1nova1 and Nova-2 RNA Binding Species

1nova1 is a protein that in humans is mutated in osteosarcoma, cutaneous squamous cell carcinoma and melanoma. It is also expressed in a number of tumours including neuroblastoma, medulloblastoma and malignant brain tumours. It regulates alternative transcription in neurons by binding impact mRNA to enhance its excision. Its loss of function leads to a progressive neurologic disorder with motor dysfunction and dementia and is associated with tumour progression and metastasis.

Nova proteins are neuron-specific RNA-binding proteins that control the sequence-specific utilization of alternative pre-mRNA splicing elements. They bind to a single RNA strand via a helix-loop-helix domain and show a strong preference for a specific sequence motif consisting of three UCAU tetrads (21, 22). To define the regions that mediate RNA-binding by these proteins, a series of UV cross-linking experiments was performed with the GnRH E1-2 riboprobe. The binding of the riboprobe with Nova-1 was not affected by excess unlabeled RNA competitors, suggesting that the first KH domains are required for sequence-specific binding.

The crystal structures of third KH domains of Nova-1 and Nova-2 have been solved by molecular substitution to a resolution of 2.6 A. The structures are a compact protease resistant domain that resembles an open-faced, sandwich-like structure. They are composed of three strands of antiparallel b sheets capped by three tri-helices. The structures reveal that the Nova KH3 binds RNA in a 22 mer tetramer, similar to the hexameric SR protein SF2/NRF2 Complex (21). As well as determining the structure, binding dissociation constants of this hexameric compound were determined by nitrocellulose filter-binding assays. In these assays 50-100 fmols of an RNA internaly labeled with a 32P were mixed in binding buffer along with Nova-1 or Nova-2 dilutions at 3-fold and incubated at 25degC for 10 minutes. The resultant nitrocellulose filter was then subjected to gel electrophoresis, and the presence of 32P-labeled RNA was confirmed by superimposition on an autoradiograph.

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