Preparation of cooperative RNA recognition complexes for crystallographic structural studies

It is essential that mRNA-binding proteins recognize specific motifs in target mRNAs to control their processing, localization, and expression. Although mRNAs are typically targets of many different regulatory factors, our understanding of how they work together is limited. In some cases, RNA-binding proteins work cooperatively to regulate an mRNA target. A classic example is Drosophila melanogaster Pumilio (Pum) and Nanos (Nos). Pum is a sequence-specific RNA-binding protein. Nos also binds RNA, but interaction with some targets requires Pum to bind first. We recently determined crystal structures of complexes of Pum and Nos with two different target RNA sequences. A crystal structure in complex with the hunchback mRNA element showed how Pum and Nos together can recognize an extended RNA sequence with Nos binding to an A/U-rich sequence 5' of the Pum sequence element. Nos also enables recognition of elements that contain an A/U-rich 5' sequence, but imperfectly match the Pum sequence element. We determined a crystal structure of Pum and Nos in complex with the Cyclin B mRNA element, which demonstrated how Nos clamps the Pum-RNA complex and enables recognition of the imperfect element. Here, we describe methods for expression and purification of stable Pum-Nos-RNA complexes for crystallization, details of the crystallization and structure determination, and guidance on how to analyze protein-RNA structures and evaluate structure-driven hypotheses. We aim to provide tips and guidance that can be applied to other protein-RNA complexes. With hundreds of mRNA-binding proteins identified, combinatorial control is likely to be common, and much work remains to understand them structurally.