The upstream open reading frame mediates constitutive effects on translation of cytochrome p-450c27 from the seventh in-frame AUG codon in rat liver

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55-kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression.     

Two forms of the Ia antigen-associated invariant chain result from alternative initiations at two in-phase AUGs

The Ia antigen-associated invariant chain (In) exists in humans as two major related forms, p33 and p35. The mRNA for In contains two in-phase AUGs, at positions 8 and 56 from the cap site. Cells transfected with a full-length cDNA clone in an expression vector synthesize both p33 and p35. Cell-free translation of mRNA synthesized in vitro from cDNA also produces both forms. When the first ATG is deleted from the cDNA clone, only the smallest form of In is produced. Mutations introduced at the second ATG lead to synthesis of the large form only. The alternative use of two in-phase AUGs on a unique mRNA is thus responsible for the synthesis of p33 and p35. This is the first documented example of such a mechanism in nonviral systems.     

Differential mRNA stability controls relative gene expression within a polycistronic operon

In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon. The polycistronic malEFG operon of E. coli contains two REP sequences (highly conserved inverted repeats) within the malE-malF intercistronic region. Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein. A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA. The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common.