Affinity Proteomic Analysis of the Human Exosome and Its Cofactor Complexes

  • Kinga Winczura
  • Michal Domanski
  • John LaCavaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)


In humans, the RNA exosome consists of an enzymatically inactive nine-subunit core, with ribonucleolytic activity contributed by additional components. Several cofactor complexes also interact with the exosome—these enable the recruitment of, and specify the activity upon, diverse substrates. Affinity capture coupled with mass spectrometry has proven to be an effective means to identify the compositions of RNA exosomes and their cofactor complexes: here, we describe a general experimental strategy for proteomic characterization of macromolecular complexes, applied to the exosome and an affiliated adapter protein, ZC3H18.

Key words

Exosome Cofactors Affinity capture Mass spectrometry Affinity proteomics 



This work is supported in part by National Institutes of Health grant R01GM126170. We gratefully acknowledge Ms. Hua Jiang and Ms. Kelly Molloy for additional proofreading.


  1. 1.
    Mitchell P, Petfalski E, Shevchenko A, Mann M, Tollervey D (1997) The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′-->5′ exoribonucleases. Cell 91(4):457–466PubMedGoogle Scholar
  2. 2.
    Koonin EV, Wolf YI, Aravind L (2001) Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach. Genome Res 11(2):240–252PubMedPubMedCentralGoogle Scholar
  3. 3.
    Kilchert C, Wittmann S, Vasiljeva L (2016) The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol 17(4):227–239PubMedGoogle Scholar
  4. 4.
    Zinder JC, Lima CD (2017) Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev 31(2):88–100PubMedPubMedCentralGoogle Scholar
  5. 5.
    Ogami K, Chen Y, Manley JL (2018) RNA surveillance by the nuclear RNA exosome: mechanisms and significance. Noncoding RNA 4(1)Google Scholar
  6. 6.
    Schmid M, Jensen TH (2018) Controlling nuclear RNA levels. Nat Rev Genet 19(8):518–529PubMedGoogle Scholar
  7. 7.
    Allmang C, Petfalski E, Podtelejnikov A, Mann M, Tollervey D, Mitchell P (1999) The yeast exosome and human PM-Scl are related complexes of 3′ --> 5′ exonucleases. Genes Dev 13(16):2148–2158PubMedPubMedCentralGoogle Scholar
  8. 8.
    Dziembowski A, Lorentzen E, Conti E, Seraphin B (2007) A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol Biol 14(1):15–22PubMedGoogle Scholar
  9. 9.
    Lejeune F, Li X, Maquat LE (2003) Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol Cell 12(3):675–687PubMedGoogle Scholar
  10. 10.
    van Dijk EL, Schilders G, Pruijn GJ (2007) Human cell growth requires a functional cytoplasmic exosome, which is involved in various mRNA decay pathways. RNA 13(7):1027–1035PubMedPubMedCentralGoogle Scholar
  11. 11.
    Tomecki R, Kristiansen MS, Lykke-Andersen S, Chlebowski A, Larsen KM, Szczesny RJ, Drazkowska K, Pastula A, Andersen JS, Stepien PP, Dziembowski A, Jensen TH (2010) The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L. EMBO J 29(14):2342–2357PubMedPubMedCentralGoogle Scholar
  12. 12.
    Staals RH, Bronkhorst AW, Schilders G, Slomovic S, Schuster G, Heck AJ, Raijmakers R, Pruijn GJ (2010) Dis3-like 1: a novel exoribonuclease associated with the human exosome. EMBO J 29(14):2358–2367PubMedPubMedCentralGoogle Scholar
  13. 13.
    Lubas M, Christensen MS, Kristiansen MS, Domanski M, Falkenby LG, Lykke-Andersen S, Andersen JS, Dziembowski A, Jensen TH (2011) Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell 43(4):624–637PubMedGoogle Scholar
  14. 14.
    Meola N, Domanski M, Karadoulama E, Chen Y, Gentil C, Pultz D, Vitting-Seerup K, Lykke-Andersen S, Andersen JS, Sandelin A, Jensen TH (2016) Identification of a nuclear exosome decay pathway for processed transcripts. Mol Cell 64(3):520–533PubMedGoogle Scholar
  15. 15.
    Andersen PR, Domanski M, Kristiansen MS, Storvall H, Ntini E, Verheggen C, Schein A, Bunkenborg J, Poser I, Hallais M, Sandberg R, Hyman A, LaCava J, Rout MP, Andersen JS, Bertrand E, Jensen TH (2013) The human cap-binding complex is functionally connected to the nuclear RNA exosome. Nat Struct Mol Biol 20(12):1367–1376PubMedPubMedCentralGoogle Scholar
  16. 16.
    Lubas M, Andersen PR, Schein A, Dziembowski A, Kudla G, Jensen TH (2015) The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis. Cell Rep 10(2):178–192PubMedGoogle Scholar
  17. 17.
    Ogami K, Richard P, Chen Y, Hoque M, Li W, Moresco JJ, Yates JR 3rd, Tian B, Manley JL (2017) An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression. Genes Dev 31(12):1257–1271PubMedPubMedCentralGoogle Scholar
  18. 18.
    Domanski M, Upla P, Rice WJ, Molloy KR, Ketaren NE, Stokes DL, Jensen TH, Rout MP, LaCava J (2016) Purification and analysis of endogenous human RNA exosome complexes. RNA 22(9):1467–1475PubMedPubMedCentralGoogle Scholar
  19. 19.
    Winczura K, Schmid M, Iasillo C, Molloy KR, Harder LM, Andersen JS, LaCava J, Jensen TH (2018) Characterizing ZC3H18, a multi-domain protein at the Interface of RNA production and destruction decisions. Cell Rep 22(1):44–58PubMedPubMedCentralGoogle Scholar
  20. 20.
    Cristea IM, Williams R, Chait BT, Rout MP (2005) Fluorescent proteins as proteomic probes. Mol Cell Proteomics 4(12):1933–1941PubMedGoogle Scholar
  21. 21.
    Domanski M, Molloy K, Jiang H, Chait BT, Rout MP, Jensen TH, LaCava J (2012) Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. BioTechniques 0((0)):1–6PubMedPubMedCentralGoogle Scholar
  22. 22.
    LaCava J, Jiang H, Rout MP (2016) Protein complex affinity capture from Cryomilled mammalian cells. J Vis Exp 118Google Scholar
  23. 23.
    Oeffinger M, Wei KE, Rogers R, DeGrasse JA, Chait BT, Aitchison JD, Rout MP (2007) Comprehensive analysis of diverse ribonucleoprotein complexes. Nat Methods 4(11):951–956PubMedGoogle Scholar
  24. 24.
    Domanski M, LaCava J (2017) RNA degradation assay using RNA exosome complexes, affinity-purified from HEK-293 cells. Bio Protoc 7(8)Google Scholar
  25. 25.
    Hakhverdyan Z, Domanski M, Hough LE, Oroskar AA, Oroskar AR, Keegan S, Dilworth DJ, Molloy KR, Sherman V, Aitchison JD, Fenyo D, Chait BT, Jensen TH, Rout MP, LaCava J (2015) Rapid, optimized interactomic screening. Nat Methods 12(6):553–560PubMedPubMedCentralGoogle Scholar
  26. 26.
    Januszyk K, Liu Q, Lima CD (2011) Activities of human RRP6 and structure of the human RRP6 catalytic domain. RNA 17(8):1566–1577PubMedPubMedCentralGoogle Scholar
  27. 27.
    O’Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251(4999):1351–1355PubMedGoogle Scholar
  28. 28.
    Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, Eriksson E (1998) Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum Gene Ther 9(13):1939–1950PubMedGoogle Scholar
  29. 29.
    Helgason CD, Miller CL (2013) Basic cell culture protocols. Methods in molecular biology, vol 946, 4th edn. Humana Press, TotowaGoogle Scholar
  30. 30.
    Freshney RI (2015) Culture of animal cells: a manual of basic technique and specialized applications, 7th edn. Wiley-Blackwell, HobokenGoogle Scholar
  31. 31.
    Katzen F (2007) Gateway((R)) recombinational cloning: a biological operating system. Expert Opin Drug Discov 2(4):571–589PubMedGoogle Scholar
  32. 32.
    LaCava J, Molloy KR, Taylor MS, Domanski M, Chait BT, Rout MP (2015) Affinity proteomics to study endogenous protein complexes: pointers, pitfalls, preferences and perspectives. BioTechniques 58(3):103–119PubMedPubMedCentralGoogle Scholar
