Figure 17.10. Model for translational control of ferritin synthesis. The 5'-UTR of ferritin mRNA contains IRE (see Fig. 17.8). When Fe3+ is scarce, a cytoplasmic IRE-binding protein (IRE-BP) interacts with IRE and prevents the 43S initiation complex from associating with the cap structure of the ferritin mRNA (see Fig. 17.8, A). When Fe3+ is abundant, the IRE-BP looses its affinity to IRE and dissociates thus allowing the initiation and translation of the ferritin mRNA.

(Reproduced, with modifications, from T.A. Roualt, R.D. Klausner & J.B. Harford, in "Translational Control", J.W.B. Hershey, M.B. Mathews & N. Sonenberg, eds., p.p. 335-362, CSHL Press, 1996, with permission).

The repression of translation using this mechanism can be artificially reproduced by constructing chimeric mRNAs bearing a 5'-proximal hairpin structure in the 5'-UTR with a specific affinity for a chosen mRNA-biding protein. For example, when the hairpin structure with a high affinity for the bacteriophage MS2 coat protein (see Fig. 16.6) was introduced into the 5'-UTR of capped chloramphenicol acetyltransferase transcript the coat protein was found to be an efficient repressor of translation of this mRNA both in vitro, in rabbit reticulocyte and wheat germ cell-free systems (Stripecke & Hentze, 1992), and in vivo, within human HeLa cells and yeast (Stripecke et al., 1994). In the same way the spliceosomal protein U1A was converted into translational repressor when the protein-binding site (weak hairpin) from the small U1 RNA was placed at the 5'-UTR of the chloramphenicol acetyltransferase mRNA (the same refs.). In both cases the translational repression seemed to be caused by the formation of a stable RNA/ protein complex near the cap structure thus preventing the association of the initiating ribosomal particle (43S initiation complex) with mRNA. The experiments have demonstrated that this type of translational repression mechanism may indeed be generally used by eukaryotic cells.

17.5.3.Regulation of Ribosomal Protein mRNA Translation

The synthesis of ribosomal proteins during oogenesis, embryogenesis and in response to changes in cellular growth rate is also regulated at the translational level by a repression mechanism. The 5'-UTRs of ribosomal protein mRNAs have been shown to be involved in regulation of their translation in mammalian cells, Xenopus embryos, insects and slime molds (for reviews, see Jacobs-Lorena & Fried, 1987; Kaspar et al., 1993; Meyuhas et al., 1996).

In vertebrates the ribosomal protein mRNAs have a common sequence motif, an oligopyrimidine tract of 7 to 14 nucleotides starting with C at capped 5'-terminus, that seems to be important for the translational regulation. No hairpin structure, however, can be generated within the short pyrimidine-rich 5'-UTRs of the ribosomal protein mRNAs. In addition to the polypyrimidine 5'-terminus, a sequence immediately downstream seems to be required for the full manifestation of the translational control. Conceivably, a repressor protein should be able to specifically bind to the 5'-terminal regulatory sequence ("translational regulatory element", or TRE) of ribosomal protein mRNA and prevent the initiating ribosomal particles from interacting with the mRNA. The affinity of such a repressor protein for TRE must change in response to cellular demands for ribosomal proteins. In any case, in contrast to prokaryotic situation, the eukaryotic ribosomal protein mRNAs are not autogenously regulated by ribosomal proteins.

No repressor protein has been unambiguously identified yet to bind to the polypyrimidine tract of the 5'-UTR in ribosomal protein mRNAs of vertebrates. Several polypyrimidine-binding proteins were suspected but without direct proof of their repressor action. Granted that such a repressor does exist, the question arises whether the sequence-specific binding of the protein to the 5'-terminal sequence can block the ribosome/mRNA association by itself, without stabilisation of a structured RNA element. In such a

ligand-free reduced state

Figure 17.11. Model for translational autoregulation of thymidylate synthase (TS) synthesis. (After E. Chu & C.J. Allegra, BioEssays 18, 191-198, 1996). The enzyme in a ligand-free (in the absence of thymidylate and folate), reduced state has an affinity to a stem-loop structure on the border between the 5'-UTR and the coding sequence of its own mRNA, as well as to a pseudoknot structure inside the coding sequence. The binding of TS to the upstream hairpin stabilises it and thus blocks the movement of the scanning 48S initiation complex to the initiation AUG codon (see Fig. 17.8, B).

ligand-free reduced state

Figure 17.11. Model for translational autoregulation of thymidylate synthase (TS) synthesis. (After E. Chu & C.J. Allegra, BioEssays 18, 191-198, 1996). The enzyme in a ligand-free (in the absence of thymidylate and folate), reduced state has an affinity to a stem-loop structure on the border between the 5'-UTR and the coding sequence of its own mRNA, as well as to a pseudoknot structure inside the coding sequence. The binding of TS to the upstream hairpin stabilises it and thus blocks the movement of the scanning 48S initiation complex to the initiation AUG codon (see Fig. 17.8, B).

case the mechanism of translational repression should be considered somewhat different from that discussed in the preceding Section. For example, a direct prevention of interactions of the capped 5'-terminus with initiation factors or initiating ribosomal complex is possible.

17.5.4.Repression by Prevention of Initiation Complex Movement along mRNA

A different repression mechanism is possible where the interaction of an RNA element of 5'-UTR with a repressor protein does not prevent the association of the ribosomal 43S complex with mRNA but forms a barrier which cannot be overcome (melted) by the 43S complex moving (scanning) from the cap structure to the initiation codon (Fig. 17.8 B). Seemingly this is the case of the feed-back translational repression of the human thymidylate synthase mRNA by the product of the translation, i.e. by the thymidylate synthase (Chu et al., 1993a). Here a 30 nucleotide stem-loop structure in the 5'-UTR specifically interacts with the thymidylate synthase probably resulting in (or contributing to) the repression of translation (Fig. 17.11). The stem-loop element is about 80 nucleotides apart from the cap and includes the initiation codon. There is also the second thymidylate synthase-binding site in the coding region of the same mRNA, the role of which is not clear. In any case the thymidylate synthase, the enzyme catalysing the conversion of dUMP into dTMP, is found to be a specific mRNA-binding protein and translational repressor of its own mRNA.

There is an indication that human dihydrofolate reductase (DHFR) is also capable of specifically binding to its own mRNA and to repress translation (Chu et al., 1993b), though no specific site for the binding of the protein within the 5'-UTR has been identified yet.

17.6. mRNA Masking

The most typical examples of masked mRNAs are the messages stored in oocytes and spermatocytes. There are also striking examples of long-term storage of mRNAs in somatic cells. The stored mRNA is really masked against any processive events, including translation, degradation and polyadenylation/ deadenylation. This can be accepted as a definition of masking, in contrast to repression where just one function, translation, is blocked. The most remarkable discovery during last years was the findings that the masking of mRNA involves primarily the 3'-UTRs of mRNAs and that the specific interactions of proteins ("masking proteins") with defined regions within the 3'-UTRs are responsible for switching off the functional activities of the respective mRNAs (Fig. 17.12) (for reviews, see Spirin, 1994, 1996).

17.6.1.Masked mRNA in Oocytes and Spermatocytes

The pioneer report in this field was made by Standart et al. (1990). It was demonstrated that oocytes of a clamp Spisula solidissima contain large amounts of masked mRNA encoding for the small subunit of ribonucleotide reductase and for cyclin A. Fertilisation triggers selective unmasking of these major stored mRNAs of the oocyte cytoplasm, while translation of a number of previously active mRNAs ceases. Under in vitro conditions, the unmasking of these two mRNAs was achieved by high salt treatment which presumably resulted in the dissociation of masking proteins. The most remarkable observation, however, was that a specific region in the middle of the 3'-UTR was responsible for binding a masking protein ("dissociable factor") (see Fig. 17.12). Those specific unmasking experiments were done with the use of antisense RNAs complementary to defined regions in the 3'-UTRs of both the mRNAs under study (the so-called "competitive unmasking assay"). The removal of the 3'-UTR sequence with the "masking box" from the mRNA also resulted in the promotion of translation. An oocyte protein of 82 kDa was found to bind specifically to the "masking element" within the 3'-UTR, and its presence in the masked mRNPs correlated with translational inactivity of the corresponding mRNAs. The evidence has been obtained that the unmasking of the maternal mRNAs is due to a maturation-dependent kinase which phosphorylates the protein (Walker et al., 1996).

