Background

RNA-binding proteins (RBPs) interact with mRNAs in cells to form ribonucleoprotein (RNP) complexes that regulate the post-transcriptional activity of these transcripts by affecting their processing, subcellular localization, translation and stability (reviewed in Lunde et al., 2007; Glisovic et al., 2008). Recent studies have expanded the number of mRNA binding proteins in mammalian cells (for example Baltz et al., 2012; Castello et al., 2012; Kwon et al., 2013); additionally, a growing number of long noncoding RNAs (lncRNAs) have been shown to bind RBPs (for example Gumireddy et al., 2013; Ng et al., 2013; McHugh et al., 2015; Yoon and Gorospe, 2016), emphasizing the importance of characterizing the roles of RNA-protein interactions. There are several methodologies available to date to identify and characterize protein binding partners of specific RNAs (reviewed in Ramanathan et al., 2019). Shortly, these include tagging of RNAs of interest with affinity aptamers (Slobodin and Gerst, 2010), using in vitro transcribed RNAs (Abdelmohsen et al., 2011; Tominaga et al., 2011; Grammatikakis et al., 2016), RNA affinity pulldown typically using a limited number of RNA or DNA oligonucleotides (Kar et al., 2011; Castello et al., 2012; Yoon et al., 2012; Abdelmohsen et al., 2014; Zhang et al., 2016) coupled with Western blot or mass spectrometry analyses (Simon et al., 2011, McHugh et al., 2015, Di Tomasso et al., 2016, Kar et al., 2017, McHugh and Guttman, 2018), or immobilized oligonucleotides to capture RNA-protein complexes under native conditions (Zeng et al., 2006).

Here, we built upon the Chromatin Isolation by RNA Purification (ChIRP) method (Chu et al., 2011 and 2012), adapted it to maximize the yield of proteins bound to specific target RNAs and coupled it with mass spectrometry analysis for thorough identification and characterization of protein binding partners of these RNA transcripts (Figure 1). Similarly to the ChIRP approach (Chu et al., 2011), to obtain high specificity in isolation of a specific target RNA we used sets of pre-biotinylated single molecule FISH probes. A typical set is comprised of 24-48 ~20 mer DNA probes, complementary to and tiled along the target RNA. As in the ChIRP protocol (Chu et al., 2011), we used glutaraldehyde crosslinking as compared to other crosslinking methods, it yielded the highest signal-to-noise ratio. However, this protocol can be adjusted to use various cross linkers, including ultraviolet light and formaldehyde, to focus on specific interactions of interest.

Typically, we grow cells in desired conditions, cross link them and freeze cell pellets for future use or use them immediately. Cell pellets are lysed and pre-cleared cell lysates are incubated with a set of 24-48 biotinylated DNA probes, followed by additional incubation with streptavidin beads (Figure 1). Eluted proteins are separated using SDS-PAGE with each lane cut into 8 equal sections and in-gel digested as per Shevchenko et al., 2006 (Figure 1). Extracted peptides are LC-MSMS analyzed (Figure 1). This method allows for the identification and characterization of protein binding partners of specific RNAs of interest.