Preparation of cooperative RNA recognition complexes for crystallographic structural studies

Abstract

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.

Keywords: Cooperative RNA binding; Nanos; PUF protein; Pumilio; X-ray crystallography.

Figure 1

Crystal structures of Pum-Nos-RNA ternary complexes. A. Schematic drawings of Pum and Nos structural domains and RNA sequences. The eight repeats forming the PUM-HD RNA-binding domain are indicated by yellow rectangles, and the two ZF domains of Nos are represented by blue rectangles. The C-terminal tail of Nos is colored red. NRE RNA sequences bound by Pum and Nos are underlined orange and blue, respectively. The PRE consensus sequences are shown in black uppercase letters at highly conserved positions and in the CycB sequence, nucleotides that do not match the consensus are red. The Nos binding sequence is A/U-rich and shown in lowercase. B. Superposition of crystal structures of Pum and Nos in complex with hb (orange, PDB ID 5KL1) and CycB (light blue, PDB ID 5KL8) RNAs. The two RNAs are shown with the protein structures from the Pum-Nos-RNA_hb complex. Pum is shown as a ribbon diagram, and Nos is shown as a surface representation. The PUM-HD and ZF domains are colored as in the schematic drawings.

Figure 2

Flowchart of protein purification and Pum-Nos-RNA ternary complex formation. Coomassie-stained SDS-PAGE gels showing samples at different stages of complex preparation are shown. In the top gel, the lanes are aliquots of total cell lysate (Total), soluble supernatant fraction (Soluble), remaining proteins flowing through the Ni column (Flow Thru), and imidazole eluate from the Ni column (Eluate). A chromatogram of the Superdex 200 gel filtration purification of the Pum-Nos-RNA ternary complex is shown, and the pooled peak fractions are indicated by the red box. Fractions from this peak are shown in the bottom SDS-PAGE gel.

Figure 3

Nos clasps the RNA nucleotides 5´ of the Pum binding site with its ZF domains and wraps around Pum with its C-terminal tail. Two orientations of the Pum-Nos-RNA_hb ternary complex are shown. Pum (yellow) is shown as a surface representation, and Nos is shown as a ribbon diagram. The ZFs and C-terminal tail are colored as in Fig. 1.