Mechanisms of translational control by the 3′ UTR in development and differentiation

Seminars in Cell & Developmental Biology 16 (2005) 49–58

Mechanisms of translational control by the 3
UTR in development and differentiation Cornelia H. de Moor∗ , Hedda Meijer, Sarah Lissenden Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, UK

Abstract Translational control plays a major role in early development, differentiation and the cell cycle. In this review, we focus on the four main mechanisms of translational control by 3
untranslated regions: 1. 2. 3. 4.

Cytoplasmic polyadenylation and deadenylation; Recruitment of 4E binding proteins; Regulation of ribosomal subunit binding; Post-initiation repression by microRNAs.

Proteins with conserved functions in translational control during development include cytoplasmic polyadenylation element binding proteins (CPEB/Orb), Pumilio, Bruno, Fragile X mental retardation protein and RNA helicases. The translational regulation of the mRNAs encoding cyclin B1, Oskar, Nanos, Male specific lethal 2 (Msl-2), lipoxygenase and Lin-14 is discussed. © 2004 Elsevier Ltd. All rights reserved. Keywords: 3
UTR; Development and differentiation; Cell cycle; Gene expression; Translation

1. Introduction Translational control is found in all stages of development and affects a wide range of mRNAs, including a large number that encode transcription factors and cell cycle regulators. Genetic analysis has revealed networks of translationally controlled developmental regulators in the fruit fly (Drosophila melanogaster) and the nematode, Caenorhabditis elegans [1]. Translational control is particularly important in animal germ cells, since transcription is absent in later stages of germ cell differentiation and does not resume until some time in early development, depending on the species [2]. In Drosophila and Caenorhabditis, an earlier transcriptional block has been shown to be essential for the formation of germ cells. In addition, an accumulation of evidence from different sources indicates that translational control plays an important role in the cell cycle. A large number of mRNAs have been reported to be translationally ∗

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regulated by specific sequences in their mRNAs, usually in the untranslated regions (UTRs). However, there are only a few cases in which mechanisms for their regulation can be proposed. The majority of instances of translational control in development are mediated by sequences in the 3
UTR. Regulation of translation by upstream open reading frames is discussed elsewhere in this issue (Proud, pp. . . .). Internal ribosome entry is also implicated in the regulation of developmentally important mRNAs. For further discussions of internal initiation, see the review by Bushell and Willis elsewhere in this issue (Bushell and Willis, pp. . . .). In this review, we will concentrate on a selection of the better characterised cases of translational control by 3
UTRs in development and differentiation.

2. Regulation of poly(A) tail length The poly(A) tail of an mRNA promotes translation in synergy with the cap. This function is mediated by the interaction

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Fig. 1. A simplified representation of translation initiation. The mRNA forms a closed loop complex with the cap-binding factor eIF4E (4E), the scaffolding factor eIF4G and poly(A) binding protein (PABP). The ternary complex (eIF2, GTP and methionyl tRNA) is bound to the small subunit of the ribosome (40S) and this complex is recruited to the mRNA through an interaction between eIF3 and eIF4G. The small ribosomal subunit scans the 5
UTR until it finds the start codon. The GTP on eIF2 is hydrolysed and the large ribosomal subunit joins. The ribosome is ready for elongation with the methionyl tRNA at the start codon of the mRNA.

of the poly(A) binding protein (PABP) with a number of translation factors, including the initiation factors eIF4A, eIF4G and the termination factor eRF-3 (for a review, see [3]). The interaction of the scaffold factor eIF4G with both the capbinding factor eIF4E and PABP circularises the mRNA into the ‘closed loop’ complex (for a summary of the relevant steps of translation initiation, see Fig. 1). This complex is thought both to stabilise the association of the cap-binding initiation factors and to facilitate the recycling of ribosomes that have terminated their translation of the mRNA. The regulation of poly(A) tail length of translationally controlled mRNAs is a recurring theme in the oogenesis and early development of many animal species. In most cases, long poly(A) tails (80–500 A residues) correlate with translation and short tails (20–50 A) with repression of translation. Many of these regulated mRNAs are maternal mRNAs, which are stored in an untranslated form in the growing oocyte and translationally activated at specific stages of oocyte or early embryo development. In the following sections, we discuss some of the better characterised examples of translational regulation by elongation and shortening of the poly(A) tail. 2.1. Cytoplasmic polyadenylation In the oocytes of the African clawed frog, Xenopus, a large number of maternal mRNAs are stored in the cytoplasm with short poly(A) tails and activated translationally during oocyte maturation, concomitantly with their polyadenylation. Most

