RNA Chaperones pp 107-120 | Cite as

Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function

  • Jennifer Porat
  • Mark A. BayfieldEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2106)


La proteins have well-established roles in the maturation of RNA polymerase III transcripts, including pre-tRNAs. In addition to protecting the 3′ end of pre-tRNAs from exonuclease digestion, La proteins also promote the native fold of the pre-tRNA using RNA chaperone activity. tRNA-mediated suppression in the fission yeast S. pombe has been an invaluable tool in determining the mechanistic basis by which La proteins promote the maturation of defective pre-tRNAs that benefit from RNA chaperone activity. More recently, tRNA-mediated suppression has been adapted to test for RNA chaperone function in the La-related proteins and in the promoting of tRNA function by tRNA modification enzymes. Thus tRNA-mediated suppression can be a useful assay for the investigation of various proteins hypothesized to promote tRNA folding through RNA chaperone related activities.

Key words

RNA chaperone assay Yeast tRNA processing Nonsense suppression Folding Northern blotting Lithium acetate transformation La and La-related proteins (LARPs) tRNA modifications 3′ end protection 



We thank R. Maraia, R. Intine, Y. Huang, A. Sakulich, S. Koduru, S. Hasson, A. Mozlin, J. Mazeika, R. Hussain, A. Vakiloroayaei, and A. Naeeni for their assistance in the development of tRNA-mediated suppression and its use in assessment of RNA chaperone function. We also thank R. Maraia for critical reading of the manuscript. This work is supported by a Discovery Grant from the Natural Science and Engineering Research Council (NSERC) of Canada to MA Bayfield and an NSERC CGS-M scholarship to J Porat.


  1. 1.
    Rajkowitsch L, Semrad K, Mayer O, Schroeder R (2005) Assays for the RNA chaperone activity of proteins. Biochem Soc Trans.
  2. 2.
    Clodi E, Semrad K, Schroeder R (1999) Assaying RNA chaperone activity in vivo using a novel RNA folding trap. EMBO J.
  3. 3.
    Phadtare S, Severinov K, Inouye M (2003) Assay of transcription antitermination by proteins of the CspA family. Methods Enzymol.
  4. 4.
    Yoo CJ, Wolin SL (1997) The yeast La protein is required for the 3′ endonucleolytic cleavage that matures tRNA precursors. Cell.
  5. 5.
    Naeeni AR, Conte MR, Bayfield MA (2012) RNA chaperone activity of human La protein is mediated by variant RNA recognition motif. J Biol Chem.
  6. 6.
    Huang Y, Bayfield MA, Intine RV, Maraia RJ (2006) Separate RNA-binding surfaces on the multifunctional la protein mediate distinguishable activities in tRNA maturation. Nat Struct Mol Biol 13:611–618CrossRefGoogle Scholar
  7. 7.
    Kucera NJ, Hodsdon ME, Wolin SL (2011) An intrinsically disordered C terminus allows the La protein to assist the biogenesis of diverse noncoding RNA precursors. Proc Natl Acad Sci U S A 108:1308–1313CrossRefGoogle Scholar
  8. 8.
    Kuehnert J, Sommer G, Zierk AW, Fedarovich A, Brock A, Fedarovich D, Heise T (2015) Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation. Nucleic Acids Res 43:581–594CrossRefGoogle Scholar
  9. 9.
    Costa-Mattioli M, Svitkin Y, Sonenberg N (2004) La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro. Mol Cell Biol.
  10. 10.
    Vakiloroayaei A, Shah NS, Oeffinger M, Bayfield MA (2017) The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets. Nucleic Acids Res 45:11341–11355CrossRefGoogle Scholar
  11. 11.
    Kohli J, Kwong T, Altruda F, Söll D, Wahl G (1979) Characterization of a UGA-suppressing serine tRNA from Schizosaccharomyces pombe with the help of a new in vitro assay system for eukaryotic suppressor tRNAs. J Biol Chem 254(5):1546–1551PubMedGoogle Scholar
  12. 12.
    Rijal K, Maraia RJ, Arimbasseri AG (2015) A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis. Gene.
  13. 13.
    Koukuntla R, Ramsey WJ, Young WB, Link CJ (2013) U6 promoter-enhanced GlnUAG suppressor tRNA has higher suppression efficacy and can be stably expressed in 293 cells. J Gene Med.
  14. 14.
    Szankasi P, Heyer WD, Schuchert P, Kohli J (1988) DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination hot spot allele ade6-M26. J Mol Biol.
  15. 15.
    Hussain RH, Zawawi M, Bayfield MA (2013) Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7. Nucleic Acids Res.
  16. 16.
    Hamada M, Sakulich AL, Koduru SB, Maraia RJ (2000) Transcription termination by RNA polymerase III in fission yeast. A genetic and biochemically tractable model system. J Biol Chem 275:29076–29081CrossRefGoogle Scholar
  17. 17.
    Intine RV, Sakulich AL, Koduru SB, Huang Y, Pierstorff E, Goodier JL, Phan L, Maraia RJ (2000) Control of transfer RNA maturation by phosphorylation of the human La antigen on serine 366. Mol Cell 6:339–348CrossRefGoogle Scholar
  18. 18.
    Van Horn DJ, Yoo CJ, Xue D, Shi H, Wolin SL (1997) The La protein in Schizosaccharomyces pombe: a conserved yet dispensable phosphoprotein that functions in tRNA maturation. RNA (New York, NY).
  19. 19.
    Arimbasseri AG, Blewett NH, Iben JR, Lamichhane TN, Cherkasova V, Hafner M, Maraia RJ (2015) RNA polymerase III output is functionally linked to tRNA dimethyl-G26 modification. PLoS Genet 11:e1005671CrossRefGoogle Scholar
  20. 20.
    Maundrell K (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127–130CrossRefGoogle Scholar
  21. 21.
    Keeney JB, Boeke JD (1994) Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136:849–856PubMedPubMedCentralGoogle Scholar
  22. 22.
    Wetzel R, Kohli J, Altruda F, Söll D (1979) Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe. Mol Gen Genet 172:221–228CrossRefGoogle Scholar
  23. 23.
    Hottinger H, Stadelmann B, Pearson D, Frendewey D, Kohli J, Söll D (1984) The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo. EMBO J 3:423–428CrossRefGoogle Scholar
  24. 24.
    Niederberger C, Gräub R, Costa A, Desgrès J, Schweingruber ME (1999) The tRNA N2,N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe. FEBS Lett 464:67–70CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of BiologyYork UniversityTorontoCanada

Personalised recommendations