Factors affecting the alternative splicing of canine glutamine synthetase gene

Abstract

Glutamine synthetase (GS) catalyzes the ATP-dependent conversion of ammonia and glutamate to glutamine. In the previous study, we found an additional exon (120 bp) in the first intron of GS gene. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5`-untranslated region (UTR) containing the additional exon. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. We have performed an analysis on the cis-acting regulatory sequences that reside in the intron immediately upstream of this mini-exon, designated here as intron 1a, using the exon-trapping system. We constructed a series of mutants, in which either whole or partial GS intron 1a was replaced with GS intron 4 which is efficiently spliced out. When this constructs were transfected into MDCK and Neuro-2A cells, we found that both the branch-site and the polypyrimidine tract in GS intron 1a serve as cis-acting elements in brain-specific alternative splicing. Furthermore, we observed that a sequence motif in the new exon has no SRp40 binding sequence in near 3``(acceptor) exon-intron junction. So we suspected that SRp40 functions as exonic trans-acting factor in the brain-specific splicing of canine GS gene. But in the exon-trapping experiment with the exonic mutant minigene constructs, it seems that the absence of SRp40 binding sequence motif in exon 1`` is not related to GS alternative splicing. On the other hand, we describe a novel method for identifying candidate trans-acting factors that are involved in regulating GS alternative splicing. We generated a cell line that stably expresses a GS minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the GS gene.