei X, Potter CJ, Luo L, Shen K. Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans. Nat Methods. 2012;9(4):391–5. Epub 2012/03/13. doi: 10.1038/nmeth.1929 22406855; PubMed Central PMCID: PMC3846601.

40. Evans TC. Transformation and microinjection. WormBook. 2006;10.

41. Glauser DA, Johnson BE, Aldrich RW, Goodman MB. Intragenic alternative splicing coordination is essential for Caenorhabditis elegans slo-1 gene function. Proc Natl Acad Sci U S A. 2011;108(51):20790–5. Epub 2011/11/16. doi: 10.1073/pnas.1116712108 22084100; PubMed Central PMCID: PMC3251113.

42. Marques F, Saro G, Lia AS, Poole RJ, Falquet L, Glauser DA. Identification of avoidance genes through neural pathway-specific forward optogenetics. PLoS Genet. 2019;15(12):e1008509. Epub 2020/01/01. doi: 10.1371/journal.pgen.1008509 31891575; PubMed Central PMCID: PMC6938339.

43. Nussbaum-Krammer CI, Neto MF, Brielmann RM, Pedersen JS, Morimoto RI. Investigating the spreading and toxicity of prion-like proteins using the metazoan model organism C. elegans. J Vis Exp. 2015;(95):52321–. doi: 10.3791/52321 25591151.


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