Role of the 3'-Untranslated Region of RPE65 mRNA in the Translational Regulation of the RPE65 Gene: Identification of a Specific Translation Inhibitory Element Su-Yan Liu and T. Michael Redmond 1 Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 Received March 9, 1998, and in revised form June 9, 1998 Previously, we demonstrated that explanted bovine retinal pigment epithelium (RPE) cells lose RPE65 protein, a major microsomal protein specific to RPE, while the RPE65 mRNA remains, suggesting posttran- scriptional regulation of RPE65 expression in vitro. Accordingly, we analyze here the effect of the 5- and 3-untranslated regions (UTRs) of RPE65 mRNA on translational efficiency using in vitro translation sys- tems. We compared the levels of translation products and mRNA stability among RPE65 transcripts contain- ing deletions of the 5- and 3-UTRs. First, the 5-UTR does not affect translational efficiency. However, the 3-UTR does influence translation efficiency. A puta- tive translation inhibitory element (TIE) is contained within the 170-nucleotide (nt) sequence downstream of the stop codon. There is also a weak destabilizing effect that is associated with the region 3 to the puta- tive TIE. But the effect of this is much less than that of the TIE. This TIE, however, does not inhibit transla- tion of the heterologous chloramphenicol acetyltrans- ferase gene, suggesting that a specific interaction with the upstream RPE65 coding sequence, or its product, may be required. Thus, the posttranscriptional regu- lation of RPE65 mRNA expression observed in cul- tured RPE may be via a mechanism of translational inhibition. Key Words: retinal pigment epithelium; translational regulation; mRNA stability; 3-untranslated region; translation inhibitory element. The importance of posttranscriptional regulation of gene expression, specifically mechanisms involving translation and stability of mRNA species, in the reg- ulation and differentiation of eukaryotic cells has be- come more widely recognized (1, 2). The role of post- transcriptional mechanisms in the regulation of retinal genes is not known, yet is likely to be involved in the regulation of some genes in this specialized neural tissue. One example may be RPE65, a conserved pro- tein highly and preferentially expressed in the RPE 2 (3, 4). Though the function of RPE65 in the RPE is still unknown, a role in retinoid metabolism is strongly suggested. First, RPE65 has been proposed to be the retinol-binding protein receptor (5). RPE65 does not, however, enhance RBP uptake in transfected cells (5). RPE65 also associates with 11-cis retinol dehydroge- nase (6), another RPE-expressed protein involved in RPE retinoid metabolism. Results from previous exper- iments (4) have suggested that RPE65 expression is posttranscriptionally regulated in primary cultured RPE cells. We found, despite the presence of abundant mRNA, that the protein was not expressed in these cultured RPE cells. A possible explanation was that translation was blocked by some factor(s), perhaps binding to the long AU-rich 3'-UTR. Accordingly, it was of interest to determine if the 3'-UTR of the RPE65 mRNA could affect its own translation. Mechanisms of posttranscriptional regulation most studied include mRNA stability and translation effi- ciency (for reviews see 1, 2). There is much evidence showing that both of these processes involve either the 5'-UTR or the 3'-UTR of the mRNA of the gene being 1 To whom correspondence should be addressed. Fax: (301) 402- 0750. E-mail: redmond@helix.nih.gov. 2 Abbreviations used: CAT, chloramphenicol acetyltransferase; IRE, iron-responsive element; ORF, open reading frame; RRL, rabbit reticulocyte lysate; RACE, rapid amplification of cDNA ends; RPE, retinal pigment epithelium; SSC, saline/sodium citrate; TIE, trans- lation inhibitory element; UTR, untranslated region; WGE, wheat germ extract; RBP, retinol-binding protein. 0003-9861/98 $25.00 37 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 357, No. 1, September 1, pp. 37– 44, 1998 Article No. BB980817