Abstract
RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.
Highlights
Over the past several decades, great strides have been made in characterizing the roles of many types of RNA-binding proteins
Different domains can be present on a variety of RNA-binding proteins (RBPs) that allow for the recognition of RNAs, some of these include: RNA Recognition Motifs (RRM), K-homology domains (KH), RGG (Arg-Gly-Gly) boxes, zinc fingers, double-stranded RNA-binding domains, or DEAD-box (Asp-Glu-Ala-Asp) helicase domains (Chen and Varani 2005; Glisovic et al 2008; Clery, Blatter, and Allain 2008; Lunde, Moore, and Varani 2007; Valverde, Edwards, and Regan 2008; Linder and Jankowsky 2011)
Downstream characterization studies between the RNA and RNA-binding protein can utilize the fluorescent tag by using single-molecule fluorescence cross-correlation spectroscopy, or other methods (Panchapakesan et al 2017). The benefits of this method are that the reactions can be performed under native conditions and with similar equipment that is used during DIG labelling
Summary
Over the past several decades, great strides have been made in characterizing the roles of many types of RNA-binding proteins. Downstream characterization studies between the RNA and RNA-binding protein can utilize the fluorescent tag by using single-molecule fluorescence cross-correlation spectroscopy, or other methods (Panchapakesan et al 2017) The benefits of this method are that the reactions can be performed under native conditions and with similar equipment that is used during DIG labelling (i.e. streptavidin beads, and desthiobiotin). The high affinity interaction between Mango and thiazole orange promotes efficient pull down of RNP complexes in nanomolar concentrations with low volumes of cell extract This approach requires a fluorophore, where the other aptamer methods do not https://mc06.manuscriptcentral.com/bcb-pubs require that additional label. The pulled-down samples can be run on an SDS-PAGE, and the RNA binding proteins can be identified using mass spectrophotometry (Beach and Keene 2008) The advantages of this method are that the washes are performed under non-denaturing conditions, and the 5-bromo-UTP RNA modification could minimize non-specific binding https://mc06.manuscriptcentral.com/bcb-pubs. One further disadvantage is that weakly bound RNA-binding proteins can be difficult to capture
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