Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective[5-H-2]uridine substitutions

Proteins that contain two or more copies of the RNA-binding domain [ribonuc leoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to b e involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, resid ue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the p resent study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct tar get RNAs derived from the tra and Sxl mRNA precursors with guanosine and ad enosine, respectively, in a position near the 5'-terminus of a uridine stre tch. First, we prepared a [5-H-2]uridine phosphoramidite, and synthesized a series of H-2-labeled RNAs, in which all of the uridine residues except on e were replaced by [5-H-2]uridine in the target sequence, GU(8)C. By observ ing the H5-H6 TOCSY cross peaks of the series of H-2-labeled RNAs complexed with the Sxl RBD1-RBD2, all of the base H5-H6 proton resonances of the tar get RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other t arget RNAs, GU(2)GU(8), AU(8), and UAU(8), were assigned by comparison with those of GU(8)C. We found that the uridine residue prior to the G or A res idue is essential for proper interaction with the protein, and that the int eraction is tighter for A than for G. Moreover, the H1' resonance assignmen ts were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the u nusual C2'-endo ribose conformation in the complex.