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Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction

  • C. Kiong Ho
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1648)

Abstract

The 5′-cap structure is an essential feature in eukaryotic mRNA required for mRNA stability and enhancement of translation. Ceratin transcripts are selectively silenced by decapping in the cytoplasm and later become translationally active again by acquiring the cap structure to regenerate translatable mRNAs. Identification of uncapped mRNA transcripts will reveal how gene expression is regulated by the mRNA recapping pathway. What follows is a sensitive method to detect and identify the uncapped mRNA from the cells. The technique consists of three parts: selective ligation of anchor RNA to the 5′-end of monophosphate RNA by double-strand RNA ligase, conversion of ligated RNA product into cDNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction.

Key words

RT-PCR 5′-cap Uncapped mRNA mRNA recapping RNA ligation RNA processing Trypanosoma brucei 

Notes

Acknowledgment

I thank Bryan Mathis (University of Tsukuba) for scientific editing service. This work was supported by the National Science Foundation under Grant Number 1050984 and the JSPS Grants-in-Aid for Scientific Research KAKENHI 16H05180.

References

  1. 1.
    Topisirovic I, Svitkin YV, Sonenberg N, Shatkin AJ (2011) Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip Rev RNA 2:277–298. doi: 10.1002/wrna.52 CrossRefPubMedGoogle Scholar
  2. 2.
    Liu H, Kiledjian M (2006) Decapping the message: a beginning or an end. Biochem Soc Trans 34:35–38. doi: 10.1042/BST20060035 CrossRefPubMedGoogle Scholar
  3. 3.
    Grudzien-Nogalska E, Kiledjian M (2017) New insights into decapping enzymes and selective mRNA decay. Wiley Interdiscip Rev RNA 8, p. e1379.  doi: 10.1002/wrna.1379.Google Scholar
  4. 4.
    Franks TM, Lykke-Andersen J (2008) The control of mRNA decapping and P-body formation. Mol Cell 32:605–615. doi: 10.1016/j.molcel.2008.11.001 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Schoenberg DR, Maquat LE (2009) Re-capping the message. Trends Biochem Sci 34:435–442. doi: 10.1016/j.tibs.2009.05.003 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ignatochkina AV, Takagi Y, Liu Y et al (2015) The messenger RNA decapping and recapping pathway in Trypanosoma. Proc Natl Acad Sci U S A 112:6967–6972. doi: 10.1073/pnas.1424909112 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang LK, Nandakumar J, Schwer B, Shuman S (2007) The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair. RNA 13:1235–1244. doi: 10.1261/rna.591807 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nandakumar J, Ho CK, Lima CD, Shuman S (2004) RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J Biol Chem 279:31337–31347. doi: 10.1074/jbc.M402394200
  9. 9.
    Ho CK, Shuman S (2002) Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc Natl Acad Sci U S A 99:12709–12714. doi: 10.1073/pnas.192184699 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Takagi Y, Sindkar S, Ekonomidis D et al (2007) Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA. J Biol Chem 282:15995–16005. doi: 10.1074/jbc.M701569200 CrossRefPubMedGoogle Scholar
  11. 11.
    Shenmin Yin, C Kiong Ho, Eric S Miller, Stewart Shuman (2004) Characterization of bacteriophage KVP40 and T4 RNA ligase 2. Virology 319(1):141–151. doi: 10.1016/j.virol.2003.10.037Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Infection Biology, Graduate School of Comprehensive Human Sciences, Faculty of MedicineUniversity of TsukubaIbarakiJapan

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