  33. 33.
    Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268(5218):1766–1769PubMedGoogle Scholar
  34. 34.
    Okerman L, Van Hende J, De Zutter L (2007) Stability of frozen stock solutions of beta-lactam antibiotics, cephalosporins, tetracyclines and quinolones used in antibiotic residue screening and antibiotic susceptibility testing. Anal Chim Acta 586(1–2):284–288PubMedGoogle Scholar
  35. 35.
  36. 36.
    Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A (2018) Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system. PLoS One 13(3):e0194887PubMedPubMedCentralGoogle Scholar
  37. 37.
    Taylor MS, LaCava J, Dai L, Mita P, Burns KH, Rout MP, Boeke JD (2016) Characterization of L1-Ribonucleoprotein particles. Methods Mol Biol 1400:311–338PubMedPubMedCentralGoogle Scholar
  38. 38.
    Domanski M, LaCava J (2017) Affinity purification of the RNA degradation complex, the exosome, from HEK-293 cells. Bio Protoc 7(8)Google Scholar
  39. 39.
    Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860PubMedGoogle Scholar
  40. 40.
    Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25(9):1327–1333PubMedGoogle Scholar
  41. 41.
    Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362PubMedGoogle Scholar
  42. 42.
    Wisniewski JR, Ostasiewicz P, Mann M (2011) High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J Proteome Res 10(7):3040–3049PubMedGoogle Scholar
  43. 43.
    Fischer R, Kessler BM (2015) Gel-aided sample preparation (GASP)—a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics 15(7):1224–1229PubMedPubMedCentralGoogle Scholar
  44. 44.
    Gillet LC, Leitner A, Aebersold R (2016) Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing. Annu Rev Anal Chem (Palo Alto, Calif) 9(1):449–472Google Scholar
  45. 45.
    Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11(12):2301–2319PubMedGoogle Scholar
  46. 46.
    Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740PubMedGoogle Scholar
  47. 47.
    Armean IM, Lilley KS, Trotter MW (2013) Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol Cell Proteomics 12(1):1–13PubMedGoogle Scholar
  48. 48.
    Greimann JC, Lima CD (2008) Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae cloning, expression, purification, and activity assays. Methods Enzymol 448:185–210PubMedPubMedCentralGoogle Scholar
  49. 49.
    Zinder JC, Wasmuth EV, Lima CD (2016) Nuclear RNA exosome at 3.1 a reveals substrate specificities, RNA paths, and allosteric inhibition of Rrp44/Dis3. Mol Cell 64(4):734–745PubMedPubMedCentralGoogle Scholar
  50. 50.
    Fernandez-Martinez J, LaCava J, Rout MP (2016) Density gradient ultracentrifugation to isolate endogenous protein complexes after affinity capture. Cold Spring Harb Protoc 2016(7)Google Scholar
  51. 51.
    Kraut A, Marcellin M, Adrait A, Kuhn L, Louwagie M, Kieffer-Jaquinod S, Lebert D, Masselon CD, Dupuis A, Bruley C, Jaquinod M, Garin J, Gallagher-Gambarelli M (2009) Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J Proteome Res 8(7):3778–3785PubMedGoogle Scholar
  52. 52.
    Sambrook J, Russell DW (2006) Preparation of denaturing polyacrylamide gels. CSH Protoc 2006(1)Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.School of Biosciences, College of Life and Environmental SciencesUniversity of BirminghamBirminghamUK
  2. 2.Department of Chemistry and BiochemistryUniversity of BernBernSwitzerland
  3. 3.Laboratory of Cellular and Structural BiologyThe Rockefeller UniversityNew YorkUSA
  4. 4.European Research Institute for the Biology of AgeingUniversity Medical Center GroningenGroningenThe Netherlands

Personalised recommendations