Stability-Based Proteomics for Investigation of Structured RNA–Protein Interactions

RNA–protein
interactions are essential to RNA
function throughout
biology. Identifying the protein interactions associated with a specific
RNA, however, is currently hindered by the need for RNA labeling or
costly tiling-based approaches. Conventional strategies, which commonly
rely on affinity pull-down approaches, are also skewed to the detection
of high affinity interactions and frequently miss weaker interactions
that may be biologically important. Reported here is the first adaptation
of stability-based mass spectrometry methods for the global analysis
of RNA–protein interactions. The stability of proteins from
rates of oxidation (SPROX) and thermal protein profiling (TPP) methods
are used to identify the protein targets of three RNA ligands, the
MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear
lysate. The 315 protein hits with RNA-induced conformational and stability
changes detected by TPP and/or SPROX were enriched in previously annotated
RNA-binding proteins and included new proteins for hypothesis generation.
Also demonstrated are the orthogonality of the SPROX and TPP approaches
and the utility of the domain-specific information available with
SPROX. This work establishes a novel platform for the global discovery
and interrogation of RNA–protein interactions that is generalizable
to numerous biological contexts and RNA targets.