In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes

  • Alexandra Zinoviev
  • Christopher U. T. Hellen
  • Tatyana V. PestovaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)


The RNA exosome is a multisubunit protein complex that exhibits a 3′ to 5′ exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.

Key words

RNA exosome Cap labeling of mRNA In vitro reconstitution of mammalian ribosomal complexes Toeprinting GTPBP1 mRNA degradation assay 



This work was supported by NIH grant GM80623 to T.V.P. and NIH grant AI123406 to C.U.T.H.


  1. 1.
    Parker R (2012) RNA degradation in Saccharomyces cerevisiae. Genetics 191:671–702PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Chlebowski A, Lubas M, Jensen TH, Dziembowski A (2013) RNA decay machines: the exosome. Biochim Biophys Acta 1829:552–560PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Łabno A, Tomecki R, Dziembowski A (2016) Cytoplasmic RNA decay pathways - enzymes and mechanisms. Biochim Biophys Acta 1863:3125–3147PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Zinder JC, Lima CD (2017) Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev 31:88–100PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Lykke-Andersen S, Tomecki R, Jensen TH, Dziembowski A (2011) The eukaryotic RNA exosome: same scaffold but variable catalytic subunits. RNA Biol 8:61–66PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Chen CY, Gherzi R, Ong SE, Chan EL, Raijmakers R, Pruijn GJ, Stoecklin G, Moroni C, Mann M, Karin M (2001) AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107:451–464CrossRefGoogle Scholar
  7. 7.
    Tran H, Schilling M, Wirbelauer C, Hess D, Nagamine Y (2004) Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU. Mol Cell 13:101–111PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Gherzi R, Lee KY, Briata P, Wegmüller D, Moroni C, Karin M, Chen CY (2004) A KH domain RNA binding protein, KSRP, promotes ARE-directed mRNA turnover by recruiting the degradation machinery. Mol Cell 14:571–583PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Anderson JS, Parker RP (1998) The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex. EMBO J 17:1497–1506PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Araki Y, Takahashi S, Kobayashi T, Kajiho H, Hoshino S, Katada T (2001) Ski7p G protein interacts with the exosome and the ski complex for 3'-to-5' mRNA decay in yeast. EMBO J 20:4684–4693PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Halbach F, Reichelt P, Rode M, Conti E (2013) The yeast ski complex: crystal structure and RNA channeling to the exosome complex. Cell 154:814–826PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    van Hoof A, Staples RR, Baker RE, Parker R (2000) Function of the Ski4p (Csl4p) and Ski7p proteins in 3'- to-5' degradation of mRNA. Mol Cell Biol 20:8230–8243PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Kowalinski E, Schuller A, Green R, Conti E (2015) Saccharomyces cerevisiae Ski7 is a GTP-binding protein adopting the characteristic conformation of active translational GTPases. Structure 23:1336–1343PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Kowalinski E, Kögel A, Ebert J, Reichelt P, Stegmann E, Habermann B, Conti E (2016) Structure of a cytoplasmic 11-subunit RNA exosome complex. Mol Cell 63:125–134PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Liu JJ, Niu CY, Wu Y, Tan D, Wang Y, Ye MD, Liu Y, Zhao W, Zhou K, Liu QS, Dai J, Yang X, Dong MQ, Huang N, Wang HW (2016) CryoEM structure of yeast cytoplasmic exosome complex. Cell Res 26:822–837PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Kalisiak K, Kuliński TM, Tomecki R, Cysewski D, Pietras Z, Chlebowski A, Kowalska K, Dziembowski A (2017) A short splicing isoform of HBS1L links the cytoplasmic exosome and SKI complexes in humans. Nucleic Acids Res 45:2068–2080PubMedPubMedCentralGoogle Scholar
  17. 17.
    Mitchell P, Tollervey D (2003) An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation. Mol Cell 11:1405–1413PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Doma MK, Parker R (2006) Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440:561–564PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Frischmeyer PA, van Hoof A, O’Donnell K, Guerrerio AL, Parker R, Dietz HC (2002) An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295:2258–2261PubMedCrossRefGoogle Scholar
  20. 20.
