TY - CHAP
T1 - LncRNA Pulldown Combined with Mass Spectrometry to Identify the Novel lncRNA-Associated Proteins
AU - Xing, Zhen
AU - Egranov, Sergey
AU - Lin, Chunru
AU - Yang, Liuqing
N1 - Publisher Copyright:
© 2021, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - Long non-coding RNAs (LncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Recent studies have revealed that nearly 80% of transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic non-coding RNAs, natural antisense transcripts, pseudogenes, and long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidence 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) and 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 lncRNA interacting protein partners in a cellular context. We provide a detailed protocol for this assay with hands-on tips based on our own experience working in the lncRNA field.
AB - Long non-coding RNAs (LncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Recent studies have revealed that nearly 80% of transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic non-coding RNAs, natural antisense transcripts, pseudogenes, and long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidence 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) and 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 lncRNA interacting protein partners in a cellular context. We provide a detailed protocol for this assay with hands-on tips based on our own experience working in the lncRNA field.
KW - Long non-coding RNAs
KW - Mass spectrometry
KW - RNA–protein interaction
UR - http://www.scopus.com/inward/record.url?scp=85113862569&partnerID=8YFLogxK
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U2 - 10.1007/978-1-0716-1697-0_1
DO - 10.1007/978-1-0716-1697-0_1
M3 - Chapter
C2 - 34417737
AN - SCOPUS:85113862569
T3 - Methods in Molecular Biology
SP - 1
EP - 10
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -