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. 2008 Nov;92(5):279-91.
doi: 10.1016/j.ygeno.2008.06.011. Epub 2008 Aug 26.

Alternative splicing and expression of human and mouse NFAT genes

Hanna Vihma  1 , Priit Pruunsild, Tõnis Timmusk
Affiliations

Alternative splicing and expression of human and mouse NFAT genes

Hanna Vihma et al. Genomics. 2008 Nov.

Abstract

Four members of the nuclear factor of activated T cells (NFAT) family (NFATC1, NFATC2, NFATC3, and NFATC4) are Ca(2+)-regulated transcription factors that regulate several processes in vertebrates, including the development and function of the immune, cardiovascular, musculoskeletal, and nervous systems. Here we describe the structures and alternative splicing of the human and mouse NFAT genes, including novel splice variants for NFATC1, NFATC2, NFATC3, and NFATC4, and show the expression of different NFAT mRNAs in various mouse and human tissues and brain regions by RT-PCR. Our results show that alternatively spliced NFAT mRNAs are expressed differentially and could contribute to the diversity of functions of the NFAT proteins. Since NFAT family members are Ca(2+)-regulated and have critical roles in neuronal gene transcription in response to electrical activity, we describe the expression of NFATC1, NFATC2, NFATC3, and NFATC4 mRNAs in the adult mouse brain and in the adult human hippocampus using in situ hybridization and show that all NFAT mRNAs are expressed in the neurons of the mouse brain with specific patterns for each NFAT.

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Figures

Supplementary Fig. 1
Supplementary Fig. 1
Aligment of the human N-terminal regions of the NFAT protein isoforms translated from alternative splice variants compiled with MULTALIN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_multalin.html). Red characters indicate identical amino acids and green characters indicate amino acids conserved 50% - 90 % among all the sequences aligned. Blue characters indicate similar amino acids between all the sequences aligned. The conserved N-terminal calcineurin binding site in the regulatory domain is highlighted with a black box. Stretch of 12 aa conservation is indicated with a red underline. Vertical green lines denote the start of exon II encoded sequences.
Supplementary Fig. 2
Supplementary Fig. 2
Aligment of the human C-terminal regions of the NFAT protein isoforms translated from alternative splice variants compiled with MULTALIN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_multalin.html). Red characters indicate identical amino acids and green characters indicate amino acids conserved 50% - 90 % among all the sequences aligned. Blue characters indicate similar amino acids between all the sequences aligned. The conserved C-terminal nuclear localisation signal (NLS) is highlighted with a black box. Conserved stretch of 13 amino acids containing a nuclear export signal (NES, blue box) is indicated with a red underline. Vertical green lines denote the end of exon VIII encoded sequences.
Supplementary Fig. 3
Supplementary Fig. 3
In situ hybridization analysis of NFATc2 mRNA expression in adult mouse brain. Dark-field emulsion autoradiographs from coronal sections at the level of striatum (A), thalamus (B), hippocampus (C and D) and cerebellum (E and F). LV, lateral ventricle; CPu, caudate putamen; CTX, cortex; Pir, piriform cortex; Th, thalamus; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; Sc, superior colliculi; Ent, entorhinal cortex; HTH, hypothalamus; PAG, periaqueductal gray; Pn, pontine nuclei; DG, dentate gyrus; CB, cerebellum; ME, medulla. Scale bar is 1 mm.
Fig. 1
Fig. 1
Structure and alternative transcripts of human NFAT genes. The structural organization of human NFATC1, NFATC2, NFATC3, and NFATC4 was determined by analyzing genomic and mRNA sequence data using bioinformatics and RT-PCR. Exons are shown as boxes and introns are shown as lines. The numbers above the exons indicate the size of the protein coding part of the exon. Protein coding sequences of the mRNAs are shown as filled boxes and open boxes indicate UTRs of the mRNAs. Numbers below the introns indicate their size. Exon numbers are shown in roman characters. Asterisks mark rarely used exons and rarely transcribed mRNA variants. NES, nuclear export signal; NLS, nuclear localization signal; DBD, DNA binding domain; RHD, Rel homology domain; Cn, calcineurin A.
Fig. 2
Fig. 2
Semiquantitative analysis of Nfatc1, Nfatc2, Nfatc3, Nfatc4, and control Hprt mRNA expression by RT-PCR in different mouse brain regions, in mouse brain at the indicated developmental time points, and in various mouse tissues.
Fig. 3
Fig. 3
Semiquantitative analysis of NFATC1, NFATC2, NFATC3, NFATC4, and controls GAPDH and HPRT mRNA expression by RT-PCR in various human tissues and brain regions.
Fig. 4
Fig. 4
In situ hybridization analysis of Nfatc1, Nfatc2, Nfatc3, and Nfatc4 mRNA expression in adult mouse brain. Dark-field emulsion autoradiographs from sagittal sections (A, C, E, and G) and coronal sections at the level of thalamus (B, D, F, and H). The sections were hybridized with a probe for Nfatc1 (A and B), Nfatc2 (C and D), Nfatc3 (E and F), or Nfatc4 (G and H). Gl, glomerular layer of olfactory bulb; GrO, granular layer of olfactory bulb; OB, olfactory bulb; Mi, mitral layer of olfactory bulb; Ctx, cortex; Cpu, caudate putamen; Hc, hippocampus; Th, thalamus; Hth, hypothalamus; Mb, midbrain; Po, pons; Cb, cerebellum; Me, medulla; Am, amygdala; DG, dentate gyrus. Scale bars, 1 mm.
Fig. 5
Fig. 5
In situ hybridization analysis of Nfatc1, Nfatc2, Nfatc3, and Nfatc4 mRNA expression in adult mouse brain. Bright-field higher magnification pictures of the olfactory bulb (A, D, G, and J), cerebellum (B, E, H, and K), and choroid plexus (C, F, I, and L) are shown. Filled arrowheads denote some positive neurons, some negative neurons are marked with unfilled arrowheads. The sections were hybridized with a probe for Nfatc1 (A, B, and C), Nfatc2 (D, E, and F), Nfatc3 (G, H, and I), or Nfatc4 (J, K, and L). Gl, glomerular cell layer of the olfactory bulb; GrO, granular cells of the olfactory bulb; Mi, mitral cell layer, PC, Purkinje cells; GC, granular cells of the cerebellum; Neg, negative cell. Scale bar, 50 μm.
Fig. 6
Fig. 6
In situ hybridization analysis of NFATC1, NFATC2, NFATC3, and NFATC4 mRNA expression in adult human hippocampus. Dark-field emulsion autoradiographs from coronal sections at the level of hippocampus. GrDG, granular cell layer of the dentate gyrus; PyCA3, CA3 pyramidal cells; hf, hippocampal fissure. Scale bar, 1 mm.
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