Abstract
Non-coding RNA sequences outnumber the protein-coding genes in the human genome, however our knowledge of their functions is still limited. RNA-binding proteins follow the transcripts, including non-coding RNAs, throughout their life, regulating not only maturation, nuclear export, stability and eventually translation, but also RNA functions. Therefore, development of sophisticated methods to study RNA-protein interactions are key to the systematic characterization of lncRNAs. Although mostly applicable to RNA-protein interactions in general, many approaches, especially the computational ones, need adjustment to be adapted to the length and complexity of lncRNA transcripts. Here we critically review all the wet lab and computational methods to study lncRNA-protein interactions and their potential to clarify the dark side of the genome.
Highlights
Over the past decade extensive efforts have been made to refine our understanding of the most complex mystery of life: the genome
The enhanced crosslinking and immunoprecipitation (CLIP) improves the library preparation step and optimizes the efficiency of the circular ligation step of the iCLIP by adding adapters in two separate steps. (i) An indexed 3′ RNA adapter is ligated to the crosslinked RNA fragment on beads during the immunoprecipitation, (ii) a 3′ singlestranded DNA adapter is ligated after reverse transcription, to determine whether two identical sequenced reads come from two unique RNA fragments or from PCR duplicates of the same RNA fragment (Van Nostrand et al, 2016b)
It was developed modeling experimental data obtained from a small scale proteomic study where 12 Halo-Tagged RNAbinding proteins (RBPs) were pulled down and their protein interactors analyzed with Multidimentional Protein Identification Technology (MudPIT) mass spectrometry
Summary
Over the past decade extensive efforts have been made to refine our understanding of the most complex mystery of life: the genome. The main limitations of RIP approaches are (i) the possible loss/gain of targets during the extraction, including non-specific interactions that may form after cell lysis (McHugh et al, 2014), (ii) the identification and exclusion of false positive hits, and (iii) the determination of the exact location of the binding site of RBPs that will require subsequent motif analysis (Li et al, 2015). To overcome these drawbacks, crosslinking of the RNA-protein complexes in the living cells was introduced.
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