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Probing RNA–Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation

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RNA Spectroscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2113))

Abstract

Recent advances in multi-wavelength analytical ultracentrifugation (MWL-AUC) combine the power of an exquisitely sensitive hydrodynamic-based separation technique with the added dimension of spectral separation. This added dimension has opened up new doors to much improved characterization of multiple, interacting species in solution. When applied to structural investigations of RNA, MWL-AUC can precisely report on the hydrodynamic radius and the overall shape of an RNA molecule by enabling precise measurements of its sedimentation and diffusion coefficients and identify the stoichiometry of interacting components based on spectral decomposition. Information provided in this chapter will allow an investigator to design experiments for probing ion and/or protein-induced global conformational changes of an RNA molecule and exploit spectral differences between proteins and RNA to characterize their interactions in a physiological solution environment.

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References

  1. Gesteland RF, Cech TR, Atkins JF (2006) The RNA world, vol 43. Cold Spring Harbor Press, New York

    Google Scholar 

  2. Fresco JR (1998) RNA structure and function, vol 35. Cold Spring Harbor Press, New York

    Google Scholar 

  3. Dethoff EA, Chugh J, Mustoe AM, Al-Hashimi HM (2012) Functional complexity and regulation through RNA dynamics. Nature 482(7385):322–330

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Mustoe AM, Brooks CL, Al-Hashimi HM (2014) Hierarchy of RNA functional dynamics. Annu Rev Biochem 83:441–466

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Mitra S (2009) Using analytical ultracentrifugation (AUC) to measure global conformational changes accompanying equilibrium tertiary folding of RNA molecules. Methods Enzymol 469:209–236

    CAS  PubMed  Google Scholar 

  6. Brautigam CA, Wakeman CA, Winkler WC (2009) Methods for analysis of ligand-induced RNA conformational changes. Methods Mol Biol 540:77–95

    CAS  PubMed  Google Scholar 

  7. Mitra S (2014) Detecting RNA tertiary folding by sedimentation velocity analytical ultracentrifugation. Methods Mol Biol 1086:265–288

    CAS  PubMed  Google Scholar 

  8. Chaires JB, Dean WL, Le HT, Trent JO (2015) Hydrodynamic models of G-Quadruplex structures. Methods Enzymol 562:287–304

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kieft JS, Costantino DA, Filbin ME, Hammond J, Pfingsten JS (2007) Structural methods for studying IRES function. Methods Enzymol 430:333–371

    CAS  PubMed  Google Scholar 

  10. Takamoto K, He Q, Morris S, Chance MR, Brenowitz M (2002) Monovalent cations mediate formation of native tertiary structure of the Tetrahymena thermophila ribozyme. Nat Struct Biol 9(12):928–933

    CAS  PubMed  Google Scholar 

  11. Chillon I, Marcia M, Legiewicz M, Liu F, Somarowthu S, Pyle AM (2015) Native purification and analysis of long RNAs. Methods Enzymol 558:3–37

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang X, Xi W, Toomey S, Chiang YC, Hasek J, Laue TM, Denis CL (2016) Stoichiometry and change of the mRNA closed-loop factors as translating ribosomes transit from initiation to elongation. PLoS One 11(3):e0150616

    PubMed  PubMed Central  Google Scholar 

  13. Luque D, Mata CP, Gonzalez-Camacho F, Gonzalez JM, Gomez-Blanco J, Alfonso C, Rivas G, Havens WM, Kanematsu S, Suzuki N, Ghabrial SA, Trus BL, Caston JR (2016) Heterodimers as the structural unit of the T=1 capsid of the fungal double-stranded RNA Rosellinia necatrix quadrivirus 1. J Virol 90(24):11220–11230

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Patel TR, Chojnowski G, Astha KA, McKenna SA, Bujnicki JM (2017) Structural studies of RNA-protein complexes: a hybrid approach involving hydrodynamics, scattering, and computational methods. Methods 118-119:146–162

    CAS  PubMed  Google Scholar 

  15. Zhang J, Pearson JZ, Gorbet GE, Colfen H, Germann MW, Brinton MA, Demeler B (2017) Spectral and hydrodynamic analysis of West Nile virus RNA-protein interactions by multiwavelength sedimentation velocity in the analytical ultracentrifuge. Anal Chem 89(1):862–870

