JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Published: October 8th, 2018

DOI:

10.3791/58100

1Department of Biology, Ball State University, 2Department of Genome Sciences, University of Washington, 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan
* These authors contributed equally

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

At least 163 distinct posttranscriptional RNA modifications have been identified, with these modifications conferring diverse and context-dependent functions to RNAs, directly influencing RNA structure, and affecting interactions of RNA with other molecules1,2. As the appreciation for the number and variety of RNA modifications increases, it is critical to develop assays that can reliably interrogate both the RNA modifications and the enzymes that catalyze them.

One of the first RNA modifications to be identified occurs at base 37 in tRNAs, adjacent to the anti-codon on the 3' s....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NOTE: The protocol was adapted from Ref. 24.

1. Obtain RNA and Enzyme of Interest

  1. Use in vitro transcribed RNAs internally labeled with 32P, and recombinant His6-Mod5 expressed in and purified from E. coli, as previously described24.
    1. Introduce in vitro transcribed RNAs (section 3) using T7 RNA polymerase in the presence of unlabeled ATP, UTP, CTP, GTP and 10 µCi of gel-purified, e.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Mod5 was incubated with a tyrosine tRNA or serine tRNA in the presence or absence of DMAPP. Following the modification reaction, products were RNase T1-digested, which cleaves the 3' end of all guanosines leaving a 3' guanosine monophosphates (GMP)24 (Figure 1). Full digestion of the RNAs produces a predictable pattern of radiolabeled fragments (Figure 2A), which are then resolved .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

RNA modifications continue to be shown to play ever more important and diverse roles in cellular and organismal function. As such, the development of assays to interrogate RNA modifying enzymes is central to better understanding the fundamental aspects of biology. This protocol describes a high-resolution in vitro assay to characterize the tRNA modification activity of Mod5.

This protocol has the distinct advantage of providing a direct, and easily interpretable readout of isopentenyl.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We would like to thank Dr. David Engelke for his guidance and helpful comments on this manuscript. PJS - Ball State University laboratory startup funds; DAB - grant 1R15AI130950-01.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Reagents
UreaGel Concentrate National Diagnostics EC-833 As part of a kit
UreaGel Diluent National Diagnostics EC-833 As part of a kit
UreaGel Buffer National Diagnostics EC-833 As part of a kit
10x TBE National Diagnostics EC-833 As part of a kit
Ammonium persulfate (APS) Sigma-Aldrich 7727-54-0
N,N,N′,N′-Tetramethylethylenediamine (TMED) Sigma-Aldrich T9281
Tris base Sigma-Aldrich T1503
Boric acid Sigma-Aldrich 10043-35-3
EDTA Sigma-Aldrich 60-00-4
ATP Sigma-Aldrich 34369-07-8
MgCl2 Sigma-Aldrich 7786-30-3
DMAPP Caymen Chemical 1186-30-7
Super RNaseIN ThermoFisher Scientific AM2694
2-mercaptoethanol Sigma-Aldrich 60-24-2
Ethanol Sigma-Aldrich 64-17-5
Sodiume acetate Sigma-Aldrich 127-09-3
Rnase T1 ThermoFisher Scientific EN0541
Glycerol Sigma-Aldrich 56-81-5
Xylene cyanol Sigma-Aldrich 2650-17-1
Equipment and Supplies
Short glass plates (20-40 cm W x 40 cm L) The Gel Company
Long glass plates (20-40 cm W x 40 cm L) The Gel Company
Vertical gel apparatus The Gel Company S2-3040
50 mL disposable syringe Fisher Scientific 03-377-26
Stainless steel binder clips Idea Scientific 1066
Phosphoscreen Sigma-Aldrich 28-9564-74
Plastic wrap (local grocery store)

