Uncovering The Mechanism Used By Drosophila Snf Protein To Specifically Bind Two RNAs

Abstract

The Drosophila SNF protein is a natural chimeric protein that evolved from the metazoan U1A and U2B” proteins. In the fly, SNF is found in both the U1 and U2 snRNPs, where it is assumed to bind to the U1 snRNA Stemloop II (the site of U1A binding) and U2 snRNA Stemloop IV (the site of U2B” binding), respectively. Like U1A and U2B” proteins, SNF has two RNA Recognition Motifs (RRM). By analogy to U1A protein, the N-terminal RRM should bind RNA, while the C-terminal RRM does not. Our experiments show that indeed the SNF C-terminal RRM does not bind RNA; NMR data show it to be soluble and stable. The SNF N-terminal RRM alone is not stable, however, and requires the C-terminal domain to maintain a folded form. NMR experiments and 15N-relaxation data show that the N-terminal RRM undergoes conformational exchange on the chemical shift timescale, while the C-terminal RRM has uniformly high order parameters characteristic of a more rigid protein.We hypothesize that the increased flexibility of the N-terminal RRM is part of its RNA binding mechanism, since SNF does indeed bind both U1 snRNA Stemloop II and U2 snRNA Stemloop IV. The affinity of SNF for these two RNAs differs by three orders of magnitude, indicating that one is preferentially bound. By comparison, however, the affinities of the human U1A protein for these two RNAs differ by more than 106-fold, indicating that SNF has found a way to bind both RNAs. This hypothesis will be tested by mutation of the N-terminal domain, with subsequent determination of protein structure, dynamics, and RNA binding.