The decisive role of the 3'-UTR in masking mRNA during developmental processes was supported by genetic analysis of the switch from spermatogenesis to oogenesis in the hermaphrodite nematode Caenorhabditis elegans (Ahringer & Kimble, 1991). During development of the hermaphrodite, the germ cell precursors differentiate into sperms at the larval stage and then into oocytes in adult animals. The translation of the so-called fem-3 mRNA is responsible for directing spermatogenesis, and the subsequent masking of this mRNA determines the switch to oogenesis. Point mutations in the middle of its 250 nt 3'-

coding sequence

o masking protein

Figure 17.12. Schematic representation of the masking induction effect on mRNA of a special protein ("masking protein") recognising a middle section ("masking box") within 3'-UTR. The eventual result of the interaction of the 3'-UTR with the "masking protein" is the prevention of translation initiation (at 5-end of the mRNA) and the stabilisation of mRNA against enzymatic attacks (at 3'-end especially). The classical examples are the masking of ribonucleotide reductase mRNA in oocytes of a clamp Spisula solidissima (N. Standart, M. Dale, E. Stewart & T. Hunt, Genes Dev. 4, 2157-212168, 1990), fem-3 mRNA during the switch from spermatogenesis to oogenesis in the hermaphrodite nematode Caenorhabditis elegans (J. Ahringer & J. Kimble, Nature 349, 346348, 1991), nanos mRNA of Drosophila eggs and early embryos (E. Gavis & R. Lehmann, Nature 369, 315-318, 1994), and erythroid 15-lipoxygenase mRNA during erythropoiesis in mammals (A. Ostareck-Lederer, D. Ostareck, N. Standart & B. Thiele, EMBO J. 13, 1476-1481, 1994). (See also M. Wickens, J. Kimble & S. Strickland, in "Translational Control", J.W.B. Hershey, M.B. Mathews & N. Sonenberg, eds., p.p. 411-450, CSHL Press, 1996, for further reading).

UTR, as well as the deletion of the central part of the 3'-UTR, abolish the capability of the fem-3 mRNA to be masked and, hence, to allow oogenesis to occur. It seems that the mutations destroy a binding site for a protein whose function is to induce masking (see Fig. 17.12).

Another sex-determining mRNA of C. elegans, tra-2, which seems to be accumulated during oogenesis in a masked form and then activated to direct sexual differentiation, was also shown to be regulated by a specific RNA-binding protein interacting with a sequence (direct repeat) in the 3'-UTR.

Spermatogenesis is another developing system where the masking/ unmasking phenomena seem to play a decisive role (see Brown, 1990; Hecht, 1990; Schaefer et al., 1995; Kleene, 1996). Here transcription ceases during meiosis or at early post-meiotic stages, but translation of mRNA encoding for the most abundant spermatozoan proteins (e.g., protamines) is delayed for many days. Thus the mRNA is stored in a masked form as cytoplasmic mRNP particles and translated only during late spermiogenesis. In particular, protamine mRNA in mice is found to be accumulated as an untranslated mRNP during the stage of round spermatid, stored for up to one week, and is translated at the stage of elongating spermatid. It is the 3'-UTR of protamine mRNA that proved to be responsible for this type of translational regulation. Though some proteins capable of specifically binding to the 3'-UTR sequences have been indicated, what is the function of the 3'-UTR-binding proteins in mRNA masking and what governs their binding or removal during spermiogenesis remains to be determined.

17.6.2.mRNA Masking and Unmasking during Embryonic Development

Early embryonic development and morphogenesis in Drosophila includes a number of interrelated events of mRNA masking and unmasking (for recent reviews, see Seydoux, 1995; Wickens et al., 1996). One of the maternal masked mRNAs in Drosophila eggs is nanos mRNA encoding for Nos protein, a morphogen governing the abdominal segmentation in embryos by blocking expression of other mRNAs. After fertilisation the nanos mRNA is localised at the posterior pole of the laid egg due to a special sequence element in its 3'-UTR which determines this anchoring. The sequences required for the posterior localisation and for the masking are overlapping. While unlocalised nanos mRNA is really masked, i.e. inactive and stable, the localisation of the mRNA induces its unmasking. Thus, a novel trigger of unmasking has been revealed. Since the nanos mRNA is active in translation only locally, at the posterior pole of the egg, the Nos protein is synthesised just at the posterior pole and diffuse to the anterior pole thus forming a gradient along the egg and then along the anterior-posterior axis of the embryo.

