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Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D.

ABSTRACT: The SLC4A10 gene, which is highly expressed in the mammalian brain, contains two known alternative splicing units, inserts A and B, and is theoretically capable of producing four NBCn2 splice variants: NBCn2-A, -B, -C, and -D. By immunoprecipitation and western blotting, a previous study showed the putative NBCn2-D to be expressed predominantly in the subcortex (SCX) and medulla (MD) of mouse brain. However, no evidence has been provided, in any species, for the existence of a full-length transcript encoding NBCn2-D. In the present study, we report for the first time the cloning of the full-length cDNAs encoding NBCn2-D from mouse SCX and MD. Based on the frequency of bacterial colonies obtained after PCR, we conclude that in SCX, the NBCn2-A transcript is dominant, whereas in MD, NBCn2-B is dominant. NBCn2-D is the least abundant transcript in each of these two brain regions. An analysis based upon the present PCR data as well as the previous immunoprecipitation/western-blot data suggests the following prevalence of NBCn2 variants in total mouse brain: NBCn2-A (~83%), NBCn2-B (~10%), NBCn2-C (~5%), and NBCn2-D (~2%). We also estimate the prevalence of each variant in each of the five brain regions (i.e., cerebral cortex, SCX, cerebellum, hippocampus, and MD). We hypothesize that the expression of different NBCn2 splice variants is characteristic of specific tissue/cells.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3201888 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D.

Liu Ying Y Xu Jiu-Ying JY Wang Deng-Ke DK Boron Walter F WF Chen Li-Ming LM

Brain research 20110323

The SLC4A10 gene, which is highly expressed in the mammalian brain, contains two known alternative splicing units, inserts A and B, and is theoretically capable of producing four NBCn2 splice variants: NBCn2-A, -B, -C, and -D. By immunoprecipitation and western blotting, a previous study showed the putative NBCn2-D to be expressed predominantly in the subcortex (SCX) and medulla (MD) of mouse brain. However, no evidence has been provided, in any species, for the existence of a full-length transc ...[more]

PMID: 21439947

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Project description:Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30-40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

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Dataset Information

Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D.

Publications

Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D.

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