Advertisement

LncRNA Pulldown Combined with Mass Spectrometry to Identify the Novel LncRNA-Associated Proteins

  • Zhen Xing
  • Chunru Lin
  • Liuqing Yang
Part of the Methods in Molecular Biology book series (MIMB, volume 1402)

Abstract

Long noncoding RNAs (LncRNAs) are nonprotein-coding transcripts longer than 200 nucleotides in length. The recent studies have revealed that at least nearly 80 % transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic noncoding RNAs, natural antisense transcripts, pseudogenes, long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidences suggesting that many lncRNAs are likely to be functional, only a small proportion has been demonstrated to be biologically and physiologically relevant due to their lower expression levels and current technique limitations. Thus, there is a greater need to design and develop new assays to investigate the real function of lncRNAs in depth in various systems. Indeed, several methods such as genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq), RNA immunoprecipitation followed by sequencing (RIP-seq) have been developed to examine the genome localization of lncRNAs and their interacting proteins in cells. Here we describe an open-ended method, LncRNA pulldown assay, which has been frequently used to identify its interacting protein partners in the cellular context. Here we provide a detailed protocol for this assay with hands-on tips based on our own experience in working in the lncRNA fields.

Key words

Long noncoding RNAs RNA-protein interaction Mass spectrometry 

References

  1. 1.
    Fatica A, Bozzoni I (2014) Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet 15:7–21CrossRefPubMedGoogle Scholar
  2. 2.
    Kung JT, Colognori D, Lee JT (2013) Long noncoding RNAs: past, present, and future. Genetics 193:651–669CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166CrossRefPubMedGoogle Scholar
  4. 4.
    Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann BM, Strein C et al (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149:1393–1406CrossRefPubMedGoogle Scholar
  5. 5.
    Lunde BM, Moore C, Varani G (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8:479–490CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071–1076CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tsai M-C, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Yang L, Lin C, Jin C, Yang JC, Tanasa B, Li W et al (2013) lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500:598–602CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Xing Z, Lin A, Li C, Liang K, Wang S, Liu Y et al (2014) lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159:1110–1125CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Cancer Biology ProgramThe University of Texas Graduate School of Biomedical SciencesHoustonUSA
  3. 3.The Center for RNA Interference and Non-Coding RNAsThe University of Texas MD Anderson Cancer CenterHoustonUSA

Personalised recommendations