    CAS  PubMed  Google Scholar 

  16. Wong CJ, Launer-Felty K, Cole JL (2011) Analysis of PKR-RNA interactions by sedimentation velocity. Methods Enzymol 488:59–79. https://doi.org/10.1016/B978-0-12-381268-1.00003-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Berke IC, Modis Y (2012) MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA. EMBO J 31(7):1714–1726

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Mayo CB, Wong CJ, Lopez PE, Lary JW, Cole JL (2016) Activation of PKR by short stem-loop RNAs containing single-stranded arms. RNA 22(7):1065–1075

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Pearson JZ, Krause F, Haffke D, Demeler B, Schilling K, Colfen H (2015) Next-generation AUC adds a spectral dimension: development of multiwavelength detectors for the analytical ultracentrifuge. Methods Enzymol 562:1–26

    CAS  PubMed  Google Scholar 

  20. Pearson J, Walter J, Peukert W, Colfen H (2018) Advanced multiwavelength detection in analytical ultracentrifugation. Anal Chem 90(2):1280–1291

    CAS  PubMed  Google Scholar 

  21. Colfen H, Laue TM, Wohlleben W, Schilling K, Karabudak E, Langhorst BW, Brookes E, Dubbs B, Zollars D, Rocco M, Demeler B (2010) The open AUC project. Eur Biophys J 39(3):347–359

    PubMed  Google Scholar 

  22. Gorbet GE, Pearson JZ, Demeler AK, Colfen H, Demeler B (2015) Next-generation AUC: analysis of multiwavelength analytical ultracentrifugation data. Methods Enzymol 562:27–47

    CAS  PubMed  Google Scholar 

  23. Johnson CN, Gorbet GE, Ramsower H, Urquidi J, Brancaleon L, Demeler B (2018) Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin. Eur Biophys J 47(7):789–797

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Demeler B, Gorbet GE (2016) Analytical ultracentrifugation data analysis with UltraScan-III. In: Uchiyama S, Arisaka F, Stafford W, Laue T (eds) Analytical ultracentrifugation. Springer, Cham, pp 119–143

    Google Scholar 

  25. Byron O, Nischang I, Patel TR (2018) European biophysics journal. In: Byron O, Nischang I, Patel TR (eds) Special issue: 23rd international analytical ultracentrifugation workshop and symposium, AUC 2017, vol 693. Springer International Publishing, Cham

    Google Scholar 

  26. Fujita H (1975) Foundations of ultracentrifugal analysis. Wiley, New York

    Google Scholar 

  27. Philo JS (2000) A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions. Anal Biochem 279(2):151–163

    CAS  PubMed  Google Scholar 

  28. Stafford WF 3rd (1994) Boundary analysis in sedimentation velocity experiments. Methods Enzymol 240:478–501

    PubMed  Google Scholar 

  29. Laue TM, Stafford WF 3rd (1999) Modern applications of analytical ultracentrifugation. Annu Rev Biophys Biomol Struct 28:75–100

    CAS  PubMed  Google Scholar 

  30. Stafford WF 3rd (1992) Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 203(2):295–301

    CAS  PubMed  Google Scholar 

  31. Correia JJ, Stafford WF (2015) Sedimentation velocity: a classical perspective. Methods Enzymol 562:49–80

    CAS  PubMed  Google Scholar 

  32. Costantino D, Kieft JS (2005) A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs. RNA 11(3):332–343

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tanford C (1961) Physical chemistry of macromolecules. Wiley, New York

    Google Scholar 

  34. Cantor CR, Schimmel PR (1980) Ultracentrifugation. In: Bartlett AC (ed) Biophysical chemistry, Part II: techniques for the study of biological structure and function, vol II. W.H. Freeman and Company, San Francisco

    Google Scholar 

  35. Scott DJ, Schuck P (2005) A brief introduction to the analytical ultracentrifugation of proteins for beginners. Analytical ultracentrifugation: techniques and methods. Royal Society of Chemistry, Cambridge, UK

    Google Scholar 

  36. Uchiyama SA (2016) Important and essential theoretical aspects of AUC. Analytical ultracentrifugation. Springer, Tokyo