  1. Boccaletto, P., et al. MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Research. 46 (D1), 303-307 (2018).
  2. Lewis, C. J., Pan, T., Kalsotra, A. RNA modifications and structures cooperate to guide RNA-protein interactions. Nature Reviews: Molecular and Cellular Biology. 18 (3), 202-210 (2017).
  3. Soll, D. Enzymatic modification of transfer RNA. Science. 173 (3994), 293-299 (1971).
  4. Schweizer, U., Bohleber, S., Fradejas-Villar, N. The modified base isopentenyladenosine and its derivatives in tRNA. RNA Biology. 14 (9), 1197-1208 (2017).
  5. Persson, B. C., Esberg, B., Olafsson, O., Bjork, G. R. Synthesis and function of isopentenyl adenosine derivatives in tRNA. Biochimie. 76 (12), 1152-1160 (1994).
  6. Urbonavicius, J., Qian, Q., Durand, J. M., Hagervall, T. G., Bjork, G. R. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO Journal. 20 (17), 4863-4873 (2001).
  7. Aubee, J. I., Olu, M., Thompson, K. M. The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation. RNA. 22 (5), 729-742 (2016).
  8. Caillet, J., Droogmans, L. Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA. Journal of Bacteriology. 170 (9), 4147-4152 (1988).
  9. Soderberg, T., Poulter, C. D. Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop. Biochemistry. 39 (21), 6546-6553 (2000).
  10. Dihanich, M. E., et al. Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae. Molecular and Cellular Biology. 7 (1), 177-184 (1987).
  11. Lemieux, J., et al. Regulation of physiological rates in Caenorhabditis elegans by a tRNA-modifying enzyme in the mitochondria. Genetics. 159 (1), 147-157 (2001).
  12. Golovko, A., Sitbon, F., Tillberg, E., Nicander, B. Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana. Plant Molecular Biology. 49 (2), 161-169 (2002).
  13. Warner, G. J., Rusconi, C. P., White, I. E., Faust, J. R. Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells. Nucleic Acids Research. 26 (23), 5533-5535 (1998).
  14. Golovko, A., Hjalm, G., Sitbon, F., Nicander, B. Cloning of a human tRNA isopentenyl transferase. Gene. 258 (1-2), 85-93 (2000).
  15. Kernohan, K. D., et al. Matchmaking facilitates the diagnosis of an autosomal-recessive mitochondrial disease caused by biallelic mutation of the tRNA isopentenyltransferase (TRIT1) gene. Human Mutations. 38 (5), 511-516 (2017).
  16. Yarham, J. W., et al. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA. PLoS Genetics. 10 (6), 1004424 (2014).
  17. Smaldino, P. J., Read, D. F., Pratt-Hyatt, M., Hopper, A. K., Engelke, D. R. The cytoplasmic and nuclear populations of the eukaryote tRNA-isopentenyl transferase have distinct functions with implications in human cancer. Gene. 556 (1), 13-18 (2015).
  18. Lamichhane, T. N., Mattijssen, S., Maraia, R. J. Human cells have a limited set of tRNA anticodon loop substrates of the tRNA isopentenyltransferase TRIT1 tumor suppressor. Molecular and Cellular Biology. 33 (24), 4900-4908 (2013).
  19. Swoboda, R. K., et al. Antimelanoma CTL recognizes peptides derived from an ORF transcribed from the antisense strand of the 3' untranslated region of TRIT1. Molecular Therapy Oncolytics. 1, 14009 (2015).
  20. Yue, Z., et al. Identification of breast cancer candidate genes using gene co-expression and protein-protein interaction information. Oncotarget. 7 (24), 36092-36100 (2016).
  21. Chen, S., et al. Association of polymorphisms and haplotype in the region of TRIT1, MYCL1 and MFSD2A with the risk and clinicopathological features of gastric cancer in a southeast Chinese population. Carcinogenesis. 34 (5), 1018-1024 (2013).
  22. Spinola, M., et al. Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer. Oncogene. 24 (35), 5502-5509 (2005).
  23. Spinola, M., et al. Ethnic differences in frequencies of gene polymorphisms in the MYCL1 region and modulation of lung cancer patients' survival. Lung Cancer. 55 (3), 271-277 (2007).
  24. Read, D. F., et al. Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene-mediated silencing. FEBS Letters. 591 (11), 1601-1610 (2017).
  25. Waller, T. J., Read, D. F., Engelke, D. R., Smaldino, P. J. The human tRNA-modifying protein, TRIT1, forms amyloid fibers in vitro. Gene. 612, 19-24 (2017).
  26. Suzuki, G., Shimazu, N., Tanaka, M. A yeast prion, Mod5, promotes acquired drug resistance and cell survival under environmental stress. Science. 336 (6079), 355-359 (2012).
  27. Soderberg, T., Poulter, C. D. Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues. Biochemistry. 40 (6), 1734-1740 (2001).
  28. Lamichhane, T. N., Blewett, N. H., Maraia, R. J. Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases. Rna. 17 (10), 1846-1857 (2011).
  29. Lamichhane, T. N., et al. Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast. Molecular and Cellular Biology. 33 (15), 2918-2929 (2013).
  30. Laten, H., Gorman, J., Bock, R. M. Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucleic Acids Research. 5 (11), 4329-4342 (1978).
  31. Etcheverry, T., Colby, D., Guthrie, C. A precursor to a minor species of yeast tRNASer contains an intervening sequence. Cell. 18 (1), 11-26 (1979).
  32. Yan, M., et al. A high-throughput quantitative approach reveals more small RNA modifications in mouse liver and their correlation with diabetes. Analytical Chemistry. 85 (24), 12173-12181 (2013).
  33. Su, D., et al. Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry. Nature Protocols. 9 (4), 828-841 (2014).
  34. Jonkhout, N., et al. The RNA modification landscape in human disease. Rna. 23 (12), 1754-1769 (2017).
  35. Dominissini, D., et al. The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature. 530 (7591), 441-446 (2016).
  36. Meyer, K. D., et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 149 (7), 1635-1646 (2012).
  37. Squires, J. E., et al. Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Research. 40 (11), 5023-5033 (2012).
  38. Schwartz, S., et al. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell. 159 (1), 148-162 (2014).
  39. Schwartz, S., et al. High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell. 155 (6), 1409-1421 (2013).
  40. Behm-Ansmant, I., Helm, M., Motorin, Y. Use of specific chemical reagents for detection of modified nucleotides in RNA. Journal of Nucleic Acids. 2011, 408053 (2011).
  41. Novikova, I. V., Hennelly, S. P., Sanbonmatsu, K. Y. Tackling structures of long noncoding RNAs. International Journal of Molecular Sciences. 14 (12), 23672-23684 (2013).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved