Iron Response ElementS

METHODS, PROCEDURE AND RESULTS

COLLECTING THE SEQUENCES

Our first objective was trying to determinate the general secondary nucleotide structure from IREs containing mRNAs. Revising the literature we found most of the iron trafficking proteins had this structure at the mature mRNA, so we started collecting sequences from these proteins with described IRES at the mRNA level. The chosen proteins were mainly ferritin and transferrin but other proteins were included as well. As the mRNA part we were interested in was the untranslated region we had to look for the sequences into an speciallized database which contains just entries from 5' and 3' untranslated regions (UTR database).

Initially we collected 29 sequences which contained almost all phylogenetic families:

- 15 ferritins from Homo sapiens (human), Mus musculus (mouse), Sus scrofa (pig), Oncorhynchus mykiss (rainbow trout), Rana catesbeiana (bullfrog), Xenopus laevis (frog), Gallus gallus (chicken), Drosophila melanogaster (fruit fly), Oncorhynchus gorbuscha (salmon), Aedes aegypti (yellow fever mosquito), Ixodes ricinus (castor) and Lymnaea stagnalis (snail).

- 9 transferrins from Homo sapiens and Rattus Norvergicus (rat).

- 5 proteins that had a high funtional homology with ferritin and transferrin

          * 2 eALAS (aminolevulinate synthase)  from Homo sapiens and Mus musculus;

          * Succinate dehydrogenase from Drosophila melanogaster;

          * 70KD S6 kinase from Drosophila melanogaster;

          * Marker protein (Ch21) from Gallus gallus.

All these sequences were divided into two groups: one that was going to be used for building the pattern (training set) and the other that, once the pattern finished, was going to assess the quality and features of it (test set). We tried that both groups not only contained ferritin and transferrin but a mixture of the different organism source as well.

- Training sequences (15 entries): included ferritin, transferrin, eALAS and succinate dehydrogenase.

- Test sequences (14 entries): contained ferritin, transferrin, S6 kinase and the marker protein.

Revising the literature we found many other proteins related to iron proteins so we decided it would be a good idea trying to determinate if all the mRNAs conected in some way to iron contained an IRE structure and how it was it: totally conserved among the proteins or with slight differences between them. We decided then to collect sequences from:

- Mitochondrial aconitase (10 entries);

- Ferroportin (Fpn1, IREG1 or MTP1) (5 entries);

- IRPs (3 entries).