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

Kathrin Theil (), Koshi Imami and Nikolaus Rajewsky ()
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Kathrin Theil: Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC)
Koshi Imami: Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC)
Nikolaus Rajewsky: Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC)

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: Abstract 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.

Date: 2019
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DOI: 10.1038/s41467-019-12050-7

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