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 approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 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)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) 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 alpha2 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 alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.
Buckanovich
R J
RJ
Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York 10021, USA.
Darnell
R B
RB
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001259:Ataxia / Q000276:immunology; D001483:Base Sequence; D016384:Consensus Sequence; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D000090682:Neuro-Oncological Ventral Antigen; D009474:Neurons / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D010257:Paraneoplastic Syndromes / Q000276:immunology; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000951:Antigens, Neoplasm; D009419:Nerve Tissue Proteins; D000090682:Neuro-Oncological Ventral Antigen; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA