Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression

Abstract

REM2 is a member of the REM, RAD, and GEM/KIR (RGK) subfamily of RAS superfamily proteins and plays an important role in brain development and function. In this study, two Rem2 isoforms were isolated from the rainbow trout (Oncorhynchus mykiss). The two genes, designated O. mykiss rem2a and rem2b, both encode 304 amino acid proteins with 61% and 62% identities to zebrafish (Danio rerio) Rem2, respectively, and each with 43% identity to mammalian (human) REM2. To our knowledge, this is the first incidence of Rem2 isoforms in a species that are the result of gene duplication. Both isoforms possessed similar tissue expression profiles with the highest levels in the brain. The rem2a gene has significantly higher expression levels than rem2b in all tissues assayed except the brain and head kidney. In the central nervous system, both isoforms showed similar expression levels with the highest levels occurring in the olfactory bulb, cerebrum, and midbrain, though rem2a expression is significantly higher in the spinal cord. Based on known functional roles of Rem2 in synapse development and stem cell proliferation, the characterization of Rem2 in rainbow trout could shed light on its role in adult vertebrate neurogenesis and brain regeneration.

Fig. 1

Nucleotide and deduced amino acid sequences of rainbow trout rem2a and rem2b. The nucleotide sequences are aligned and shown in the 5′ to 3′ direction and numbered to the right. The deduced amino acid sequences are in the single letter amino acid code and numbered to the right in italics. Where amino acids differences occur due to codon differences, the Rem2a amino acid is listed first, followed by a slash (/), followed by the Rem2b amino acid. The start and stop codons are boxed. Nucleotides highlighted in gray ( ) indicate where trout rem2a and rem2b differ. The location of the primers and probes used in gene expression assays are underlined.

Fig. 2

Amino acid sequence alignment of the rainbow trout Rem2a (GenBank accession number JN175327) and Rem2b (JN175328) isoforms. Included in the alignment are the pufferfish unnamed protein (CAG10942.1), zebrafish Rem2 (NM_001123046), human REM2 (NP_775798.2), human RAD (NP_004156.1), human REM (AAC33132.1), and human GEM (NP_005252.1). Shades of gray indicate the corresponding level of identity across sequences with the darker shades indicating increased levels identity ( >50%, > 75%, = 100%). The arrow (▾) indicates where the trout isoforms differ. Consensus sequences for GTP-binding regions (G1–G5) and the conserved C7 sequence motif are indicated below the alignments (Finlin et al., 2000). The G3 consensus shown is that which is unique to the RGK subfamily. The binding sites for the 14-3-3 protein are indicated with an asterisk (Correll et al., 2009). Bold lines below the alignment indicate N-terminal and C-terminal extensions (Splingard et al., 2007). The solid boxes indicate the mammalian REM2 extended N-terminus and C-terminus following Seu and Pitt (2006). The dashed box indicates the SH3 (Src homology 3) binding motif (Splingard et al., 2007). Underlined methionines (M) in Human REM2 (M¹¹), Rem (M¹) and GEM (M¹) indicate where these amino acids have been previously aligned (Seu and Pitt, 2006; Splingard et al., 2006). Amino acids are numbered to the right. Percent identity of Rem2a and Rem2b with each sequence is indicated at the end of the alignment.

Fig. 3

Tissue specificity of rainbow trout rem2a and rem2b using Real-Time PCR with TaqMan® probes. The overall relative gene expression profile in the different tissues for each isoform was similar (Two-way ANOVA; not significant). There was a significant difference between the two isoforms regarding overall relative expression among the tissues (Two-way ANOVA; P < 0.0001). There was a significant difference in relative expression between all tissues for both isoforms (Two-way ANOVA; P < 0.0001). Significantly greater expression of a particular isoform in a tissue is indicated by an asterisk (t-test, P < 0.05). Each isoform showed differential expression between the different tissues (One-way ANOVA; P < 0.0001) and the relative expression in the brain of each isoform (indicated by “a” for rem2a and “b” for rem2b) was significantly greater than its respective expression in all other tissues (Tukey’s multiple comparison; P < 0.01).

Fig. 4

Regionalization of rem2a and rem2b in the rainbow trout CNS using Real-Time PCR with TaqMan® probes. The overall pattern of relative gene expression in the CNS of the rainbow trout brain for each isoform was similar (Two-way ANOVA; not significant). There was no significant expression difference between the isoforms among the different regions. There was a significant difference in relative expression between all regions of the CNS assayed for both isoforms (Two-way ANOVA; P < 0.0001). Significantly greater expression of a particular isoform in a region is indicated by an asterisk (t-test, P < 0.05). Each isoform showed differential expression in the CNS (One-way ANOVA; rem2a and rem2b; P < 0.0002). The relative expression of each isoform in the cerebrum (indicated by “a¹” for rem2a and “b¹” for rem2b) was significantly greater than its respective expression in the cerebellum, hindbrain, and spinal cord, and in the case of rem2a, the olfactory bulb as well (Tukey’s multiple comparison; P < 0.05). Both the midbrain and the olfactory bulb had greater relative expression levels than the cerebellum (indicated by “a²” for rem2a and “b²” for rem2b), except rem2a relative expression in the midbrain was also significantly greater than its expression in the hindbrain as well (P < 0.05).