Risc

An miRNA function

Perfect base pairing; target cleavage

Incomplete base pairing; translation repression

▲ FIGURE 12-27 Model for miRNA translational repression and RNA interference mediated by the RNA-induced interfering complex (RISC). Processing of both miRNA precursors into miRNAs and long double-stranded RNAs into short interfering RNAs (siRNAs) requires the Dicer ribonuclease. In both cases, cleavage by Dicer yields a double-stranded RNA intermediate containing 21-23 nucleotides per strand with two-nucleotide 3' single-stranded tails. One strand of this intermediate assembles with multiple proteins to form an RNA-induced silencing complex (RISC). In siRNA function, the target mRNA hybridizes perfectly with the RISC RNA, leading to cleavage of the target. In miRNA function, the RISC RNA forms a hybrid with the target mRNA that contains some base-pair mismatches; in this case translation of the target mRNA is blocked. This is the situation for the lin-4 and let-7 miRNAs in C. elegans. [Adapted from G. Hutvagner and P D. Zamore, 2002, Science 297:2056; see also V. Ambrose, 2001, Cell 107:823.]

sequence differs at a few bases from the let-7 sequence. This finding has led to the hypothesis that RISC complexes have two distinct functions: (1) an siRNA function (i.e., RNA interference) leading to cleavage of precisely complementary mRNAs and (2) an miRNA function leading to translational repression of mRNAs with a few base-pair mismatches (see Figure 12-27).

Mutations in the fragile-X gene (FMR1), which encodes a protein containing a KH RNA-binding motif, are associated with the most common form of heritable mental retardation. Recent results indicate that the FMR1 protein is a subunit of human RISC complexes.

Consequently, the mental retardation associated with FMR1 mutations may result from a defect in translational repression of specific target mRNAs during development of the central nervous system. I

RNA interference is believed to be an ancient cellular defense against certain viruses and mobile genetic elements in both plants and animals. Plants with mutations in the genes encoding Dicer and RISC proteins exhibit increased sensitivity to infection by RNA viruses and increased movement of transposons within their genomes. The double-stranded RNA intermediates generated during replication of RNA viruses are thought to be recognized by the Dicer ribonucle-ase, inducing an RNAi response that ultimately degrades viral mRNAs. During transposition, transposons are inserted into cellular genes in a random orientation, and their transcription from different promoters produces complementary RNAs that can hybridize with each other, initiating the RNAi system that then interferes with the expression of transposon proteins required for additional transpositions.

In plants and C. elegans the RNAi response can be induced in all cells of the organism by introduction of double-stranded RNA into just a few cells. Such organism-wide induction requires a protein produced in plants and in C. el-egans that is homologous to the RNA replicases of RNA viruses. This finding suggests that double-stranded siRNAs are replicated and then transferred to other cells in these organisms. In plants, transfer of siRNAs might occur through plasmodesmata, the cytoplasmic connections between plant cells that traverse the cell walls between them (see Figure 6-34). Organism-wide induction of RNA interference does not occur in Drosophila or mammals, presumably because their genomes do not encode RNA replicase homologs. In plants, double-stranded RNA also induces the DNA methylation of genes with the same sequence by an unknown mechanism. Such gene methylation in response to RNAi inhibits transcription of the gene, probably through the binding of histone deacetylases. This type of RNAi-induced gene methylation does not occur in animals.

Cytoplasmic Polyadenylation Promotes Translation of Some mRNAs

In addition to repression of translation by micro RNAs, protein-mediated translational control helps regulate expression of some genes. Regulatory sequences, or elements, in mRNAs that interact with specific proteins to control translation generally are present in the untranslated region (UTR) at the 3' or 5' end of an mRNA. Here we discuss a type of protein-mediated translational control involving 3' regulatory elements. A different mechanism involving RNA-binding proteins that interact with 5 ' regulatory elements is discussed later.

Sequence-specific translation-control proteins may bind cooperatively to neighboring sites in 3' UTRs and function in a combinatorial manner, similar to the cooperative binding of transcription factors to regulatory sites in an enhancer

Translationally dormant Translationally active

CPE Poly(A) signal

▲ FIGURE 12-28 Model for control of cytoplasmic polyadenylation and translation initiation. Left: In immature oocytes, mRNAs containing the U-rich cytoplasmic polyadenylation element (CPE) have short poly(A) tails. CPE-binding protein (CPEB) mediates repression of translation through the interactions depicted, which prevent assembly of an initiation complex at the 5' end of the mRNA. Right: Hormone stimulation of oocytes activates a protein kinase that phosphorylates CPEB, causing it to release Maskin. The cleavage/polyadenylation region of a gene. In most cases studied, translation is repressed by protein binding to 3' regulatory elements. The mechanism of such repression is best understood for mRNAs that must undergo cytoplasmic polyadenylation before they can be translated.

Cytoplasmic polyadenylation is a critical aspect of gene expression in the early embryo. The egg cells (oocytes) of multicellular organisms contain many mRNAs, encoding numerous different proteins, that are not translated until after the egg is fertilized by a sperm cell. Some of these "stored" mRNAs have a short poly(A) tail, consisting of only «20-40 A residues, to which just a few molecules of cytoplasmic poly(A)-binding protein (PABPI) can bind. As discussed in Chapter 4, multiple PABPI molecules bound to the long poly(A) tail of an mRNA interact with the eIF4G initiation factor, thereby stabilizing the interaction of the mRNA 5' cap with eIF4E, which is required for translation initiation (see Figure 4-31). Because this stabilization cannot occur with mRNAs that have short poly(A) tails, such mRNAs stored in oocytes are not translated efficiently. At the appropriate time during oocyte maturation or after fertilization of an egg cell, usually in response to an external signal, approximately 150 A residues are added to the short poly(A) tails on these mRNAs in the cytoplasm, stimulating their translation.

Recent studies with mRNAs stored in Xenopus oocytes have helped elucidate the mechanism of this type of transla-tional control. Experiments in which short-tailed mRNAs are injected into oocytes have shown that two sequences in their 3' UTR are required for their polyadenylation in the cytoplasm: the AAUAAA poly(A) signal that is also required for the nuclear polyadenylation of pre-mRNAs, and one or more copies of an upstream U-rich cytoplasmic polyadenyla-

specificity factor (CPSF) then binds to the poly(A) site, interacting with both bound CPEB and the cytoplasmic form of poly(A) polymerase (PAP). After the poly(A) tail is lengthened, multiple copies of the cytoplasmic poly(A)-binding protein I (PABPI) can bind to it and interact with eIF4G, which functions with other initiation factors to bind the 40S ribosome subunit and initiate translation. [Adapted from R. Mendez and J. D. Richter, 2001, Nature Rev. Mol. Cell Biol. 2:521.]

tion element (CPE). This regulatory element is bound by a highly conserved CPE-binding protein (CPEB) that contains an RRM domain and a zinc-finger domain.

According to the current model, in the absence of a stimulatory signal, CPEB bound to the U-rich CPE interacts with the protein Maskin, which in turn binds to eIF4E associated with the mRNA 5' cap (Figure 12-28, left). As a result, eIF4E cannot interact with other initiation factors and the 40S ri-bosomal subunit, so translation initiation is blocked. Signal-induced phosphorylation of CPEB at a specific serine causes the displacement of Maskin, allowing cytoplasmic forms of the cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase to bind to the mRNA. Once the poly(A) polymerase catalyzes the addition of A residues, PABPI can bind to the lengthened poly(A) tail, leading to the stabilized interaction of all the participants needed to initiate translation (see Figure 12-28, right; see also Figure 4-25). In the case of Xenopus oocyte maturation, the protein kinase that phosphorylates CPEB is activated in response to the hormone progesterone. Thus timing of the translation of stored mRNAs encoding proteins needed for oocyte maturation is regulated by this external signal.

Considerable evidence indicates that a similar mechanism of translational control plays a role in learning and memory. In the central nervous system, the axons from a thousand or so neurons can make connections (synapses) with the den-drites of a single postsynaptic neuron (see Figure 7-48). When one of these axons is stimulated, the postsynaptic neuron "remembers" which synapse was stimulated. The next time that synapse is stimulated, the strength of the response triggered in the postsynaptic cell differs from the first time. This change in response has been shown to result largely from the translational activation of mRNAs stored in the region of the synapse, leading to the local synthesis of new proteins that increase the size and alter the neurophysiologi-cal characteristics of the synapse. The finding that CPEB is present in neuronal dendrites has led to the proposal that cy-toplasmic polyadenylation stimulates translation of specific mRNAs in dendrites, much as it does in oocytes. In this case, presumably, synaptic activity (rather than a hormone) is the signal that induces phosphorylation of CPEB and subsequent activation of translation.

mRNAs Are Degraded by Several Mechanisms in the Cytoplasm

The concentration of an mRNA is a function of both its rate of synthesis and its rate of degradation. For this reason, if two genes are transcribed at the same rate, the steady-state concentration of the corresponding mRNA that is more stable will be higher than the concentration of the other. The stability of an mRNA also determines how rapidly synthesis of the encoded protein can be shut down. For a stable mRNA, synthesis of the encoded protein persists long after transcription of the gene is repressed. Most bacterial mRNAs are unstable, decaying exponentially with a typical half-life of a few minutes. For this reason, a bacterial cell can rapidly adjust the synthesis of proteins to accommodate changes in the cellular environment. Most cells in multicellular organisms, on the other hand, exist in a fairly constant environment and carry out a specific set of functions over periods of days to months or even the lifetime of the organism (nerve cells, for example). Accordingly, most mRNAs of higher eukaryotes have half-lives of many hours.

However, some proteins in eukaryotic cells are required only for short periods of time and must be expressed in bursts. For example, certain signaling molecules called cy-

▲ FIGURE 12-29 Pathways for degradation of eukaryotic mRNAs. In the deadenylation-dependent (middle) pathways, the poly(A) tail is progressively shortened by a deadenylase (orange) until it reaches a length of 20 or fewer A residues at which the interaction with PABPI is destabilized, leading to weakened interactions between the 5' cap and translation initiation factors. The deadenylated mRNA then may either (1) be decapped and tokines, which are involved in the immune response of mammals, are synthesized and secreted in short bursts. Similarly, many of the transcription factors that regulate the onset of the S phase of the cell cycle, such as c-Fos and c-Jun, are synthesized for brief periods only (Chapter 21). Expression of such proteins occurs in short bursts because transcription of their genes can be rapidly turned on and off and their mRNAs have unusually short half-lives, on the order of 30 minutes or less.

Cytoplasmic mRNAs are degraded by one of the pathways shown in Figure 12-29. For most mRNAs, the length of the poly(A) tail gradually decreases with time through the action of a deadenylating nuclease. When it is shortened sufficiently, PABPI molecules can no longer bind and stabilize interaction of the 5' cap and initiation factors (see Figure 4-31). The exposed cap then is removed by a decapping enzyme, and the unprotected mRNA is degraded by a 5' ^ 3' exonuclease. Removal of the poly(A) tail also makes mRNAs susceptible to degradation by cytoplasmic exosomes containing 3' ^ 5' exonucleases. The 5' ^ 3' exonucleases predominate in yeast, and the 3' ^ 5' exosome apparently predominates in mammalian cells.

For mRNAs degraded in these deadenylation-dependent pathways, the rate at which they are deadenylated controls the rate at which they are degraded. The rate of deadenylation varies inversely with the frequency of translation initiation for an mRNA: the higher the frequency of initiation, the slower the rate of deadenylation. This relation probably is due to the reciprocal interactions between initiation factors and PABPI that stabilize the binding of PABPI to the poly(A) tail, thereby protecting it from the deadenylation exonuclease.

Many short-lived mRNAs in mammalian cells contain multiple, sometimes overlapping, copies of the sequence AUUUA in their 3' untranslated region. Specific RNA-binding degraded by a 5' ^ 3' exonuclease or (2) be degraded by a 3' ^ 5' exonuclease In cytoplasmic exosomes. Some mRNAs (right ) are cleaved Internally by an endonuclease, and the fragments degraded by an exosome. Other mRNAs (left ) are decapped before they are deadenylated, and then degraded by a 5' ^ 3' exonuclease. [Adapted from M. Tucker and R. Parker, 2000, Ann. Rev. Biochem. 69:571.]

Decapping pathway (deadenylation-independent)

Deadenylation-dependent pathways

Endonucleolytic pathway

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