Advertisement

Discovery, Identification, and Functional Characterization of Plant Long Intergenic Noncoding RNAs After Virus Infection

  • Ruimin Gao
  • Peng Liu
  • Nadia Irwanto
  • De Rong Loh
  • Sek-Man WongEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1933)

Abstract

Long intergenic noncoding RNAs (lincRNAs), which possess diverse features such as remodeling chromatin and genome architecture, RNA stabilization, and genome architecture, are important regulatory factors in plant genomes. They serve to fine-tune the expression of neighboring genes. Here, we describe a procedure of discovery, identification, and functional characterization of plant lincRNAs after virus infection. From high-throughput RNA-Seq transcriptome analysis, the noncoding RNA transcripts with significant fold changes (upregulation or downregulation) will be discovered and identified. The lincRNA of interest will be further confirmed and validated using rapid amplification of cDNA ends (RACE). In addition, functional characterization of the lincRNA will be followed up through overexpression and knockdown strategies.

Key words

Long noncoding RNAs Long intergenic noncoding RNAs (lincRNAs) RNA-Seq Transcriptome analysis Rapid amplification of cDNA ends (RACE) 

Notes

Acknowledgments

This work was supported by the Ministry of Education Tier 1 research grant R-154-000-A34-114 through the National University of Singapore (NUS) and NUS High School of Mathematics and Science.

References

  1. 1.
    Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43(6):904–914.  https://doi.org/10.1016/j.molcel.2011.08.018CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Au PC, Zhu QH, Dennis ES, Wang MB (2011) Long non-coding RNA-mediated mechanisms independent of the RNAi pathway in animals and plants. RNA Biol 8(3):404–414CrossRefGoogle Scholar
  3. 3.
    Liu J, Wang H, Chua NH (2015) Long noncoding RNA transcriptome of plants. Plant Biotechnol J 13(3):319–328.  https://doi.org/10.1111/pbi.12336CrossRefGoogle Scholar
  4. 4.
    Ransohoff JD, Wei Y, Khavari PA (2018) The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol 19:143–157.  https://doi.org/10.1038/nrm.2017.104CrossRefGoogle Scholar
  5. 5.
    Ulitsky I, Bartel DP (2013) lincRNAs: genomics, evolution, and mechanisms. Cell 154(1):26–46.  https://doi.org/10.1016/j.cell.2013.06.020CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Deniz E, Erman B (2017) Long noncoding RNA (lincRNA), a new paradigm in gene expression control. Funct Integr Genomics 17(2–3):135–143.  https://doi.org/10.1007/s10142-016-0524-xCrossRefGoogle Scholar
  7. 7.
    Cai L, Chang H, Fang Y, Li G (2016) A comprehensive characterization of the function of LincRNAs in transcriptional regulation through long-range chromatin interactions. Sci Rep 6:36572.  https://doi.org/10.1038/srep36572CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ding X, Zhu L, Ji T, Zhang X, Wang F, Gan S, Zhao M, Yang H (2014) Long intergenic non-coding RNAs (LincRNAs) identified by RNA-seq in breast cancer. PLoS One 9(8):e103270.  https://doi.org/10.1371/journal.pone.0103270CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tan G, Liu K, Kang J, Xu K, Zhang Y, Hu L, Zhang J, Li C (2015) Transcriptome analysis of the compatible interaction of tomato with Verticillium dahliae using RNA-sequencing. Front Plant Sci 6:428.  https://doi.org/10.3389/fpls.2015.00428CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zuluaga AP, Vega-Arreguin JC, Fei Z, Matas AJ, Patev S, Fry WE, Rose JK (2016) Analysis of the tomato leaf transcriptome during successive hemibiotrophic stages of a compatible interaction with the oomycete pathogen Phytophthora infestans. Mol Plant Pathol 17(1):42–54.  https://doi.org/10.1111/mpp.12260CrossRefGoogle Scholar
  11. 11.
    Jin J, Liu J, Wang H, Wong L, Chua NH (2013) PLncDB: plant long non-coding RNA database. Bioinformatics 29(8):1068–1071.  https://doi.org/10.1093/bioinformatics/btt107CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7(9):1494–1496.  https://doi.org/10.1093/mp/ssu044CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ruimin Gao
    • 1
  • Peng Liu
    • 1
  • Nadia Irwanto
    • 2
  • De Rong Loh
    • 2
  • Sek-Man Wong
    • 1
    • 3
    • 4
    Email author
  1. 1.Department of Biological SciencesNational University of SingaporeSingaporeSingapore
  2. 2.NUS High School of Mathematics and ScienceSingaporeSingapore
  3. 3.Temasek Life Sciences LaboratorySingaporeSingapore
  4. 4.National University of Singapore Suzhou Research InstituteSuzhouChina

Personalised recommendations