Studying RNA-Protein Interactions of Pre-mRNA Complexes by Mass Spectrometry.

RNA-protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique.

Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.

RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins.

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA.