title: Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.
abstract: Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.
authors.affiliation: Department of Nutritional Sciences, University of Arizona, Tucson 85721, USA. helen.nichol@usask.ca
authors.forename: Helen
authors.initials: H
authors.lastname: Nichol
authors.forename: Joy
authors.initials: J
authors.lastname: Winzerling
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D019476:Insect Proteins / Q000378:metabolism; D007313:Insecta / Q000235:genetics*; D035925:Iron-Regulatory Proteins / Q000235:genetics*; D007915:Lepidoptera / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
chemical_list: D020121:5' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D039562:Eukaryotic Initiation Factor-4F; D019476:Insect Proteins; D035925:Iron-Regulatory Proteins; D012313:RNA