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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 thus 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 selected mRNAs 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 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 will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

Identification of proteins and miRNAs that specifically bind an mRNA in vivo