Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins - PubMed (original) (raw)

Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins

M Ramming et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5' coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3-7 and 16-29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

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Figures

Figure 1

Figure 1

Identification of alternative spliced regions C5–C7. (A) Schematic representation of the gephyrin cDNA and clones p8–p10 containing novel nucleotide sequences. Exonic regions are displayed as boxes, intronic sequences are shown as a line. Dotted lines indicate only partially sequenced introns. Alternatively spliced regions are represented by hatched boxes, and initiation and stop codons are marked by arrows and asterisks, respectively. The alternatively spliced region C4b has been identified by Heck et al. (28). (B) Deduced amino acid sequences of regions C5–C7. The first aa shown is encoded by the preceding exon.

Figure 2

Figure 2

Structure of the murine gephyrin gene and domain analysis of the encoded protein. (A) Exon positions and relevant restriction sites of murine gephyrin genomic DNAs. (Upper) The exon borders of the gephyrin gene are projected onto a schematic representation of the gephyrin cDNA. An arrow marks the initiation codon, an asterisk the stop signal. Gray boxes indicate the alternatively spliced exons C1–C7. (Lower) Positions and lengths of genomic clones. Arrows indicate the relative positions of the exonic sequences on the isolated λ phages (solid lines) and P1 clones (dashed lines). Clone numbers and _Eco_RI restriction sites (vertical bars) used for mapping are indicated. Note different scales of size bars for both representations. (B) Comparison of the exon-intron boundaries and domains of gephyrin with sequence similarities to bacterial, invertebrate, and plant Moco proteins. Boxes and numbers within cinnamon and gephyrin correspond to the exons of the respective genes. Dashed and gray boxes indicate regions with sequence similarities to the bacterial proteins MoaB/MogA and MoeA, respectively. The black box in the central region of gephyrin indicates the region displaying sequence similarity to microtubule associated proteins encoded by exon 14. Dashed lines mark exon/intron boundaries conserved between cinnamon and gephyrin. Crossed arrows indicate swapping of the MoaB/MogA and MoeA homology domains in cnx 1 as compared with cinnamon and gephyrin. (C) Alignment of the amino acid motifs encoded by exons 13 and 14 of the murine gephyrin gene with the core repeat motif of MAP2 and tau and the oligomerization domain of keratin 1B. The motif encoded by exon 14 aligns well with the second imperfect octadecapeptide repeat of the microtubule-associated proteins MAP2 from mouse (mmap2), MAP2 from rat (rmap2) and tau from rat (rtau) and the C-terminal sequence predicted from exon 13 aligns well with the oligomerization domain of keratin 1B. Identical residues are indicated by dashes, and isofunctional ones by two dots.

Figure 3

Figure 3

Distribution of alternatively spliced gephyrin sequences in murine non-neuronal tissues and brain. Oligonucleotide probes specific for the alternatively spliced exons 3 (C2: A), 13 (C5: B), 19 (C6: C), and the invariant 3′ region of the murine gephyrin mRNA (T1: D Upper) as well as for β actin transcripts (D Lower) were hybridized to a multiple tissue Northern blot. The exon 3 probe revealed a predominant 3.8-kb transcript in liver in addition to differentially spliced mRNAs in heart, brain, liver, kidney, and testis (A). The exon 13 probe hybridized to a mRNA of 6.5 kb in muscle and heart (B). Expression of transcripts containing exon 19 (C) was seen in liver (3.8 and 2.2 kb), skeletal muscle (6.5 and 3.6 kb), and possibly testis (≈9 kb and 2.2 kb). In addition, higher molecular weight mRNAs were detected in heart (6.5 kb). In brain, heterogeneous bands 5–9 kb were labeled. The T1 probe detected 3.6- to 3.8-kb RNAs in all tissues examined except spleen (D Upper). Control hybridizations obtained with a probe specific for β actin transcripts also are shown (D Lower).

Figure 4

Figure 4

In situ hybridization analysis. Autoradiographs of parasagittal (Upper) and horizontal (Lower) sections of adult mouse brain were hybridized to 35S-labeled oligonucleotides specific for exons 2 (C1), 13 (C5), 21 (C7), and the T1 region (exon 28) of the gephyrin mRNA. Note similar labeling patterns of the C1, C5, and C7 probes.

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