of these mRNAs encode cell cycle regulators including the mitotic cyclins, cdk2, wee1 and Aurora A (reviewed in [4]). The regulatory sequences governing their translational activation and polyadenylation are primarily found in the 3
untranslated regions of the mRNAs. The best characterised of these sequences is the cytoplasmic polyadenylation element (CPE), which is found in mRNAs encoding the mitotic cyclins and other cell cycle regulators [4]. CPE-dependent cytoplasmic polyadenylation requires two different elements in the 3
UTR of the mRNA: the hexanucleotide polyadenylation signal (AAUAAA) and the CPE, which is a U-rich element. In Xenopus, the CPE usually is U4–5 A1–2 U, although some variations may be tolerated in the context of specific mRNAs [4]. CPEs only mediate polyadenylation efficiently if situated 100 nt or less from the poly(A) signal. The CPE is bound by CPE binding protein (CPEB), a conserved RNA binding protein, containing a zinc finger and two RNA recognition motifs (RRMs) [5,6]. Progesterone induces meiotic maturation in Xenopus oocytes through a membrane-bound receptor [7,8]. Induction of cytoplasmic polyadenylation is mediated by the activation of the serine/threonine kinase Aurora A/Eg2, possibly through a repression of glycogen synthase kinase 3 [9]. As the Aurora A mRNA is also itself a target of cytoplasmic polyadenylation, this may constitute a positive feedback loop. Aurora A phosphorylates CPEB on serine 174 and phosphorylated CPEB recruits cleavage and polyadenylation specificity factor (CPSF) to the poly(A) signal [10,11]. As in nuclear polyadenylation, CPSF is thought to bring in

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the poly(A) polymerase (PAP) to the mRNA and the addition of A residues ensues. XGef is a CPEB-interacting guanine nucleotide exchange factor that stimulates cytoplasmic polyadenylation of the c-mos mRNA. Injection of antibodies against XGef block polyadenylation, indicating that this factor plays an unknown, but important, role in cytoplasmic polyadenylation [12]. The function of CPEB in meiotic maturation is conserved in mice, where the cyclin B1 and c-mos mRNAs are also regulated by cytoplasmic polyadenylation [13,14]. Both male and female CPEB knockout mice have defects early in germ cell differentiation, indicating an early role for CPEB in germ cell development in addition to the later functions in meiotic maturation [15]. In addition to its function in meiosis, CPE-mediated cytoplasmic polyadenylation is also required for the early embryonic cell cycles in Xenopus and may play a role in mammalian somatic cell division as well [16,17]. Strikingly, cytoplasmic polyadenylation has also been implicated in the cell cycle of Schizosaccharomyces pombe. Cid1 and Cid13 are involved in the cell cycle checkpoint activated by blocking DNA polymerase activity and were recently shown to be cytoplasmic poly(A) polymerases [18,19]. Cid1 and Cid13 are only distantly related to PAP, but have closer relatives in higher eukaryotes that have also been shown to have poly(A) polymerase activity, including the C. elegans cytoplasmic poly(A) polymerase Gld-2 and mammalian homologs [20,21]. Gld-2 and its binding partner the KH domain RNA binding protein Gld-3 are both required for meiosis in C. elegans, providing a counterbalance to the translational repressor FBF, a Puf family protein [1,22,23]. More about the Puf family proteins can be founding the section on the translational repression of cyclin B1. 2.2. Deadenylation In most somatic cells, deadenylation of mRNAs is linked to their degradation by decapping and/or exosome activity [24]. However, in oocytes and early embryos mRNAs with very short poly(A) tails survive intact, both in vertebrates and in Drosophila. In this situation, short poly(A) tails correlate with translational repression. Both specific and default deadenylation pathways are active in oogenesis and early development (reviewed in [25]). The most extensively studied specific deadenylation element in developmental systems is the embryonic deadenylation element (EDEN), which has been characterised in a number of Xenopus mRNAs that are activated by cytoplasmic polyadenylation in the oocyte, including those encoding the kinases Aurora A (Eg2) and cmos. This regulation is probably necessary to allow normal cell cycle progression in the embryo. The translational repression of the c-mos mRNA in the embryo is necessary to avoid cell cycle arrest after fertilisation [26]. The presence of an EDEN in an RNA conveys rapid and specific deadenylation in fertilised eggs and extracts [27]. The consensus sequence for the EDEN is proposed to be an extended UR

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repeat (R for purine), but additional AU rich sequences are likely to be important for efficient deadenylation [28,29]. An EDEN-binding protein (EDEN-BP) has been identified and is necessary for EDEN specific deadenylation in extracts and embryos [27,30]. EDEN-BP contains three RNA recognition motifs and is a member of the elav family of RNA binding proteins. Human CUG-BP and Drosophila Bruno-like protein 3 (Bru-3) are close homologues of EDEN-BP and they bind similar sequences. CUG-BP can substitute for EDENBP in in vitro deadenylation reactions [30–32]. Although a short poly(A) tail is predicted to reduce translational efficiency, it is unlikely to abrogate it completely. For instance, the Xenopus mRNA for histone B4 mRNA is stored in the oocyte with a short poly(A) tail, but despite this, a fraction of the mRNA is found in polysomes and the protein accumulates during oogenesis [33]. It, therefore, seems likely that EDEN-BP and/or other proteins binding to EDEN containing mRNAs provide additional mechanisms to achieve complete translational repression.

3. Translation repression by eIF4E binding complexes As described above, eIF4G is recruited to the 5
end of the mRNA by the binding of eIF4E. This binding is mediated by a 4E binding motif in eIF4G, YxxxxL␾, where ␾ is an aliphatic residue, usually F, L or M. Interference with this association can be expected to greatly reduce normal, cap-dependent translation. Indeed small, heat-stable proteins called eIF4E-binding protein (4E-BPs), which also contain the 4E binding motif can repress general cap-dependent translation by excluding eIF4G from the cap-binding initiation complex. The affinity of 4E-BPs for eIF4E is modulated by their phosphorylation (for a review, see [34]). Whereas the 4E-BPs have effects on a large population of mRNAs by sequestering the majority of eIF4E molecules in a cell, mRNA associated proteins containing eIF4E binding motifs are expected to be specific translational repressors as long as their concentration is well below that of eIF4E. Several such translational regulators have been proposed to play a role in development. 3.1. Maskin, Pumilio and Xp54 in the translational repression of cyclin B1 In full grown but immature Xenopus oocytes, the mRNA for cyclin B1 is translationally repressed by sequences in its 3
untranslated region that include the multiple cytoplasmic polyadenylation elements (CPEs) [35,36]. Three synthetic CPEs are sufficient for full repression, but other sequences in the cyclin B13
UTR are also likely to contribute [36]. This translational repression requires initiation of translation to be cap-dependent. The CPE binding protein CPEB binds a protein called Maskin, which contains an eIF4E binding motif, and so can repress translation by excluding eIF4G

