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Requirements for the translational repression of ferritin transcripts in wheat germ extracts by a 90-kDa protein from
P H Brown1, S Daniels-McQueen, W E Walden
1Department of Biology, Washington University, St. Louis, Missouri 63130.
Abstract:
A specific repressor of ferritin mRNA translation originally detected in rabbit reticulocyte lysates has now been purified to homogeneity from rabbit liver, as described in a companion paper (Walden, W. E., Patino, M. M., and Gaffield, L. (1989) J. Biol. Chem. 264, 13765-13769). This repressor is a 90-kDa protein that binds to a sequence in the 5'-untranslated region of ferritin mRNA. In this communication we describe the molecular features of a ferritin light chain transcript that are required for the repression of its translation by this protein. Addition of small amounts of the 90-kDa ferritin repressor protein (FRP) completely inhibited translation of ferritin transcripts in a wheat germ system. This repression did not require mRNA sequences contained in the 3'-untranslated region or in the majority of the ferritin coding region. In contrast, the first 130 nucleotides of the 5'-untranslated region, which contains the 28-nucleotide "iron responsive element" (IRE), was required for the repressive effect. Moreover, repression of full length transcripts was relieved by addition of a molar excess of a 92-nucleotide transcript of the 5'-untranslated region which also contained the IRE. These results suggest that no sequence information other than a portion of the 5'-untranslated region containing the IRE sequence is required for action of the 90-kDa FRP. In addition, a quantitative comparison of the repression of transcript with that of poly(A+) RNAs indicates that no post-transcriptional modifications of the latter (other than cap addition) are involved in the action of the 90-kDa FRP.
Insights
A purified 90-kDa ferritin repressor protein (FRP) inhibits ferritin mRNA translation. Only the 5'-untranslated region containing the iron-responsive element (IRE) is necessary for this repression, indicating minimal sequence requirements for FRP action.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein-RNA Interactions
Background:
- A 90-kDa repressor protein (FRP) controls ferritin mRNA translation.
- This repressor binds to a specific sequence within the 5 eal-untranslated region of ferritin mRNA.
Purpose of the Study:
- To identify the molecular features of ferritin mRNA required for repression by the 90-kDa FRP.
- To determine the minimal sequence elements necessary for FRP-mediated translational inhibition.
Main Methods:
- Purification of the 90-kDa ferritin repressor protein (FRP) from rabbit liver.
- In vitro translation assays using a wheat germ system with modified ferritin transcripts.
- Competition assays with synthetic mRNA fragments.
Main Results:
- The 90-kDa FRP completely inhibited ferritin mRNA translation in vitro.
- Repression required the 5 eal-untranslated region, specifically the 28-nucleotide iron-responsive element (IRE).
- Sequences outside the 5 eal-untranslated region, including the coding and 3 eal-untranslated regions, were not essential for repression.
Conclusions:
- The 90-kDa FRP requires only a portion of the 5 eal-untranslated region containing the IRE for its translational repression activity.
- Post-transcriptional modifications, beyond cap addition, are not involved in the action of the 90-kDa FRP on poly(A+) RNAs.