This proposal aims to reveal the structure of the RNA binding domain of the human LRP130 protein, along with the RNA binding specificity for this protein, and the structure of the RNA bound complex.LRP130 is an RNA binding protein that associates with spli ced mRNAs. The protein does not contain any sequence homology with known RNA binding motifs, and therefore is likely to bind RNA through a novel RNA binding fold. LRP130 contains several pentatricopeptide repeat (PPR) motifs; other PPR containing proteins have been shown to bind RNA, and several have been implicated in organellar RNA metabolism. In vivo the majority of the total LRP130 protein is located within mitochondria, where it is directly bound to polyadenylated RNAs. There is also a small fraction w ithin nuclei, bound to mRNAs. In vitro, the RNA binding activity maps to the C-terminal region, and when expressed in E.coli this protein displays a preferential affinity for polypyrimidines. The RNA binding domain of LRP130 will be expressed in E.coli p urified by standard chromatographic techniques, and the structure solved by X-ray crystallography. In addition, the RNA binding specificity of this protein will be determined using in vitro evolution (SELEX) experiments. This will provide a high affinity l igand for crystallisation in complex with the protein, as well as suggesting a consensus sequence for RNA binding that can be used to identify sites of interaction in potential target mRNAs. The structure of the protein-RNA complex will then be solved, aga in by crystallography.LRP130 is the first identified mammalian protein directly bound to mitochondrial RNA in vivo. The structure of the RNA binding domain, with and without RNA bound, will be an important step towards understanding the structural basis fo r the functions of PPR motif proteins, and will potentially provide a molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.
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