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from the mRNP complex [37]. The eIF4E-binding motif of Maskin is somewhat atypical in that the tyrosine is replaced with a threonine (TEADFLL), but its binding to eIF4E has been clearly demonstrated. Reduction of Maskin activity in oocytes or embryos leads to elevated cyclin B1 levels [17,38]. The translational activation of CPE-containing mRNAs during oocyte maturation requires cytoplasmic polyadenylation [36]. In concordance with this fact, Maskin dissociates from eIF4E during meiotic maturation and this separation requires poly(A) binding protein (PABP) and a functional interaction between eIF4G and PABP [38]. These data indicate that Maskin is involved in translational repression of the cyclin B1 mRNA by competitively blocking eIF4G recruitment in Xenopus laevis, as depicted in Fig. 2B. However, the Maskin homologs in other species, the transforming acidic coiled coil domain proteins (TACCs), all lack the eIF4E binding motif. Even the close Xenopus relative Silurana (Xenopus) tropicalis has not retained this sequence (TDADFFP, accession AL887399 and AL894198). TACCs are centrosomal proteins that have a conserved function in mitotic spindle formation in Caenorhabditis, Drosophila and mammals [39]. Depletion of the closest human homolog of Maskin, TACC3, leads to a delay in mitosis in HeLa cells, similar to the effect of Maskin knockdown in Xenopus embryos [17,40]. It is therefore difficult to be certain if this phenomenon is due to translational control of cyclin B1, to the microtubule organising role of Maskin/TACC3, or to both. Translational repression by CPEs is, however, definitely conserved, as these elements also mediate translational repression in immature mouse oocytes, where they also have been linked to deadenylation [41,42]. In addition to Maskin, two other CPEB binding proteins have been implicated in translational repression: the Puf repeat protein Pumilio and the RNA helicase Xp54 [43,44]. Pumilio is part of a highly conserved family of RNA binding proteins (Puf family) that have roles in translational control in many organisms, including animals and yeast [1,23,45]. Puf proteins are involved in regulating multiple mRNAs in the cell cycle and in germ cell determination in Drosophila and Caenorhabditis. In fact, Pumilio is involved in the translational control of cyclin B in the Drosophila germ plasm [46]. In Xenopus oocytes, Pumilio overexpression inhibits cyclin B1 translation and anti-Pumilio antibody injection induces it, providing the first evidence that the function of these proteins is conserved in vertebrates too [47]. In addition to interacting with CPEB, Pumilio also has binding sites of its own in the cyclin B13
untranslated region, which contribute to the translational repression. The mechanism of action of Puf protein mediated translational repression is at present unknown, but Puf protein binding to mRNAs has been linked to deadenylation in two cases [48,49]. The Xp54 RNA helicase, a member of the RCK family that also includes Drosophila Me31B (see below), was found to associate with RNA-bound CPEB in both Spisula (clam p47) and Xenopus [43]. Tethering of the helicase to an heterol-

Fig. 2. Models for the repression of translation. (A) Drosophila caudal mRNA repression by binding of a complex between Bicoid and eIF4E (4E), excluding eIF4G from mRNP. (B) Xenopus cyclin B1 mRNA repression by a complex consisting of CPEB, Maskin, eIF4E which excludes eIF4G from the mRNA. The RNA helicase Xp54 and the RNA binding protein Pumilio both bind to CPEB and repress via less well characterised mechanisms. (C) Rabbit lipoxygenase mRNA repression by hnRNPK and hnRNP E1 somehow prevents the joining of the 60S ribosomal subunit. (D) Drosophila oskar mRNA binds a complex of eIF4E, CUP and Bruno, which excludes eIF4G. Binding of Hrp48 to the 5 and 3 UTR enhances repression. Polysomal association of the message and mutational analysis suggest an additional mechanism of repression exists, possibly involving microRNA binding (RISC) and/or the nascent polypeptide associated complex (NAC). For detailed discussions, see under the subheadings in the text. Note that the description of the regulation of the oskar mRNA occurs under two headings.

ogous mRNA using a heterologous RNA binding domain results in translational repression in injected oocytes. This repression is dependent on helicase activity. A poly(A) tail cancels the effect of tethered Xp54, consistent with the activation by cytoplasmic polyadenylation of the cyclin B1 mRNA. Strikingly, eIF4E immunoprecipitates with the MS2-Xp54

