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A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels

Nature volume 410, pages 936–939 (2001)Cite this article

Abstract

Calcium regulation of gene expression is critical for the long-lasting activity-dependent changes in cellular electrical properties that underlie important physiological functions such as learning and memory1. Cellular electrical properties are diversified through the extensive alternative splicing of ion channel pre-messenger RNAs2; however, the regulation of splicing by cell signalling pathways has not been well explored. Here we show that depolarization of GH3 pituitary cells represses splicing of the STREX exon3 in BK potassium channel transcripts through the action of Ca2+/calmodulin-dependent protein kinases (CaMKs). Overexpressing constitutively active CaMK IV, but not CaMK I or II, specifically decreases STREX inclusion in the mRNA. This decrease is prevented by mutations in particular RNA repressor sequences. Transferring 54 nucleotides from the 3′ splice site upstream of STREX to a heterologous gene is sufficient to confer CaMK IV repression on an otherwise constitutive exon. These experiments define a CaMK IV-responsive RNA element (CaRRE), which mediates the alternative splicing of ion channel pre-mRNAs. The CaRRE presents a unique molecular target for inducing long-term adaptive changes in cellular electrical properties. It also provides a model system for dissecting the effect of signal transduction pathways on alternative splicing.

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Figure 1: Depolarization-induced changes in STREX splicing depend on CaMK.
Figure 2: CaMK IV overexpression represses STREX exon inclusion in HEK cells.
Figure 3: STREX RNA elements responsive to CaMK IV repression in HEK cells.
Figure 4: The CaRRE responds to depolarization in GH3 cells.
Figure 5: CaMK IV repression and a CaRRE in other ion channel exons. a, Exons SloII87, and 5 (E5) and (E21) of NMDAR1 inserted into pDUP4-1.

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Data deposits

The genomic DNA sequences for mouse STREX and SloII87 have been deposited in GenBank under accession numbers AF295094 and AF295095, respectively.

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Acknowledgements

We thank T. Chatila for the CaMK I and IV-dCT plasmids, and suggesting the use of KN93; M. Greenberg for the CaMK IV-dCTK75E plasmid; R. Maurer for the CaMK II-dCT plasmid; T. Soderling for the CaMK IV (HMDT→DEDD) plasmid; B. Howard for PC12 cells. We are grateful to R. Harris-Warrick, D. McCobb, K. Lynch, G. Chanfreau, P. Boutz, J. Underwood, K. Martin, M. Barad and H. Herschman for comments on the manuscript. This work is supported by the Howard Hughes Medical Institute.

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  1. Douglas L. Black: Correspondence and requests for materials should be addressed to D.L.B.

Authors and Affiliations

  1. Howard Hughes Medical Institute,

    Jiuyong Xie & Douglas L. Black

  2. Department of Microbiology & Molecular Genetics, University of California Los Angeles, 90095-1662, California, USA

    Douglas L. Black

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Correspondence to Douglas L. Black.

Supplementary information

Figure 1.

CaMK IV expression and activity. a. Blot of GH3 and HEK proteins probed with a CaMK IV-specific antibody. U1 70K is a protein loading control. b. Western blots with anti-Flag tag antibody of proteins from untransfected HEK 293 cells (lane 1), or cells transfected with Flag-tagged CaMK IV-dCT (lane 2), CaMK IV-dCTK75E (lane 3) or the pcDNA3.1(+) vector (lane 4). U1 70K and anti-phospho-CREB-Ser133 antibodies were used to probe the same membrane. All the CaMK-dCT plasmids including CaMK I-dCT and CaMK II-dCT were active as measured by increased Ser133 phosphorylation of co-expressed CREB proteins. (JPG 16.4 KB)

Figure 2.

STREX intron sequences and the 3' splice site affect STREX exon inclusion. a. pDUPST2 is similar to pDUPST1 but with much shorter intron sequences. Black bar: STREX exon. The M1 and M2 mutations are the same as in Fig. 3c. b. Primer extension assay of RNA from HEK cells transfected with pDUPST1 (ST1, lane 1), nothing (NT, lane2), wild type pDUPST2 (WT, lanes 3-5) and its mutants D56 (lanes 6-8), M1 (lanes 9-11), M2 (lanes 12-14), and double mutants D56+M1( lanes 15-17) or D56+M2 (lanes 18-20). These were cotransfected with CaMK IV-dCT(IV, lanes 4, 7, 10, 13, 16, and 19) or CaMK IV-dCTK75E( IVm, lanes 5, 8, 11, 14, 17 and 20). DUPST2 (lane 3) shows reduced exon inclusion compared to DUPST1 (lane 1), due to the loss of intron sequences. c. Percent decrease in STREX inclusion by CaMK IV-dCT for each plasmid in b (average ± SD, n = 3), relative to the same plasmid without CaMK IV-dCT. (JPG 29.5 KB)

Figure 3.

A full length but Ca++-independent CaMK IV (HMDT->DEDD) represses exon inclusion through the CaRRE element similarly to CaMK IV-dCT. Primer extension assay of RNA from HEK cells transfected with pDUP175 (lanes 1-3), pDUP175ST (lanes 4-6), and its M2 mutant (lanes 7-9). These cells were cotransfected with CaMK IV(HMDT->DEDD) (IV, lanes 2, 5 and 8 ), or its vector pME18S (Vec, lanes 3, 6 and 9) as indicated. Splicing of the pDUP175ST central exon with the CaRRE upstream is strongly repressed by CaMK IV(HMDT->DEDD), whereas splicing of the pDUP175 and pDUP175STM2 central exons is not affected. (JPG 12.6 KB)

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Xie, J., Black, D. A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature 410, 936–939 (2001). https://doi.org/10.1038/35073593

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