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

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.

Control of alternative pre-mRNA splicing by Ca++ signals

“…Besides the Slo gene, similar regulation of the alternative splicing of NMDAR1 and other genes by membrane depolarization/CaMK IV has also been observed [84,104,105,107].…”

“…These stimuli can either promote or repress the inclusion of an exon (Table 1). Importantly, their effects on splicing can be inhibited by the L-type Ca ++ channel blockers verapamil, nifedipine or nimodipine [95,96,102,103,107], by the NMDA receptor antagonist AP5 or MK-801 [84,95,96,103], by the Ca ++ /calmodulin-dependent protein kinase inhibitors KN93 or KN62 [84,95,96,104,105], or by the cell permeable Ca ++ chelator BAPTA-AM [101,108]. Together, these observations strongly support the regulation of alternative pre-mRNA splicing by variations in intracellular Ca ++ levels.…”

“…These factors include the ionophore ionomycin [93], the neurotransmitter glutamate or its analogue NMDA (N-methyl-D -aspartate) [94][95][96][97][98], the muscarinic acetylcholine receptor agonist pilocarpine (inducing seizure) [99], the ionotropic glutamate receptor agonist kainate, the inhibitor of the sarco/endoplasmic reticulum Ca 2+ ATPase thapsigargin [100][101][102], and depolarizing concentrations of KCl [84,98,101,[103][104][105][106] (for a more complete list of the pharmacological agents and their descriptions, see Table 2). These stimuli can either promote or repress the inclusion of an exon (Table 1).…”

“…Of the various Ca ++ signals that regulate splicing, those through the CaMK IV pathway have been the most intensively studied [104,105,107]. A prime example of the effect on splicing by this pathway is the BK channel STREX exon, one of the more than 10 alternative exons of the vertebrate Slo gene [19,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] (Fig.…”

“…Moreover, PP2A is also shown to inactivate CaMK IV by directly dephosphorylating the Thr196 of CaMK IV [187]. Since the CaMK IV activity is essential for its role in the control of splicing [104,105,107], it is possible that the PP2A dephosphorylation of CaMK IV at Thr196 has an effect on CaMK IV-regulated alternative splicing.…”