The Length of 5′-Untranslated Leader Sequences Influences Distribution of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase mRNA in Polysomes: Effects of Lovastatin, Oxysterols, and Mevalonate

Abstract

Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5′-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5′-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D, M, Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5′-extension analysis under the same conditions, levels of transcripts with 5′-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5′-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5′-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5′-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5′-extension analysis. Under all three conditions, transcripts with 5′-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5′-UTL sequences.