Cloning and sequencing of the cDNA for the 4.2 kb mRNA of eIF-2 alpha, reveal the presence of four consensus polyadenylation sites. In human tissues only two sites are recognized in vivo. Secondary structure analysis of the region containing the two silent sites show an area of highly predicted secondary structure encompassing these two sites. Northern blot analysis of mouse and human tissues show the presence of both the 1.6 and 4.2 kb mRNAs, establishing conservation of the poly A sites in the 4.2 mRNA 3'UTR. The 4.2 kb mRNA was highly expressed in skeletal muscle. Interestingly, a third mRNA was strongly expressed in mouse testes and to a weaker extent in human testis tissue. Expression of this third polyadenylation site in a specific tissue, which is normally unrecognized in other tissues, may provide us with a system for studying poly A site selection in specific tissues and allow us to assess whether RNA structure plays a role in recognition of cleavage and polyadenylation. Corresponding eIF-2 alpha protein levels and activities are being assessed for the different tissues. We have demonstrated that there is a difference between the stability in vivo of the 1.6 and 4.2 kb mRNAs of eIF-2 alpha. We are in the process of developing in vitro assays to ascertain the importance of sequence and/or secondary structure of the 3'UTR responsible for this difference. Deletion mutants are being prepared, to map the sequence and/or structure elements responsible for the increased stability of the 4.2 kb mRNA.
