OBJECTIVES


The aim of our project was to try to predict new genes containing IRE (Iron Responsive Elements) motifs, as well as trying to define its secondary structure pattern. The prediction was based on 60,770 sequences of mouse cDNA which were extracted from the article Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. The transcriptome was analized in order to improve our predictions.


INTRODUCTION


The 30 nucleotides IRE motif is located in the untranslated regions at the 5' end of ferritin, and the 3' end of transferrin receptor mRNAs. Under conditions of low intracellular iron, two classes of iron-regulatory proteins bind IRE motifs with high affinity. This RNA protein complexes regulate expression of the gene products, depending upon their location in the mRNA. The minimal consensus sequence for the IRE consists of two helical stems connected by a bulge , where this bulge is separated from a six-nucleotide loop by five base-pairs. This loop is highly conserved in naturally occurring and in vitro selected IREs and has the sequence 5'-CAGUGX-3', where X can be any nucleotide except G. The relative positioning of an hairpin loop and bulge appears critical for precognition of the IRE by IRP. The strict requeriments for position and spatial relationship of the bulge and loop suggests that the bulge may orient the stem-loop regions for optimal protein binding. The absence of lon-range structural constraints in the standard NMR solution structure determination of the IRE led to a poorly defined global structure for this RNA.


IRE-containing mRNAs have been identified in vertebrates, invertebrates, and bacteria. Besides IRE motifs are located in the UTR of ferritin, they encode proteins that function in iron uptake, storage, and export (mammals, birds, amphibia, insects,and bacteria) and for heme synthesis or the trichloroacetic acid cycle/ATP production (mammals, amphibia, fish, insects, and bacteria). No IREs have been detected in plants, although an IRE-hybridizable, nonferritin sequence in soybean has been observed. IREs present in the 59 or 39 noncoding regions of mRNA were originally thought to be structurally the same, based on the predicted secondary structure of the stem loop and the similarity of IRP1 binding. However, based on comparisons among larger numbers of IRE sequences from different mRNAs coupled with additional studies of structure and binding with purified IRPs it is now apparent that the mRNA-specific divergences in IRE sequence and structure define isoforms of the IREs.


To reach our objectives the project was divided in four separated but coordinated working groups:


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