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fusion protein and this interaction becomes RNA-dependent upon oocyte maturation, suggesting that a protein-dependent interaction is removed at this time [50]. However, so far it is unclear whether the eIF4E interaction with Xp54 is direct, mediated by other proteins or simply due to RNAse resistance of the repressed mRNP. Direct evidence for a requirement of Xp54 activity in the translational repression of cyclin B1 is also lacking. 3.2. Regulation of caudal translation by Bicoid The homeo-domain containing protein Bicoid forms a morphogenic gradient in Drosophila embryos, acting both at the level of transcription and translation (reviewed in [51]). The Bicoid gradient represses the translation of the uniformly distributed caudal mRNA, which encodes a transcription factor necessary for posterior segmentation. The translational repression is mediated by the Bicoid-binding region (BBR) in the 3
UTR of the caudal mRNA [52,53]. The binding of Bicoid to RNA requires its DNA binding homeo domain and is required for translational repression of caudal [52,54,55]. Bicoid contains a eIF4E binding motif that is necessary both for association with eIF4E and for translation repression, but that is not required for the transcriptional activation activity of the protein [54]. This leads to a model in which Bicoid binding to the caudal mRNA allows it to bind to the eIF4E bound to the cap of this mRNA and prevents the recruitment of eIF-4G, as depicted in Fig. 2A. In addition to the homeodomain and the 4E binding motif, a protein–protein interaction domain (PEST) in Bicoid is also required for translational repression of caudal, indicating that the actual mechanism is probably somewhat more complex than the current model suggests [56]. 3.3. Regulation of oskar and nanos translation by Cup Localisation of mRNAs in the oocyte and early embryo plays a large role in the early development of Drosophila. In most cases, the mRNA is synthesised in the nurse cells and transported to its final location in the oocyte in a translationally repressed form. One such mRNA encodes the germline determinant Oskar. The translational repression sequences in the 3
UTR of the oskar mRNA are bound by the RNA binding protein Bruno, which is required for translational repression of oskar [57]. A novel Bruno-binding protein, Cup, binds to eIF4E using an eIF4E binding motif, and this motif is required for the action of Cup. These data result in a model in which Bruno recruits Cup to the mRNA and Cup blocks eIF4G recruitment through its interaction with eIF4E [58–60]. Activation of oskar requires the CPEB homologue Orb and a long poly(A) tail, indicating that the activation of translation of localised oskar mRNA may be mediated by cytoplasmic polyadenylation [61,62]. These features make the translational control of oskar remarkably similar to the proposed translational regulation of cyclin B1 by CPEB and maskin in Xenopus oocytes.

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However, this model is certainly too simple, as outlined below. Hrp48, an RNA binding protein related to mammalian hnRNP A/B, binds to the 5
and 3
UTR of oskar mRNA and is also required for translational repression [63–65]. A dimerisation of the Hrp48 bound to the ends of the mRNA is proposed to aid the association of Cup with eIF4E. In addition, among the many genes that are necessary for normal translational silencing of oskar are those encoding the Y box protein Yps and the RNA helicase Me31B [63–67]. Because these proteins are found in oskar mRNA containing complexes, it is likely that their effect is direct. Nanos protein is a translational regulator involved in posterior patterning in partnership with Pumilio. Its mRNA is only translated in the posterior of the embryo. Translational repression of nanos mRNA in the rest of the embryo is mediated by sequences in its 3
UTR that bind the SAM domain protein Smaug [68,69]. Smaug binds Cup and Cup is necessary for nanos translational repression, indicating a similar model as outlined for oskar translational repression by Bruno and Cup [70]. A single domain in Smaug binds either Oskar or Cup. Consequently, the localisation of the Oskar protein in the posterior pole could mediate the activation of the nanos mRNA in this region, by an exchange of Oskar for Cup on the Smaug protein [71]. There are some problems with the notion that translational control of oskar and nanos occurs at the level of translation initiation, as the Cup model suggests, and these are discussed in the section on post-initiation repression.

4. Regulation of ribosomal subunit recruitment Although the cap-binding translational repressors are currently enjoying a surge of interest, there are clear indications that this is not the only step in translation initiation that can be regulated developmentally. Two cases, one affecting 40S recruitment and the other affecting 60S recruitment, are described below. The exact molecular mechanisms of these modes of regulation are as yet unknown. 4.1. Translational control of msl-2 mRNA by Sex-Lethal In Drosophila, the upregulation of transcription from the single X chromosome in the male (dosage compensation) is mediated by the Male Specific Lethal (MSL) transcription complexes [72]. The msl genes are equally transcribed in males and females, but the crucial component msl-2 is repressed post-transcriptionally in females by the RNA recognition motif (RRM) protein Sex-Lethal (SXL). SXL mediates the retention of an intron in the 5
UTR of the msl-2 mRNA [73,74]. Full translational repression of this mRNA requires SXL binding to U rich sequences in the retained intron, as well as in the 3
UTR of the mRNA [73–75]. The 3
UTR sequences probably recruit additional proteins that are necessary for this action [76]. As the translation of uncapped

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or unpolyadenylated mRNAs is still repressed by these sequences, the repression does not interfere with the formation of the closed loop complex [75,77]. Sucrose gradient analysis has demonstrated that SXL binding to both UTRs prevents the stable association of the small ribosomal subunit with the mRNA [77]. 4.2. Translational control of rabbit 15 lipoxygenase During the final steps of erythroid differentiation, the enzyme 15 lipoxygenase (LOX) destroys the mitochondrial membrane and this is partially controlled at the level of translation [78]. The 3
UTR of the rabbit LOX mRNA contains the differentiation control element (DICE), consisting of 10 contiguous repeats of the sequence C4 RC3 UCU2 C4 A2 G, with slight variations [79,80]. The binding of hnRNP K and hnRNP E1 to this element blocks the joining of the 60S large ribosomal subunit to the small subunit at the initiation codon, as depicted in Fig. 2C [81]. HnRNP K is usually a predominantly nuclear protein, but phosphorylation by ERK increases the cytoplasmic concentration and enhances DICE-mediated translational repression in HeLa cells, adding a potential layer of regulation [82]. Activation of the translationally repressed messenger can be achieved by c-Src mediated phosphorylation of hnRNP K, which leads to the release of this RNA binding protein from the DICE [83].

5. Post-initiation repression Classically, translational control of specific mRNAs has been thought to occur at the level of translation initiation. However the number of repressed mRNAs found in the polyribosomes has been growing steadily, and the discoveries described below seem set to drive a large increase in the near future. The mechanisms for translational repression in polyribosomes are unclear, but it seems likely that the elongation or termination steps of translation are affected. However, there is evidence that, at least in some cases, repression is mediated by degradation of the nascent protein as well. 5.1. microRNAs During the larval development of C. elegans the mRNA for the transcription factor LIN-14 is repressed through the action of the lin-4 gene, which encodes a 22 nt RNA. The lin-4 RNA mediates this repression through imperfect basepairing with several target sequences in the 3
UTR of the lin-14 mRNA [84–86]. The repression of LIN-14 expression must be controlled in a post-initiation step as the lin-14 mRNP is polyribosomal as judged by its migration in sucrose gradients and its buoyant density [87]. Recently, hundreds more of such endogenous RNA regulators, now called microRNAs (miRNAs), have been found, both by bioinformatic analysis and experimentally, in plants and animals, including humans (reviewed in [88,89]). miR-

NAs are processed from imperfectly base-paired hairpin precursors and display considerable evolutionary conservation, both in the sequence of the miRNAs as well as in the genomic organisation of their precursor encoding regions [90,91]. miRNA encoding genes may account for as much as1% of the genes in plants and animals, indicating that they are likely to be a major class of regulators of gene expression [89]. miRNA processing and repressor complex assembly requires similar enzymes and RNP components as those required for RNA interference (RNAi) (reviewed in [88,89]). The major difference between the effectors of RNAi, the short interfering RNAs (siRNAs), and miRNAs seems to be that siRNAs are derived from exogenously provided perfectly double stranded RNA instead of from imperfectly hybridised stems from endogenously transcribed RNAs. However, even this processing is mediated in part by the same enzyme, Dicer [88,89]. Indeed, whereas originally siRNAs were thought to direct RNA degradation through cleavage and miRNAs to mediate translational repression, it now appears that each can mediate the function originally ascribed to the other, depending on the degree of complementarity with the target mRNA. Perfectly base-paired si/miRNAs mediate cleavage, and imperfectly basepaired si/miRNAs mediate repression [92–94]. For cleavage, only one perfectly complementary site is required in the mRNA, but for repression several miRNA binding sites are required, with a high degree of complementarity in the 5
region of the miRNAs being crucial for targeting [95]. Because of these similarities between the biochemistry and genetics of siRNAs and miRNAs, their effector complexes are now collectively called the RNA-induced silencing complexes (RISCs) [88]. RISC components have been shown to be essential for early development, including germ cell maintenance. However, only a few validated target mRNAs for miRNAs exist, most of them developmental regulators in C. elegans [88]. A wealth of potential targets have been identified computationally in Drosophila and vertebrates, implicating a large variety of biological functions. The way in which RISCs mediate repression of their mRNA targets is still obscure. The data from study of lin-14 regulation indicate that the repressed mRNAs are polyribosomal [87], but the lack of data on puromycin treatment or ribosome run-off of this mRNA (see below), makes it impossible to decide between inhibition of translation or degradation of nascent protein. The core protein components of the RISC appear to be the Argonaute proteins (reviewed in [88,89]). These proteins contain conserved PAZ and Piwi domains and are present as multiple orthologs in all species that have RNAi. The function of the Piwi domain is unknown, but the PAZ domain forms a nucleic acid binding fold and is proposed to bind to the RNA [96,97]. A mammalian Argonaute orthologue was first cloned as the translation initiation factor eIF2C, a factor that stabilises the ternary initiation complex (eIF2, GTP and methionyl-tRNA) and the methionyl-tRNA interaction with the 40S ribosome [98,99]. Interference with the forma-

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tion of these complexes would lead to inhibition of ribosome subunit recruitment (see Fig. 1), which is inconsistent with a polyribosomal association of the mRNA. RISC mediated RNA degradation has been reported to require a number of RNA helicases from different families, at least one of which is required for RISC assembly, but none has been directly implicated in mRNA repression so far [100]. Interestingly, a homologue of Fragile X Mental Retardation Protein (FMRP) has also been found in the RISC in Drosophila [101]. FMRP and its relatives FXR1P and FXR2P are also found in RISC in human cells and FMRP overexpression mutants interact genetically with Argonaute mutants in Drosophila, indicating that FMRP is a conserved and functional component of RISC [102]. In humans, reduction of FMRP expression leads to Fragile X linked mental retardation and FMRP affects synaptic plasticity [103]. FMRP is associated with polyribosomal mRNAs and ribosome runoff experiments indicate that these associated ribosomes are elongating at least to some extent [104]. It, therefore, seems likely that FMRP mediated mRNA repression is related or possibly identical to miRNA mediated repression and that this repression is not efficient in preventing translation elongation. This is similar to the situation described for Oskar and Nanos synthesis, as discussed below. 5.2. Polyribosomal association of oskar and nanos mRNA Mutations affecting the components of the RNA silencing complex are also implicated in repression as well as the activation of oskar mRNA translation, opening the possibility of the involvement of microRNAs in this increasingly complex regulatory system [100,105–107]. This hypothesis better explains the properties of the endogenous mRNPs than the Cup model, as the oskar and nanos mRNA are found in the polyribosome fractions in different types of mutant flies that do not translate these mRNAs [108,109]. Puromycin treatment drives these mRNAs from the polyribosomes. Since puromycin acts by binding to the ribosome like an aminoacyltRNA and releasing the peptide chain after translocation, this suggests that the ribosomes are actively translating the mRNA. Consistent with this notion, ribosomes run-off the nanos mRNA in extracts in which Nanos protein expression is repressed [109]. These data indicate that a 3
UTR mediated degradation of the nascent peptide is involved in nanos and oskar post-transcriptional control. Strikingly, the intact nascent polypeptide-associated complex (NAC) is required for proper post-transcriptional control of oskar mRNA [108]. The known molecular function of NAC is to prevent spurious interactions of the signal recognition particle with nascent proteins by binding them, which could provide a potential link to nascent protein stability and/or to ribosomal pausing. Confusingly, there is also good evidence that Nanos and Oskar expression are regulated by the eIF4E-binding protein Cup and its recruiting RNA binding proteins (see above), leading to the model depicted in Fig. 2D. One explanation

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is that these two mechanisms could regulate translational repression at different stages in oogenesis [59,60,105]. Alternatively, the translation of these mRNAs is repressed by their 3
UTRs at both the initiation and elongation steps (to account for the observed polysomal association), as well as at the level of nascent protein stability.

6. Concluding remarks Translational control by the 3
untranslated region can affect formation of the closed loop translation initiation complex (cytoplasmic polyadenylation, deadenylation and cap-binding repressors), ribosome binding (Sex-lethal and lipoxygenase) or a post-initiation step (lin-14, oskar, nanos, FMRP associated mRNAs). A large number of translational regulators are evolutionarily conserved including the Puf protein family, CPEB/Orb, Bruno-like proteins, RNA helicases and components of RISC, including microRNAs. Many regulated mRNAs contain multiple translational control elements, indicating a much greater complexity of regulation than previously expected.

Acknowledgements Our research has been supported by the Wellcome Trust and the BBSRC.

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