a. Iron metabolism: Transferrin (Tf) is a plasma protein that transports iron. Tf binds to cell-surface receptors (transferrin receptors [TfR]) that get internalized and provide cells, such as erythroblasts, with iron. The mRNA for the TfR has several cis-acting iron-responsive elements (IRE) in its 3′-UTR. IRE have a short stem-loop structure that can be bound by trans-acting iron regulatory proteins (IRP), as shown in Figure 33.14. When the iron concentration in the cell is low, the IRP bind to the 3′-IRE and stabilize the mRNA for TfR, allowing TfR synthesis. When intracellular iron levels are high, the IRP dissociate. The lack of IRP bound to the mRNA hastens its destruction, resulting in decreased TfR synthesis. [Note: The mRNA for ferritin, an intracellular protein of iron storage, has a single IRE in its 5′-UTR. When iron levels in the cell are low, IRP bind the 5′-IRE and prevent the use of the mRNA, and less ferritin is made. When iron accumulates in the cell, the IRP dissociate, allowing synthesis of ferritin molecules to store the excess iron. Aminolevulinic acid synthase 2, the regulated enzyme of heme synthesis (see p. 278) in erythroblasts, also contains a 5′-IRE.] (See Chapter 29 for a discussion of iron metabolism.) b. RNA interference: RNAi is a mechanism of gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. It plays a key role in such fundamental processes as cell proliferation, differentiation, and apoptosis. RNAi is mediated by short (~22 nucleotides), noncoding RNA called microRNA (miRNA). The miRNA arise from far longer, genomically encoded nuclear transcripts, primary miRNA (pri-miRNA), that are partially processed in the nucleus to pre-miRNA by an endonuclease (Drosha) then transported to the cytoplasm. There, an endonuclease (Dicer) completes the processing and generates short, double-stranded miRNA. A single strand (the guide or antisense strand) of the miRNA associates with a cytosolic protein complex known as the RNA-induced silencing complex (RISC). The guide strand hybridizes with a complementary sequence in the 3′-UTR of a full-length target mRNA, bringing RISC to the mRNA. This can result in repression of translation of the mRNA or its degradation by an endonuclease (Argonaute/Ago/Slicer) of the RISC. The extent of complementarity appears to be the determining factor (Fig. 33.15). RNAi can also be triggered by the introduction of exogenous double-stranded short interfering RNA (siRNA) into a cell, a process that has enormous therapeutic potential.
The first clinical trial of RNAi-based therapy involved the neovascular form of age-related macular degeneration (AMD), which is triggered by overproduction of vascular endothelial growth factor (VEGF), leading to the sprouting of excess blood vessels behind the retina. The vessels leak, clouding and often entirely destroying vision (therefore, neovascular AMD is also referred to as wet AMD). An siRNA was designed to target the mRNA of VEGF and promote its degradation. Although considerable effort and resources have been expended to develop RNAi-based therapeutics, especially for the treatment of cancer, no products have gone from trials to the market. The research applications of RNAi, however, have grown rapidly.