Background

DNA is the fundamental molecule containing the genetic information of the cell. However, RNA and its interactions with proteins play an essential functional role in using this genetic information and translating it to biological implication(s). RNA is less stable than DNA, is often single-stranded, and forms secondary and tertiary structures such as hairpins, helices, stem-loops, and others. The structure and sequence of an RNA molecule are essential for the formation of specific protein–RNA complexes (Singh et al., 2019). Further, there are thousands of proteins in the cell responsible for recognizing specific RNAs, and these interactions may be critical for carrying out their role: they may promote or repress RNA degradation, as well as regulate the splicing, transport, and translation of mRNA strands (Corley et al., 2020).

Many proteins are subjected to dynamic post-translational modifications (PTMs), which impact the structure and function of the proteins being modified (Ramazi and Zahiri, 2021). These PTMs include, but are not limited to, phosphorylation, acetylation, ubiquitination, and methylation. Further, any mutation in protein sequence may block PTMs and prevent protein–RNA interactions (Zhang et al., 2020). Similarly, RNA may be subjected to chemical modifications that impact its structure. These RNA modifications include methylation of adenosine (N6-methyladenosine), guanine carbonyl addition (8-oxo-7,8-dihydroguanosine), and acetylation of cytidine (N4-acetylcytidine), among others (Boo and Kim, 2020). For these reasons, the study of protein–RNA interactions is necessary for improving our understanding of how RNA plays a role in vital cellular processes.

Traditionally, electrophoretic mobility shift assays (EMSA), fluorescence anisotropy, and filter binding have been used to study protein–RNA interactions (Majumder and Palanisamy, 2021). In contrast to traditional techniques, the proposed RNA pull-down method obtains in vitro binding affinity measurements (i.e., K_D) for protein–RNA interactions (Figure 1A and 1B). Furthermore, RNA pull-down offers an approach in which an RNase-free environment is easy to maintain and uses standard immunoblotting for detection of bound protein. This contrasts with other protocols such as fluorescence anisotropy, which depend on specialized equipment, or other techniques, which are difficult to maintain an RNase-free environment. RNA pull-down can provide valuable information regarding protein–RNA binding affinities and analyze the effect of chemical modifications on these interactions. We have successfully applied this protocol to study the binding of nuclear polyadenylated RNA-binding (Nab) 3 protein RNA-recognition motif (RRM) with RNA and how point mutations to the lysine-363 residue affect this protein–RNA interaction (Lee et al., 2020).