Welcome guest, Sign in

Home

X
加载中

Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Biochemical Assays for MTase Activity

Microbiology > Microbial biochemistry > Protein > Activity
Authors: Yu Chen
Yu ChenAffiliation: College of Life Sciences, Wuhan University, Wuhan, China
For correspondence: chenyu@whu.edu.cn
Bio-protocol author page: a1107
 and Deyin Guo
Deyin GuoAffiliation: College of Life Sciences, Wuhan University, Wuhan, China
For correspondence: dguo@whu.edu.cn
Bio-protocol author page: a1108
Vol 4, Iss 2, 1/20/2014, 1824 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1023

[Abstract] Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.

Keywords: Methyltransferase, S-adenosyl-L-methionine, RNA capping, Cap structure

Materials and Reagents

  1. Bodicon m7G capping system (Bodicon, catalog number: CS0130)
  2. S-adenosyl methionine (SAM) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
  3. Bodicon Capping Enzyme (10 U/µl) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
    Note: Because the sale of this kit was low, the previous companies which provided this capping kit were out of service. This capping kit was provided by a new company in China as custom-made products (contact e-mail: service@bodicon.cn, phone:+86-13628662011). In fact the similar capping kit from any other companies (such as EPICENTRE biotechnologies, ScriptCap m7G capping system, catalog number: SCCE0610) is suitable for this experiment, and people can also contact with us to get the related protein or kit.
  4. Inorganic pyrophospatase (YIPP) (New England Biolabs, catalog number: M2403S)
  5. S-adenosyl [methyl-3H] methionine ([3H]-SAM) (PerkinElmer, catalog number: NET155H001MC)
  6. DEAE Sephadex (GE Healthcare, catalog number: 17-0170-01)
  7. GTP (Thomas Scientific, catalog number: R0461)
  8. RNase inhibitor (Thomas Scientific, catalog number: EO0381)
  9. RNase free water
  10. Phenol-chloroform (pH 4.8-5.2 for RNA only)
  11. Ethanol (RNase free)
  12. RNase free water
  13. Sodium Dodecyl Sulfonate (SDS)
  14. Ethylene Diamine Tetraacetic Acid (EDTA)
  15. NH4HCO3
  16. NaCl
  17. 10x MTase Buffer (see Recipes)
  18. Cap-0 cap structure (m7GpppN-RNA) (see Recipes)
  19. Non-methylated Cap-0 cap structure (GpppN-RNA) (see Recipes)
  20. MTase assay reaction mix (see Recipes)

Equipment

  1. Bechtop
  2. Water bath
  3. Centrifuge
  4. Liquid scintillation counter

Procedure

  1. Synthesis of RNA cap structure as substrates.
    The nascent RNA transcribed in vitro possesses a 5' triphosphate end. Two RNA capping systems are used to synthesize cap structure as the substrates of MTase.
    1. Combine 1-10 µg RNA and RNase free water up to 12.5 µl of total reaction volume.
    2. To heat to denature the in vitro transcribed RNA, incubate the tube at 65 °C for 10 min, and transfer the tube to ice immediately.
    3. Combine the following reaction components in the order given:
      Cap-0 cap structure (m7GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      3 mM SAM   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
      Non-methylated Cap-0 cap structure (GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      Inorganic pyrophospatase 0.1 U/µl   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
    4. Incubate at 37 °C for 2 h.
    5. Purify the RNA substrates by using phenol-chloroform extraction and ethanol precipitation methods.
  2. Prepare 10x MTase Buffer. The MTase buffer may be changed depending on different interested MTases.
  3. Combine the following reaction components in the order given for MTase assays:
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water
    up to 30 μl
    Total
    30 μl
  4. Incubate the reaction at 30-37 °C depending on different MTases for 1.5 h.
  5. Transfer the tubes onto ice and add equal volume (30 μl) of 0.2% SDS, 20 mM EDTA.
  6. Keep the tubes on ice, add 1 ml of 10 mM NH4HCO3 (pH 8.5).
  7. Prepare 1 ml DEAE Sephadex column and equilibrated with 10 ml 10 mM NH4HCO3 (pH 8.5).
  8. Load the samples onto the equilibrated column.
  9. Wash the column with 10 ml of 10 mM NH4HCO3 (pH 8.5), 100 mM NaCl.
  10. Elute the samples with 1.5 ml of 10 mM NH4HCO3 (pH 8.5), 400 mM NaCl.
  11. Add equal volume scintillation liquid, mix well by vortexing and measure the signal using Liquid scintillation counter. The counting signal of [3H], which is transformed from [3H]-SAM to RNA substrates, represents the activity of tested MTases.

Recipes

  1. 10x MTase Buffer
    0.5 M Tris-HCl (pH 7.5 or 8.0)
    50 mM KCl
    20 mM MgCl2
    20 mM DTT
  2. Cap-0 cap structure (m7GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    3 mM SAM
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  3. Non-methylated Cap-0 cap structure (GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    Inorganic pyrophospatase 0.1 U/µl
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  4. MTase assay reaction mix
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water up to
    30 μl
    Total
    30 μl

Acknowledgments

We thank Dr. Tero Ahola from University of Helsinki for the kindly help and advices for setting up this MTase assay. We thankfully acknowledge the University of Helsinki for providing research facilities and support during our visits. This work was supported by the China NSFC grants (81130083, 31170152 and 81271817).

References

  1. Chen, Y., Tao, J., Sun, Y., Wu, A., Su, C., Gao, G., Cai, H., Qiu, S., Wu, Y., Ahola, T. and Guo, D. (2013). Structure-function analysis of severe acute respiratory syndrome coronavirus RNA cap guanine-N7-methyltransferase. J Virol 87(11): 6296-6305.
  2. Chen, Y., Su, C., Ke, M., Jin, X., Xu, L., Zhang, Z., Wu, A., Sun, Y., Yang, Z., Tien, P., Ahola, T., Liang, Y., Liu, X. and Guo, D. (2011). Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog 7(10): e1002294.
  3. Chen, Y., Cai, H., Pan, J., Xiang, N., Tien, P., Ahola, T. and Guo, D. (2009). Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc Natl Acad Sci U S A 106(9): 3484-3489.


How to cite: Chen, Y. and Guo, D. (2014). Biochemical Assays for MTase Activity. Bio-protocol 4(2): e1023. DOI: 10.21769/BioProtoc.1023; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook