Alternative splicing generates a novel non-secretable cytosolic isoform of NELL2

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Abstract

NELL2 is as a neuron-specific secreted glycoprotein. The present study provides evidence of an alternatively spliced variant of the rat NELL2 gene that yields cytosolic NELL2 (cNELL2). cNELL2 was initially detected in the thymus and subsequently found to be ubiquitously expressed in many other tissues. The absence of the sequences corresponding to the third exon, which contains the terminal portion of the signal peptide, accounts for the uniform distribution of cNELL2 throughout the cytoplasm. This is in contrast to NELL2, which is preferentially located at distinct subcellular structures involved in the secretary process, such as endoplasmic reticulum and Golgi apparatus. Western blot analysis showed that cNELL2 was not present in the medium but only in lysates, while NELL2 was detected as a glycosylated larger form in both lysates and media. Immunoprecipitation analysis revealed that cNELL2 interacts with PKCβ1. These results suggest that cNELL2 is involved in PKCβ1-mediated intracellular signaling.

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Materials and methods

Antibodies. Monoclonal antibodies specific for EGFP (B-2) and a rabbit anti-PKCβI antibody (C-16) were obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-actin antibody was purchased from Sigma.

RNA extraction and RT-PCR. Total RNA was isolated from tissues of adult male Sprague–Dawley rats (SD, 14 weeks old) using TRIZOL (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse-transcribed using SuperScript II RT (Invitrogen), random hexamers (50 

A novel alternative splicing variant of the rat NELL2 gene

During RT-PCR analysis of NELL2 expression in the rat thymus, two different NELL2 transcripts were detected: (1) the expected NELL2 mRNA (468 bp), which corresponds to the known secreted NELL2 mRNA [4], [9], [10], and (2) a shorter 339-bp PCR fragment (Fig. 1A). Since PCRs were carried out with specific primers corresponding to exons 2 and 5 of rat NELL2 cDNA, alternative splicing was proposed to account for the presence of this fragment. Fig. 1A shows the genomic structure of the rat NELL2 gene

Discussion

Here we report a novel cytosolic form of rat NELL2 (cNELL2) that is generated by alternative splicing. In several previous studies in which human and rat tissues [2], [3], [4], [11] were analyzed by either Western or Northern blotting, only the secreted form of NELL2 was detected. Two technical problems have made studies of cNELL2 difficult: First, the expression level of cNELL2 is much lower than that of secreted NELL2, as demonstrated by our RT-PCR results which showed differences in the

Acknowledgments

This work was supported by a grant from the Basic Research Program of the Korean Science & Engineering Foundation (R13-2005-012-01003-0), and the Korean Research Foundation (KRF-2002-CS0045 and KRF-2006-005-J04204). The nucleotide sequences reported in this paper have been submitted to the GenBank™ databases under Accession No. EF110908.

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These authors contributed equally to this work.

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