    Google Scholar 

  37. Demeler B, Brookes E, Wang R, Schirf V, Kim CA (2010) Characterization of reversible associations by sedimentation velocity with UltraScan. Macromol Biosci 10(7):775–782

    CAS  PubMed  Google Scholar 

  38. MacGregor IK, Anderson AL, Laue TM (2004) Fluorescence detection for the XLI analytical ultracentrifuge. Biophys Chem 108(1–3):165–185

    CAS  PubMed  Google Scholar 

  39. Lawson CLH, Hanson RJ (1974) Solving least squares problems. Automatic computation. Prentice-Hall, Englewood Cliffs

    Google Scholar 

  40. Brookes E, Cao W, Demeler B (2010) A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur Biophys J 39(3):405–414

    PubMed  Google Scholar 

  41. Brookes E, Demeler B (2007) Parsimonious regularization using genetic algorithms applied to the analysis of analytical ultracentrifugation experiments. In: GECCO ACM proceedings of the 9th annual conference on genetic and evolutionary computation, pp 361–368

    Google Scholar 

  42. Demeler B, Brookes E (2008) Monte Carlo analysis of sedimentation experiments. Colloid Polym Sci 268(2):129–137

    Google Scholar 

  43. Beckert B, Masquida B (2011) Synthesis of RNA by in vitro transcription. Methods Mol Biol 703:29–41

    CAS  PubMed  Google Scholar 

  44. Shcherbakova I, Gupta S, Chance M, Brenowitz M (2004) Monovalent ion-mediated folding of the Tetrahymena thermophila ribozyme. J Mol Biol 342(5):1431–1442

    CAS  PubMed  Google Scholar 

  45. Mitra S, Laederach A, Golden BL, Altman RB, Brenowitz M (2011) RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways. RNA 17(8):1589–1603

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Shcherbakova I, Mitra S (2009) Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure. Methods Enzymol 468:31–46

    CAS  PubMed  Google Scholar 

  47. Kwok L, Shcherbakova I, Lamb J, Park H, Andresen K, Smith H, Brenowitz M, Pollack L (2006) Concordant exploration of the kinetics of RNA folding from global and local perspectives. J Mol Biol 355(2):282–293

    CAS  PubMed  Google Scholar 

  48. Williams TL, Gorbet GE, Demeler B (2018) Multi-speed sedimentation velocity simulations with UltraScan-III. Eur Biophys J 47(7):815–823

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Cao W, Demeler B (2008) Modeling analytical ultracentrifugation experiments with an adaptive space-time finite element solution for multicomponent reacting systems. Biophys J 95(1):54–65

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Cao W, Demeler B (2005) Modeling analytical ultracentrifugation experiments with an adaptive space-time finite element solution of the Lamm equation. Biophys J 89(3):1589–1602

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Philo JS (2006) Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques. Anal Biochem 354(2):238–246

    CAS  PubMed  Google Scholar 

  52. Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320(1):104–124

    CAS  PubMed  Google Scholar 

  53. Brautigam CA (2011) Using Lamm-equation modeling of sedimentation velocity data to determine the kinetic and thermodynamic properties of macromolecular interactions. Methods 54(1):4–15

    CAS  PubMed  Google Scholar 

  54. Behlke J, Ristau O (1997) Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles. Biophys J 72(1):428–434

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Philo JS (1997) An improved function for fitting sedimentation velocity data for low-molecular-weight solutes. Biophys J 72(1):435–444

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Schuck P, MacPhee CE, Howlett GJ (1998) Determination of sedimentation coefficients for small peptides. Biophys J 74(1):466–474

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Brown PH, Schuck P (2008) A new adaptive grid-size algorithm for the simulation of sedimentation velocity profiles in analytical ultracentrifugation. Comput Phys Commun 178(2):105–120

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Sherwood PJ Stafford WF (2016) SEDANAL: model-dependent and model-independent analysis of sedimentation data. In: Uchiyama S, Arisaka F, Stafford W, Laue T (eds) Analytical ultracentrifugation. Springer, Tokyo, pp 81–102

