rnA-protein complexes play pivotal roles in many central biological processes. Although methods based on highthroughput 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 crosslinking, 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 workflow can be applied to any rnA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mrnA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct rnA-binding proteins. the open-source software pipeline developed for this purpose, rnP xl , is available as part of the openms project.RNA molecules bind to proteins to form ribonucleoprotein complexes (RNPs). These are indispensable for the synthesis, stability, transport and activity of mRNAs 1 and noncoding RNAs 2,3 . RNA-binding proteins (RBPs) assume numerous functions in RNPs. RBPs can modulate or stabilize RNA structures, thereby making RNA catalytically active, for example, during pre-mRNA splicing 4 . RNA can also guide a catalytically active RBP to its destination; examples of this are microRNA-or long noncoding RNA-mediated translational control and epigenetic modulation 5,6 . RBPs are also involved in splicing and can recruit or repel other proteins, induce hydrolysis of RNA or protect RNA from degradation.

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. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed b propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

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