    van Hoof A, Frischmeyer PA, Dietz HC, Parker R (2002) Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science 295:2262–2264PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Schmidt C, Kowalinski E, Shanmuganathan V, Defenouillère Q, Braunger K, Heuer A, Pech M, Namane A, Berninghausen O, Fromont-Racine M, Jacquier A, Conti E, Becker T, Beckmann R (2016) The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Science 354:1431–1433PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Woo KC, Kim TD, Lee KH, Kim DY, Kim S, Lee HR, Kang HJ, Chung SJ, Senju S, Nishimura Y, Kim KT (2011) Modulation of exosome-mediated mRNA turnover by interaction of GTP-binding protein 1 (GTPBP1) with its target mRNAs. FASEB J 25:2757–2769PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Atkinson GC (2015) The evolutionary and functional diversity of classical and lesser-known cytoplasmic and organellar translational GTPases across the tree of life. BMC Genomics 16:78PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Zinoviev A, Goyal A, Jindal S, LaCava J, Komar AA, Rodnina MV, Hellen CUT, Pestova TV (2018) Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2. Genes Dev 32:1226–1241PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    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 0:1–6PubMedPubMedCentralGoogle Scholar
  26. 26.
    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 22:1467–1475PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Pisarev AV, Unbehaun A, Hellen CU, Pestova TV (2007) Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol 430:147–177PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Kolupaeva VG, de Breyne S, Pestova TV, Hellen CU (2007) In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry. Methods Enzymol 430:409–439PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Pestova TV, Hellen CU (2005) Reconstitution of eukaryotic translation elongation in vitro following initiation by internal ribosomal entry. Methods 36:261–269PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Pestova TV, Borukhov SI, Hellen CU (1998) Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature 394:854–859PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Pestova TV, Hellen CU, Shatsky IN (1996) Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol 16:6859–6869PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Lomakin IB, Hellen CU, Pestova TV (2000) Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol Cell Biol 20:6019–6029PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU (2000) The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403:332–335PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Lomakin IB, Shirokikh NE, Yusupov MM, Hellen CU, Pestova TV (2006) The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH. EMBO J 25:196–210PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Bullard JM, Cai YC, Demeler B, Spremulli LL (1999) Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase. J Mol Biol 288:567–577PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Pestova TV, Hellen CU (2001) Preparation and activity of synthetic unmodified mammalian tRNAi(met) in initiation of translation in vitro. RNA 7:1496–1505PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Pestova TV, Hellen CU (2003) Translation elongation after assembly of ribosomes on the cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. Genes Dev 17:181–186PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Unbehaun A, Borukhov SI, Hellen CU, Pestova TV (2004) Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP. Genes Dev 18:3078–3093PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Kolupaeva VG, Unbehaun A, Lomakin IB, Hellen CU, Pestova TV (2005) Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association. RNA 11:470–486PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Yu Y, Ji H, Doudna JA, Leary JA (2005) Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes. Protein Sci 14:1438–1446PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Abaeva IS, Pestova TV, Hellen CU (2016) Attachment of ribosomal complexes and retrograde scanning during initiation on the Halastavi árva virus IRES. Nucleic Acids Res 44:2362–2377PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Raychaudhuri P, Maitra U (1986) Identification of ribosome-bound eukaryotic initiation factor 2.GDP binary complex as an intermediate in polypeptide chain initiation reaction. J Biol Chem 261:7723–7728PubMedPubMedCentralGoogle Scholar
  43. 43.
    Alkalaeva EZ, Pisarev AV, Frolova LY, Kisselev LL, Pestova TV (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell 125:1125–1136PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Anthony DD, Merrick WC (1992) Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J Biol Chem 1267:1554–1562Google Scholar
  45. 45.
    Schenborn ET, Mierendorf RC Jr (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res 13:6223–6236PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Chamberlin M, Ring J (1973) Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem 248:2235–2244PubMedPubMedCentralGoogle Scholar
  47. 47.
    van Houten V, Denkers F, van Dijk M, van den Brekel M, Brakenhoff R (1998) Labeling efficiency of oligonucleotides by T4 polynucleotide kinase depends on 5'-nucleotide. Anal Biochem 265:386–389PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Alexandra Zinoviev
    • 1
  • Christopher U. T. Hellen
    • 1
  • Tatyana V. Pestova
    • 1
    Email author
  1. 1.Department of Cell BiologySUNY Downstate Health Sciences UniversityBrooklynUSA

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