CLIP Identifies Nova-Regulated RNA Networks in the Brain

Abstract

Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP).Thirty-four transcripts were identified multiple times by Nova CLIP.Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition.Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets.Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.

The human RBPome: From genes and proteins to human disease

“…Hence, it becomes important to understand the structural and functional characteristics of RBPs in humans. Increasing interest in RBPs has led to the development of various experimental protocols like SELEX (systematic evolution of ligands by exponential enrichment) [9,10], CLIP [11], PAR-CLIP [12,13], iCLIP [14] and RNA compete [15], to identify the binding specificities of RBPs; thus adding context and dynamics to the regulation of gene expression at post-transcriptional level.…”

High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

“…[19][20][21] En masse characterization of targets, target sites and binding motifs has been described for a selected set of RBPs in a range of organisms. [22][23][24] Unfortunately, due to the complex nature of RBP-mediated regulation, studies employing only a single method fall short on providing a clear assessment of RBP function, as discussed in.…”

PQBP1, a factor linked to intellectual disability, affects alternative splicing associated with neurite outgrowth

“…2A; Lunde et al 2007). We used the CLIP (cross-linking and immunoprecipitation) method to determine whether PQBP1 directly binds its targets in vivo (Ule et al 2003). Live HeLa cells were exposed to UV light to cross-link RNA-protein complexes, and then PQBP1 was immunopurified from cell lysates (Fig.…”