Depolarization and CaM Kinase IV Modulate NMDA Receptor Splicing through Two Essential RNA Elements

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Abstract
Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity. Multiple mechanisms direct changes in neuronal activity in response to external stimuli, ranging from short-acting modifications of membrane proteins to longer-acting changes in gene expression. A frequently regulated step in gene expression is the pre-mRNA splicing reaction in which the inclusion of exons (protein-coding sequences) or the position of splice sites produces alternatively spliced mRNA isoforms encoding functionally different proteins. Here, we study splicing of the NMDA receptor, which responds to the neurotransmitter glutamate to modify neuronal activity. We show that the splicing of an important exon (E21) in the NMDA receptor subunit NR1 mRNA is repressed by cell depolarization and activation of the intracellular signaling molecule, CaMK IV. We find that this splicing repression is mediated by two regulatory sequences within the exon itself. One sequence is similar to a previously described regulatory element that had not been known to function in an exon. The other is a new element. The characterization of these elements as a family of degenerate sequences allowed the identification of a group of exons sharing responsiveness to cell depolarization and CamK IV. These results define a new set of gene expression changes that may occur in modulating neuronal activity.