Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Abstract

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT's advantages and applications and discuss some of its limitations.

Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA

“…It is noteworthy that formaldehyde crosslinking has been used to map points of protein contact on DNA (Solomon and Varshavsky 1985), and can be similarly applied to RNAprotein complexes as well. Nevertheless, our current and previous work (Singh et al 2014;Mabin et al 2018;Woodward et al 2019) shows that formaldehyde crosslinking combined with RIPiT-seq is a powerful approach to isolate and analyze RNA footprints of multisubunit RNP assemblies, which are often heterogeneous in nature.…”

“…This two-step purification approach is an extension of RIP and involves sequential IP of a pair of proteins to enrich an RNP of a particular composition, whose RNA footprints can then be identified by high-throughput sequencing (Supplemental Fig. S1; Singh et al 2012Singh et al , 2014Woodward et al 2019). Previously, we used RIPiT-seq to obtain footprints of EJCs containing CASC3 and RNPS1 to describe the mutually exclusive nature of the two complexes (Mabin et al 2018).…”

Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins

“…These pathways (along with MLKL mediated necroptosis) lead to PANoptosis or inflammatory cell death (78)(79)(80)(81)(82)(83)(84)(85). (39,(45)(46)(47)(48). It also has the advantage to be reversible upon incubation of samples at high temperature, which can facilitate RNA recovery and downstream processing such as reversetranscription.…”