Identification of proteins and miRNAs that specifically bind an mRNA in vivo [small RNA]

Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were so far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to a selected mRNA in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a novel and specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR provides a platform for investigating RNA in vivo regulation in diverse biological systems. small RNA sequencing of 16 vIPR RNA pulldown samples (C. elegans), with 3 replicates of the gfp control pulldown, 2 replicates each of pulldowns of the transcripts gld-1::gfp, gld-1, alg-1, lin-41, and 1 replicate of gfp::lin-41