Cooh

Translated ferritin

Translated ferritin

Low iron

—1 1-1 1-1 Codmg reg^n |- An —-//-—► No translation initiation

High iron

Inactive IRE-BP Active IRE-BP

Low iron

IREs

IREs

High iron

Inactive IRE-BP Active IRE-BP

Low iron

AU-rich region

Degraded mono-nucleotides

QQQ An

■> Little degradation

▲ FIGURE 12-30 Iron-dependent regulation of mRNA translation and degradation. The iron response element-binding protein (IRE-BP) controls translation of ferritin mRNA (a) and degradation of transferrin-receptor (TfR) mRNA (b). At low intracellular iron concentrations IRE-BP binds to iron-response elements (IREs) in the 5' or 3' untranslated region of these mRNAs. At high iron concentrations, IRE-BP undergoes a conformational change and cannot bind either mRNA. The dual control by IRE-BP precisely regulates the level of free iron ions within cells. See the text for discussion.

by receptor-mediated endocytosis (Chapter 17). The 3'-untranslated region of TfR mRNA contains IREs whose stems have AU-rich destabilizing sequences (Figure 12-30b). At high iron concentrations, when the IRE-BP is in the inactive, nonbinding conformation, these AU-rich sequences are thought to promote degradation of TfR mRNA by the same mechanism that leads to rapid degradation of other shortlived mRNAs, as described previously. The resulting decrease in production of the transferrin receptor quickly reduces iron import, thus protecting the cell. At low iron concentrations, however, IRE-BP can bind to the 3' IREs in TfR mRNA. The bound IRE-BP is thought to block recognition of the destabilizing AU-rich sequences by the proteins that would otherwise rapidly degrade the mRNAs. As a result, production of the transferrin receptor increases and more iron is brought into the cell.

Other regulated RNA-binding proteins may also function to control mRNA translation or degradation, much like the dual-acting IRE-BP. For example, a heme-sensitive RNA-binding protein controls translation of the mRNA encoding aminolevulinate (ALA) synthase, a key enzyme in the synthesis of heme. And in vitro studies have shown that the mRNA encoding the milk protein casein is stabilized by the hormone prolactin and rapidly degraded in its absence.

Nonsense-Mediated Decay and Other mRNA Surveillance Mechanisms Prevent Translation of Improperly Processed mRNAs

Translation of an improperly processed mRNA could lead to production of an abnormal protein that interferes with functioning of the normal protein encoded by the mRNA. (This effect is equivalent to that resulting from dominant-negative mutations, discussed in Chapter 9, although the cause is different.) Several mechanisms collectively termed mRNA surveillance help cells avoid the translation of improperly processed mRNA molecules. We have previously mentioned two such surveillance mechanisms: the recognition of improperly processed pre-mRNAs in the nucleus and their degradation by the exosome, and the general restriction against nuclear export of incompletely spliced pre-mRNAs that remain associated with a spliceosome.

Another mechanism called nonsense-mediated decay causes degradation of mRNAs in which one or more exons have been skipped during splicing. Except for pre-mRNAs that normally undergo alternative splicing, such exon skipping often will alter the open reading frame of the mRNA 3' to the improper exon junction, resulting in introduction of an incorrect stop codon. For nearly all properly spliced mRNAs, the stop codon is in the last exon. Nonsense-mediated decay results in the rapid degradation of mRNAs with stop codons that occur before the last splice junction in the mRNA.

A search for possible molecular signals that might indicate the positions of splice junctions in a processed mRNA led to the discovery of exon-junction complexes. As noted already, these complexes stimulate export of mRNPs from the nucleus. Analysis of yeast mutants suggests that some of the proteins in exon-junction complexes function in nonsensemediated decay. One proposal based on these and other findings is that exon-junction complexes interact with a deadenylase that rapidly removes the poly(A) tail from an associated mRNA, leading to its rapid decapping and degradation by a 5 ^ 3 exonuclease (see Figure 12-29). In the case of properly spliced mRNAs, the exon-junction complexes are thought to be dislodged from the mRNA by passage of the first "pioneer" ribosome to translate the mRNA, thereby protecting the mRNA from degradation. For mRNAs with a stop codon before the final exon junction, however, one or more exon-junction complexes remain associated with the mRNA, resulting in nonsense-mediated decay.

Nonsense-mediated decay occurs in the cytoplasm of yeast cells. Remarkably, in mammalian cells, there is evi dence that the pioneer ribosome translates the mRNA while its 5' end is associated with the nuclear cap-binding complex and its poly(A) tail is associated with nuclear PABPII. This finding and other results raise the possibility that in cells of higher organisms, the first round of translation may occur in the nucleus as part of the nonsense-mediated decay mechanism of mRNA surveillance.

Localization of mRNAs Permits Production of Proteins at Specific Regions Within the Cytoplasm

Many cellular processes depend on localization of particular proteins to specific structures or regions of the cell. In later chapters we examine how some proteins are transported after their synthesis to their proper cellular location. Alternatively, protein localization might be achieved by localization of mRNAs to specific regions of the cell cytoplasm in which their encoded proteins function. In most cases examined thus far, such mRNA localization is specified by sequences in the 3' untranslated region of the mRNA.

A well-documented example of mRNA localization occurs in mammalian myoblasts (muscle precursor cells) as they differentiate into myotubes, the fused, multinucleated cells that make up muscle fibers. Myoblasts are motile cells that extend cytoplasmic regions, called lamellipodia, from the leading edge in the direction of movement. Extension of lamellipodia during cell movement requires polymerization of p-actin (Chapter 19). Sensibly, p-actin mRNA is concentrated in the leading edges of myoblasts, the region of the cell cytoplasm where the encoded protein is needed for motility. When myoblasts fuse into syncytial myotubes, p-actin expression is repressed and the muscle-specific a-actin is induced. In contrast to p-actin mRNA, a-actin mRNA is restricted to the perinuclear regions of myotubes. When cultured myoblasts in the process of differentiating are stained with fluorescent probes specific for a- or p-actin mRNA, both mRNAs are localized to their respective cellular regions.

To test the ability of actin mRNA sequences to direct the cytoplasmic localization of an mRNA, fragments of a- and p-actin cDNAs were inserted into separate plasmid vectors that express p-galactosidase from a strong viral promoter. The resulting plasmids then were transfected into cultured cells, which were assayed for p-galactosidase activity. These experiments showed that inclusion of the 3' untranslated end of a- or p-actin cDNAs directs localization of the expressed p-galactosidase, whereas the 5' untranslated and coding regions do not (Figure 12-31).

Treatment of cultured myoblasts with cytochalasin D, which disrupts actin microfilaments, leads to rapid delocal-ization of actin mRNAs, indicating that cytoskeletal actin microfilaments participate in the localization process. Disruption of other cytoskeletal components, however, does not alter the localization of actin mRNAs. Other types of evidence also implicate the actin cytoskeleton in mRNA localization. Presumably certain RNA-binding proteins interact

TRANSFECTED ACTIN SEQUENCES „ v ß-Actin a-Actin

(a)

5' | |

■BfI

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