Mutational analysis of the DST element in tobacco cells and transgenic plants: identification of residues critical for mRNA instability

DST (downstream element), an approximately 40-base sequence derived from the 3' untranslated region (UTR) of SAUR (small auxin up RNA) genes, represents one of only a few sequence elements that have been demonstrated directly to target transcripts for rapid decay in plant cells. Substitution mutations were made in conserved regions of the DST element containing the sequences ATAGAT and GTA, which are invariant among several SAUR genes. The mutant DST elements were inserted into the 3' UTR of a beta-globin reporter gene and then assessed for their ability to destabilize the reporter transcript in stably transformed BY-2 tobacco cells. Their effect on reporter mRNA accumulation in both intact transgenic tobacco plants and stably transformed BY-2 cells was also measured. Five- and six-base substitutions in the ATAGAT and GTA regions of DST, respectively, resulted in inactivation of the element as an instability determinant in all systems tested. Smaller, two-base substitution mutations within the ATAGAT and GTA regions had varying effects on DST function in BY-2 cells, ranging from little or no effect to significant increases in reporter mRNA half-life and accumulation. In contrast, all two-base substitution mutations tested resulted in inactivation of DST in intact tobacco leaves. Together, these results indicate that bases within both the ATAGAT and GTA regions of DST are required for its function as an mRNA instability determinant in both BY-2 cells and leaves of transgenic plants, and that the sequence requirements for DST to function in leaves are more stringent.