Alternative RNA splicing of the NMDA receptor NR1 mRNA in the neurons of the teleost electrosensory system - PubMed (original) (raw)

Alternative RNA splicing of the NMDA receptor NR1 mRNA in the neurons of the teleost electrosensory system

D Bottai et al. J Neurosci. 1998.

Abstract

The sequence for cDNA encoding the NMDA receptor subunit 1 (aptNR1) of the weakly electric fish Apteronotus leptorhynchus has been determined. The deduced amino acid sequence is approximately 88% identical to other vertebrate NR1 proteins, with sequence homology extending to the alternatively spliced cassettes N1 and C1. The fish and mammalian N1 and C1 splice cassettes are identical at 20 of 21 and 30 of 37 amino acid positions, respectively. We did not detect a C2 splice cassette in aptNR1 mRNA, but we did find two novel C-terminal alternative splice cassettes labeled C1' and C1". The relative levels of NR1 transcripts containing the N1 and C1 splice cassettes were determined by using RNase protection and in situ hybridization analysis. N1-containing mRNAs are more abundant in caudal brain regions, similar to the patterns reported for mammalian brain. In contrast, the relative levels of transcripts containing the C1 splice cassette are much lower in fish than in mammals, averaging only 9% for the whole brain. The levels of C1 splicing increased in more rostral brain regions. In situ hybridizations with N1- and C1-specific probes demonstrated that N1 cassette splicing occurs in most neurons but that C1 splicing is heterogeneous and is restricted to a subset of neuronal types in the electrosensory system.

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Figures

Fig. 1.

Sequence comparison of the NR1 proteins. The_Apteronotus_ NR1 protein sequence is shown and compared with NR1 sequences of Xenopus (Soloviev et al., 1996), rat (Anantharam et al., 1992), and human (Foldes et al., 1993; Karp et al., 1993; Planells-Cases et al., 1993). Residues that are identical to the aptNR1 sequence are omitted in the other sequences. Positions of deletions are indicated by dashes. The amino acid positions are indicated on the right.TM1, TM3, and TM4 indicate the putative transmembrane segments, and P indicates the proposed pore segment. Glycosylation (filled squares), protein kinase C (filled diamonds), and protein kinase A (filled star) are indicated above the aptNR1 sequence.N1, C1, C1′, and_C1"_ indicate the positions of the alternatively spliced cassettes.

Fig. 2.

Analysis of aptNR1 mRNAs. RNA samples (20 μg) from liver and brain were fractionated by formaldehyde agarose gel electrophoresis, and the Northern blot was probed with a 1.3 kb fragment of the aptNR1 cDNA. RNA size markers are indicated on the_left_.

Fig. 3.

Estimation of the relative amounts of N1+ and N1− NR1 mRNA in various regions of the Apteronotus CNS. A, Schematic representation of the RNase protection assay. AptNR1 mRNA is shown at the top as a double line. The structures of the probe and products are shown below as_single lines_. The length of each fragment is given in nucleotides. B, The products of RNase protection analysis with RNA isolated from different regions of the_Apteronotus_ brain were analyzed by electrophoresis on a 5% polyacrylamide gel. RNA (5 μg) was used in each assay, except for ELL and cerebellum, which used 20 μg each. DNA size markers are indicated on the left. The splice isoforms to which the bands correspond are indicated on the right.

Fig. 4.

RNase protection analysis confirms the presence of novel NR1 splice isoforms containing cassettes C′ and C".A, Schematic representation of the RNase protection assay. AptNR1 mRNA is shown at the top as a_double line_. The structures of the probe and products are shown below as single lines. The length of each fragment is given in nucleotides. B, The products of RNase protection analysis with RNA (20 μg) isolated from_Apteronotus_ brain and liver were analyzed by electrophoresis on a 5% polyacrylamide gel. DNA size markers are indicated on the left. The splice isoforms to which the bands correspond are indicated on the right.

Fig. 5.

Estimation of the relative amounts of C1+ and C1− NR1 mRNA in various regions of the Apteronotus CNS. A, Schematic representation of the RNase protection assay. AptNR1 mRNA is shown at the top as a double line. The structures of the probe and products are shown below as_single lines_. The length of each fragment is given in nucleotides. B, The products of RNase protection analysis with RNA isolated from different regions of the_Apteronotus_ brain were analyzed by electrophoresis on a 5% polyacrylamide gel. RNA (5 μg) was analyzed in each assay, except for ELL, which used 20 μg. DNA size markers are indicated on the_left_. The splice isoforms to which the bands correspond are indicated on the right.

Fig. 6.

Localization of aptNR1 alternatively spliced transcripts in neurons of the electrosensory system. I,In situ hybridization of NR1 splice variants in the electrosensory lateral line lobe. A, D, Both pyramidal cells (Pyr. cells) and granular interneurons (Gr. cells) of the ELL are labeled with the NR1 pan probe. B, E, Pyramidal cells and interneurons also express the N1 splice cassette. C, F, The C1 splice cassette is expressed in pyramidal cells but appears to be absent from ELL interneurons. II,In situ hybridization of NR1 splice variants in layers 7–8A of the torus semicircularis dorsalis. A, The pan probe reveals strong labeling in cells of this region; as previously reported (Bottai et al., 1997), this region of the torus has the highest density of NR1 expression. B, The N1 probe also appears to label most cells, although at a much lower level.C, There does not appear to be any specific hybridization with the C1 probe to neurons in the torus.III, In situ hybridization of NR1 splice variants in the pacemaker nucleus. Pacemaker and relay cells were identified on the basis of somatic diameter in cases in which they were sectioned through their nuclei (relay cells are far larger). A, D, Both pacemaker and relay cells are labeled with the NR1 pan probe. B, E, Both pacemaker and relay cells also strongly express the N1 splice cassette. C, F, The C1 splice cassette appears to be absent from pacemaker cells but is strongly expressed in relay cells.

Fig. 7.

Phylogenetic tree comparing the ancestral relationships among the NR1 protein sequences from_Apteronotus_, Xenopus (Soloviev et al., 1996), duck (Kurosawa et al., 1994), rat (Moriyoshi et al., 1991), and human (Planells-Cases et al., 1993). The length of each branch is proportional to evolutionary distance. The sequences were aligned by using the multiple sequence alignment program Clustal V in the DNASTAR sequence analysis package.

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Alternative RNA splicing of the NMDA receptor NR1 mRNA in the neurons of the teleost electrosensory system - PubMed (original) (raw)