Regulation of Eukaryotic Gene Expression: Messenger RNA processing and use

Eukaryotic mRNA undergoes several processing events before it is exported from the nucleus to the cytoplasm for use in protein synthesis. Capping at the 5′-end , polyadenylation at the 3′-end , and splicing  are essential for the production of a functional eukaryotic messenger from most pre-mRNA. Variations in splicing and polyadenylation can affect gene expression. In addition, messenger stability also affects gene expression.
1. Alternative splicing: Tissue-specific protein isoforms can be made from the same pre-mRNA through alternative splicing, which can involve exon skipping (loss), intron retention, and use of alternative splice-donor or -acceptor sites (Fig. 1). For example, the pre-mRNA for tropomyosin (TM) undergoes tissue-specific alternative splicing to yield a number of TM isoforms. [Note: Over 90% of all human genes undergo alternative splicing.]

Figure 1:  Tissue-specific alternative splicing produces different proteins, or isoforms, from a single gene. mRNA = messenger RNA.
2. Alternative polyadenylation: Some pre-mRNA transcripts have more than one site for cleavage and polyadenylation. Alternative polyadenylation (APA) generates mRNA with different 3′-ends, altering the untranslated region (UTR) or the coding (translated) sequence. [Note: APA is involved in the production of the membrane-bound and secreted forms of immunoglobulin M.]
The use of alternative splicing and polyadenylation sites, as well as alternative transcription start sites explains, at least in part, how the ~20,000 to 25,000 genes in the human genome can give rise to well over 100,000 proteins.
3. Messenger RNA editing: Even after mRNA has been fully processed, it may undergo an additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing. An important example in humans occurs with the transcript for apolipoprotein (apo) B, an essential component of chylomicrons  and very-low-density lipoproteins ([VLDL] ). Apo B mRNA is made in the liver and the small intestine. However, in the intestine only, the cytosine (C) base in the CAA codon for glutamine is enzymatically deaminated to uracil (U), changing the sense codon to the nonsense or stop codon UAA, as shown in Figure 2. This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL).

Figure 2:  Editing of apolipoprotein (apo) B pre-mRNA in the intestine and generation of the apo B-48 protein needed for chylomicron synthesis. Gln = glutamine; mRNA = messenger RNA; A = adenine; C = cytosine; G = guanine; U = uracil.
4. Messenger RNA stability: How long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. Regulation of iron metabolism and the gene-silencing process of RNA interference (RNAi) illustrate the importance of mRNA stability in the regulation of gene expression.
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 3. 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  in erythroblasts, also contains a 5′-IRE.] 

Figure 3:  Regulation of transferrin receptor (TfR) synthesis. [Note: The IRE are located in the 3′-UTR (untranslated region) of TfR messenger RNA (mRNA).] 7-CH3-G = 7-methylguanosine cap; (A)n = polyadenylate tail.
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 RNAinduced 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. 4). 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.

Figure 4:  Biogenesis and actions of microRNA (miRNA). [Note: The extent of complementarity between the target messenger RNA (mRNA) and the miRNA determines the final outcome, with perfect complementarity resulting in mRNA degradation.] Pri = primary; RISC = RNA-induced silencing complex.
1) RNA interference–based therapeutics
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.
5. Messenger RNA translation: Regulation of gene expression can also occur at the level of mRNA translation. One mechanism by which translation is regulated is through phosphorylation of the eukaryotic translation initiation factor, eIF-2 (Fig. 5). Phosphorylation of eIF-2 inhibits its function and so inhibits translation at the initiation step . [Note: Phosphorylation of eIF-2 prevents its reactivation by inhibiting GDP-GTP exchange.] Phosphorylation is catalyzed by kinases that are activated in response to environmental conditions, such as amino acid starvation, heme deficiency in erythroblasts, the presence of doublestranded RNA (signaling viral infection), and the accumulation of misfolded proteins in the rough endoplasmic reticulum .

Figure 5:  Regulation of translation initiation in eukaryotes by phosphorylation of eukaryotic translation initiation factor, eIF-2. RER = rough endoplasmic reticulum; ADP = adenosine diphosphate; Pi = inorganic phosphate; = phosphate.

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