Characterization of multiple transcripts of the hamster dolichol-P-dependent N-acetylglucosamine-1-P transferase suggests functionally complex expression.

Abstract

The evolutionarily conserved dolichol-P-dependent N-acetylglucosamine-1-P transferase gene, ALG7, functions by initiating the dolichol pathway of protein N-glycosylation. In yeast, ALG7 has a complex expression pattern and plays a critical role in diverse cellular functions, including proliferation and morphological response. In Chinese hamster ovary cells (CHO), ALG7 gives rise to three mRNAs of 1.5, 1.9 and 2.2 kb. We report results of RNA blotting assays, ribonuclease protection, PCR-amplification and sequencing of the CHO ALG7 transcripts 5' and 3' ends which suggest that the 1.5 and 1.9 kb transcripts are produced as a consequence of initiation at 2 distinct start sites, 350-379 bp apart. The transcriptional start site for the 1.5 kb mRNA is positioned between the first two in frame ATGs, while that of the 1.9 kb species is located upstream of these two in-frame ATGs. In order to test the translational competence of the 1.5 and 1.9 kb mRNAs, we constructed DNA templates specifying these transcripts and used them for in vitro transcription/translation. Our data show that the 1.9 kb mRNA served in the synthesis of 36 and 24 kDa species, as well as a low-abundance 32 kDa protein. The 1.5 kb transcript gave rise to a translation product of 32 kDa. The latter is synthesized in CHO cells and hamster submandibular glands. These results suggest the possibility that the 1.5 and 1.9 kb transcripts give rise to related protein isoforms with different lengths of their NH2-terminal regions.