Background

Post-transcriptional regulation of gene expression enables rapid systematic cellular responses to various stimuli and stressors (Chua et al., 2020; Weskamp et al., 2021). Post-transcriptional regulation can be mediated by various cis- and trans-acting elements, including regulatory elements on the mRNA itself as well as microRNAs and RNA-binding proteins (RBPs) (Theil et al., 2018; Furlan et al., 2021; Ma et al., 2022). In cells, RBPs often exist within large complexes, known as ribonucleoprotein (RNP) complexes, that include the RBPs themselves alongside their mRNA binding partner(s) and other proteins/molecules. Through these interactions in RNP complexes, RBPs can regulate the expression, translation, and/or localization of the mRNA species to which they bind (Dvir et al., 2018; Formicola et al., 2019).

Importantly, a single RBP can bind to and regulate the expression of many mRNAs (Hentze et al., 2018). Thus, deregulation of RBP expression has wide-ranging consequences for cellular health and survival. Notably, deregulation of RBP activity has been implicated in numerous human pathologies, including neurological disorders, kidney disease, and various types of cancer (Chatterji and Rustgi, 2018; Wang et al., 2018; Shi et al., 2020; Kinoshita et al., 2021; Weskamp et al., 2021). In order to begin probing the mechanism via which deregulation of RBP expression leads to disease, it is essential to first identify the group of mRNA binding partners to which the RBP binds. By identifying a list of mRNA binding partners for a given RBP, researchers can begin identifying candidate mRNA partner(s) from the list with potential links to the observed phenotype(s) and can thus begin elucidating the mechanism via which deregulated RBP expression leads to a diseased state (Fakhraldeen et al., 2022).

Given the importance of identifying mRNA binding partners of RBPs, research efforts have focused on developing methodologies that can reproducibly purify and identify mRNAs from RNP complexes (Zhao et al., 2022). The most common methodologies developed thus far rely upon cross-linking of often over-expressed RBPs prior to immunoprecipitation (IP). Once the RNP complexes are purified via manual IP, the cross-links are reversed, and the RNA is extracted and then analyzed via the researchers’ preferred method (e.g., qRT-PCR, microarray analysis, Northern blot, or RNA-Seq). Multiple versions of this so-called ribonucleoprotein immunoprecipitation (RIP) procedure have been developed, including RIP-Chip (manual RIP followed by microarray analysis), CLIP (cross-linking and immunoprecipitation), iCLIP (individual nucleotide resolution CLIP), PAR-CLIP (photoactivatable ribonucleoside-enhanced CLIP), HiTS-RAP (high throughput sequencing–RNA affinity profiling), and various adaptations therefrom (Galgano and Gerber, 2011; Jain et al., 2011; Danan et al., 2016, 2022; Haecker and Renne, 2014; Spitzer et al., 2014; Guerreiro et al., 2014; Ozer et al., 2015; Sutandy et al., 2016; Diaz-Munoz et al., 2017; George et al., 2017; Perconti et al., 2019; Lewinski et al., 2020; Benhalevy and Hafner, 2020; Baldini and Labialle, 2021). These approaches to RIP have several drawbacks. First, introduction of exogenously expressed RBPs can affect the mRNA binding profile of the RBP. Second, the processes of cross-linking and reverse cross-linking themselves can introduce artifacts by inducing interactions that were previously absent/not physiologically relevant, or by not capturing all interactions. Third, performing the IP procedure manually is time consuming and may promote irreproducibility based on the handler. Finally, any added benefits of potentially being able to identify specific binding sites of RBPs with their mRNA binding partners using these techniques are overshadowed by the complex computational approaches required in order to perform such analyses (Jens, 2016; Huessler et al., 2019; Busch et al., 2020; Zhao et al., 2022).

The method described here relies on exclusion-based sample preparation (ESP) technologies to perform RIP (Berry et al., 2015; Pezzi et al., 2018; Fakhraldeen et al., 2022). Specifically, we describe a procedure for performing RIP using the EXTRACTMAN device, which is an adaptation of the ESP-based Sliding Lid for Immobilized Droplet Extraction (SLIDE) technology. Not only does this method require less starting material, but it also offers significantly faster processing times, which is of particular relevance to RIP given the labile nature of RNA. Finally, the use of this sensitive method precludes the need to over-express the RBP of interest or to cross-link the lysate and instead allows for purification of the endogenous RBP in its native state. We describe the successful application of this method to identify mRNA binding partners of the RBP IGF2BP1 in the human embryonic kidney cell line HEK293T as well as in the human breast cancer cell lines MCF7 and MDA-MB-231 (Fakhraldeen et al., 2022). The extracted RNA was analyzed by qRT-PCR, but it can be analyzed using microarray analysis, RNA-Seq, or any endpoint of interest for RNA identification/profiling.