The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of similar to 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (K-d of similar to 2 nM). The loop region of these RNAs harbors a similar to 15-bp pyrimidine-rich element [UCAU(N)(0-2)](3) which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)](3) element as necessary for in vitro binding, Consensus [UCAU (N)(0-2)](3) elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha 2 (GlyR alpha 2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha 2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha 2 pre-mRNA may underlie the motor dysfunction seen in POMA.