    Google Scholar 

  59. Memon S, Riedel M, Janetzko F, Demeler B, Gorbet G, Marru S, Grimshaw A, Gunathilake L, Singh R, Attig N, Lippert T (2014) Advancements of the UltraScan scientific gateway for open standards-based cyberinfrastructures. Concurr Comput Pract Exp 26(13):2280–2291

    Google Scholar 

  60. Pierce M, Marru S, Demeler B, Singh R, Gorbet G (2014) The apache airavata application programming interface: overview and evaluation with the UltraScan science gateway. In: 9th gateway computing environments workshop (GCE 2014), New Orleans, LA, USA, 2014. IEEE Press, Piscataway

    Google Scholar 

  61. Kieft JS, Batey RT (2004) A general method for rapid and nondenaturing purification of RNAs. RNA 10(6):988–995

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Takamoto K, Das R, He Q, Doniach S, Brenowitz M, Herschlag D, Chance M (2004) Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations. J Mol Biol 343(5):1195–1206

    CAS  PubMed  Google Scholar 

  63. Sclavi B, Sullivan M, Chance MR, Brenowitz M, Woodson SA (1998) RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279(5358):1940–1943

    CAS  PubMed  Google Scholar 

  64. Chauhan S, Woodson SA (2008) Tertiary interactions determine the accuracy of RNA folding. J Am Chem Soc 130(4):1296–1303

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Nelson TG, Ramsay GD, Perugini MA (2016) Fluorescence detection system. In: Uchiyama S, Arisaka F, Stafford W, Laue T (eds) Analytical ultracentrifugation. Springer, Tokyo, pp 39–61

    Google Scholar 

  66. Pan J, Woodson SA (1998) Folding intermediates of a self-splicing RNA: mispairing of the catalytic core. J Mol Biol 280(4):597–609

    CAS  PubMed  Google Scholar 

  67. Wilkinson KA, Merino EJ, Weeks KM (2006) Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1(3):1610–1616

    CAS  PubMed  Google Scholar 

  68. Grohman J, Del Campo M, Bhaskaran H, Tijerina P, Lambowitz A, Russell R (2007) Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA. Biochemistry 46(11):3013–3022

    CAS  PubMed  Google Scholar 

  69. Carey MF, Peterson CL, Smale ST (2013) The RNase protection assay. Cold Spring Harb Protoc 2013(3):pdb.prot071910

    PubMed  Google Scholar 

  70. Herschlag D, Cech TR (1990) Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry 29(44):10159–10171

    CAS  PubMed  Google Scholar 

  71. Russell R, Das R, Suh H, Travers KJ, Laederach A, Engelhardt MA, Herschlag D (2006) The paradoxical behavior of a highly structured misfolded intermediate in RNA folding. J Mol Biol 363(2):531–544

    CAS  PubMed  Google Scholar 

  72. Wan Y, Mitchell D 3rd, Russell R (2009) Catalytic activity as a probe of native RNA folding. Methods Enzymol 468:195–218

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Mitra S, Brenowitz M (2008) Metal ions and RNA folding kinetics. In: Hud NV (ed) Nucleic-acid metal ion interactions. Royal Society of Chemistry, Cambridge, pp 221–265

    Google Scholar 

  74. Hud NV (2008) Nucleic acid-metal ion interactions. Royal Society of Chemistry, Cambridge

    Google Scholar 

  75. Chen C, Mitra S, Jonikas M, Martin J, Brenowitz M, Laederach A (2013) Understanding the role of three-dimensional topology in determining the folding intermediates of group I introns. Biophys J 104(6):1326–1337

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

S.M. is grateful to the Chemistry Department of New York University to host him as a faculty during the preparation of this manuscript. The Twort intron work described here was originally funded by 1RO1-GM085130 from the National Institute of General Medical Sciences of the National Institutes of Health to Prof. Michael D. Brenowitz at the Albert Einstein College of Medicine. B.D. wishes to credit NIH-NIGMS grant RO1-120600 and the Canada Research Chairs program for financial support of this work.

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Authors and Affiliations

  1. Department of Chemistry, New York University, New York, NY, USA

    Somdeb Mitra

  2. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada

    Borries Demeler

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  1. Somdeb Mitra
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  2. Borries Demeler
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Correspondence to Somdeb Mitra .

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Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

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Mitra, S., Demeler, B. (2020). Probing RNA–Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_19

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  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_19

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