Question 1 . Ferritin is a protein involved in the storage of iron inside cells.

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Question 1. Ferritin is a protein involved in the storage of iron inside cells. To prevent toxic accumulation of too much iron inside cells, the intracellular level of ferritin is tightly regulated. To study the regulation of ferritin synthesis, mammalian cells are grown with or without iron in the culture medium. Note that iron in the culture medium is rapidly transported inside cells.

a) Upon addition of iron to the culture medium, the intracellular concentration of ferritin mRNA is unchanged but the concentration of ferritin protein increases. How do you think ferritin expression is regulated? Briefly explain.

The regulatory sequence given below is found in the ferritin mRNA between the cap structure and the start codon.

Mutations within in this sequence are used to study the regulation of ferritin expression. The following observation are made:

- Ferritin expression is high, independent of the iron concentration, when (i) the entire region is deleted, or (ii) the region located upstream of the underlined sequence is deleted or (iii) the underlined sequence is replaced with a random sequence.

-Ferritin expression remains iron-dependent when this region is replaced by the following sequence:

Note that the sequence differences are indicated in bold.

b) Explain why these observations suggest that both sequence and structure of the 5’ end of ferritin mRNA are important for the regulation of ferritin expression.

c) Ferritin translation becomes iron-independent when the regulatory sequence is moved from the 5’ side (upstream of the open reading frame) to the 3’ side (downstream of the open reading frame) of ferritin mRNA. Which step of ferritin translation do you think is affected by the intracellular level of iron?

d) IRP is a protein involved in the regulation of ferritin expression. Anti-IRP antibodies attached to sepharose beads are added to a cell extract, then the extract is centrifuged to separate the pellet fraction (containing the sepharose beads ) from the supernatant fraction.
If the cells are cultured in the absence of iron, ferritin mRNA is found together with IRP in the pellet. In contrast when cells are cultured in the presence of iron ferritin mRNA remains in the supernatant fraction while IRP alone is found in the pellet. Briefly explain the likely role of IRP in the regulation of ferritin expression.

Explanation / Answer

a)Upon iron addition,ferritin mRNA remains same but ferritin concentration increases due to increased translation of ferritin mRNA.In the ferritin mRNA sequence shown, the bold sequence is of Iron Response Elements (IRE) regulate translation of ferritin mRNAs.These IRE can be in 5'UTR or 3'UTR (Untranslated regions). When iron concentration inside a cell is low , IRP or Iron regulatory proteins , bind to 5' UTR IRE and inhibit ferritin mRNA translation by hinderings its interaction with the ribosome. This prevents sequestering iron away for storage.When iron is high (as in iron medium), IRPs cannot bind to 5'UTR IRE since its interacts with Iron-Sulphur complex, rendering it unsuitable to to bind to IREs, resulting in high translation rate of ferritin mRNA.

b) IRE sequence in ferritin mRNA is a conserved stem loop (CAGUG).Ferritin expression would be iron independent of the entire CAGUG sequence is deleted or replaced or upstream sequence is deleted, since IRP wouldnt be able to bind to IREs and prevent translation inhibition.

If CUCAG sequence (in bold) replaces the native sequence, ferritin translation remains iron dependent, since IRP binding to IRE is not affected by this replacement, and IRE is intact.

c) Ferritin translation is iron independent when IRE/regulatory sequence is moved to 3' side, since IRP (IRP2, similar to IRP1 but lacking aconitase activity) binding to 3'side IRE stabilizes ferritin mRNA, leading to free translation. When iron levels are high, IRPs are removed from IREs , resulting in 5'UTR IRE being free and enables translation.

d) In absence of iron, ferritin mRNA and IRP are found together since IRP binds to 5'UTR IRE and inhibits ferriting mRNA translation. While in presence of iron, IRP is dissociated from IRE and found alone.

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