Title

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

Author

Buckanovich RJ; Darnell RB

Address

Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York 10021, USA

Source

Mol Cell Biol, 17(6):3194-201 1997 Jun

Abstract

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.

Language

English

Unique Identifier

97299669

MESH Headings

Animal ; Antigens, Neoplasm *ME ; Ataxia IM ; Base Sequence ; Consensus Sequence ; In Vitro ; Mice ; Molecular Sequence Data ; Nerve Tissue Proteins *ME ; Neurons *CH ; Nucleic Acid Conformation ; Paraneoplastic Syndromes IM ; Polymerase Chain Reaction ; Ribonucleoproteins *ME ; RNA *ME ; RNA-Binding Proteins *ME ; Structure-Activity Relationship ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S.

Publication Type

JOURNAL  ARTICLE

ISSN

0270-7306

Country of Publication

UNITED STATES

CAS Registry Number

0 (Antigens, Neoplasm); 0 (Nerve Tissue Proteins); 0 (Nova antigen); 0 (Ribonucleoproteins); 0 (RNA-Binding Proteins); 63231-63-0 (RNA)