Post-transcriptional regulation of transferrin receptor (TfR) mRNA levels by iron is mediated by a portion of the 3' untranslated region (UTR) of the mRNA. Within the 3' UTR, a minimal regulatory element of 678 nt has been identified; this regulatory region contains 5 iron responsive elements (IRE-BP). The IRE-BP binds to IRE's with high affinity in the absence of iron; the affinity of this interaction is much lower in the presence of iron. In the absence of iron, the high affinity binding of the IRE-BP prevents rapid degradation of the TfR mRNA; in the presence of iron TfR mRNA is rapidly degraded. The mRNAs coding for the TfR, as well as the group of rapidly degraded mRNAs containing an AU-rich instability determinant (e.g. gmcsf and c-fos mRNAs) are stabilized by the addition of protein synthesis inhibitors. We have been able to establish that a labile trans-acting protein rather than cis-acting protein component of the translation machinery is required for the turnover of these mRNAs. In order to further our understanding of the events leading to the degradation of the TfR mRNA (and possibly many other mRNAs), we plan to use a somatic cell genetic approach. This approach has been used to gain new information about other multistep cellular processes such as the secretory pathway, endocytosis, cholesterol biosynthesis and carbohydrate biosynthesis. This approach involves the development of a method for separating clones of cells which have been rendered defective in the function of interest by mutagenesis from their wild type counterparts. Isolation of mutant clones is followed by phenotypic and genotypic analyses. Ultimately, cells can be rescued by complementation of function using cDNA libraries constructed in vectors designed to allow cloning of the cDNA's coding for the complementing polypeptide(s). In this way, we hope to define the process and identify the participants leading to the degradation of the TfR (and possibly many other) mRNA(s) in the cytoplasm.