The Nos protein is found to be an RNA-binding protein recognising similar sequences, the so-called NRE (for "Nos Response Element"), in the 3'-UTRs of two other maternal mRNAs, namely hunchback mRNA uniformly distributed in the egg, and bicoid mRNA localised at the anterior pole. The interaction of Nos with the NREs of these mRNAs blocks their translation. The consequence is that the expression of hunchback mRNA is gradually decreased from the anterior pole to the posterior, this being the main factor in the proper abdominal segmentation of the embryo. Normally the bicoid mRNA should not meet Nos because of the anterior localisation of this mRNA, but in the case of its delocalisation it will be blocked by Nos. It is not clear, however, if the blocking effect of Nos is real masking of the mRNAs, or just a different type of translational inactivation without mRNA conservation. As to hunchback mRNA, its rapid degradation after the translational inactivation was reported.

17.6.3.mRNA Masking and Unmasking during Cell Differentiation

The best studied case of mRNA masking/unmasking during final stages of cell differentiation, rather than at germ cell maturation and activation, or at early embryonic development, is the fate of the mRNA encoding for erythroid 15-lipoxygenase (LOX) (Ostareck-Lederer et al., 1994). The LOX mRNA is synthesised at the early stages of erythropoiesis and becomes masked, i.e. stored in the form of untranslatable and stable cytoplasmic mRNPs, for all the subsequent stages, until the late stage of peripheral reticulocytes. The unmasking of the LOX mRNA and the synthesis of the enzyme takes place during maturation of reticulocytes into erythrocytes. The enzyme attacks phospholipids thus inducing the degradation of mitochondria during final erythrocyte maturation. The long 3'-UTR of the reticulocyte LOX mRNA contains a characteristic sequence where a pyrimidine-rich 19 nucleotide motif is repeated 10 times. It is the 3'-UTR repeat region that was found to be responsible for the masking of the LOX mRNA through specific binding of a 48 kDa protein. The 48 kDa protein (LOX-BP) is a part of the translationally inactive (masked) LOX-mRNP in bone marrow cells and reticulocytes. The protein is also capable of selectively inhibiting translation of hybrid foreign mRNAs, such as chloramphenicol acetyltransferase or luciferase mRNAs, containing the same 3'-UTR regulatory element. The minimal binding site for LOX-BP which is sufficient for the effect is 2 to 4 repeats. It is noteworthy that the LOX-BP seems to acts independently of the 5'-cap and 5'-UTR, so that the involvement of a "cross-talk" between the two ends of mRNA (see below) is unlikely in this case.

17.6.4.Masking and Unmasking of mRNA in Differentiated Cells

There are also other examples of the involvement of the 3'-UTR in translational control (reviewed in Spirin, 1994, 1996). Creatine kinase B mRNA was shown to be regulated due to its 3'-UTR, and a specific RNAase-resistant, gel-retarded complex was demonstrated to be formed from this mRNA or its 3'-UTR and some components (presumably proteins) of the cell extract. This suggests that some portion of the 3'-UTR of creatine kinase B mRNA binds to a protein resulting in block of translation, and that the block can be relieved in response to stage-specific, tissue-specific, or hormonal regulatory signals.

Translation of human interferon p mRNA was also reported to be dependent on its 3'-UTR. The translation was inhibited in animal cell extracts but not in the wheat germ extract thus suggesting the existence of a specific 3'-UTR-binding "inhibitor" in animal cells. The sequence responsible for the translational inhibition was found to be rich in uridines and adenosines and to contain several AUUUA repeats. It is remarkable that the sequence is effective in mRNA repression independent of its position within the 3'-UTR, but is no longer effective when inserted upstream from the AUG initiation codon, i.e. in the 5'-UTR.

Similar AU-rich elements (ARE) in the 3'-UTRs were reported to be recognition signals for selective rapid degradation of several other mRNAs, such as lymphokine, cytokine and protooncogene mRNAs. Correspondingly, all these mRNAs also seem to be subjected to the translational control under consideration. The translational control imparted by the presence of the AU-rich element in the 3'-UTR seems to be regulated (released) by inducing stimuli. For example, the tumour necrosis factor (TNF) mRNA or some artificial chimeric mRNAs can exist within macrophages in a translationally inactive, or stored form, seemingly due to the presence of the AU-rich element in the 3'-UTR. Endotoxin (LPS) specifically induces their translation. Just as it is important in suppressing translation, the AU-rich element has been found to be critical for response to endotoxin. All this suggests the existence of cytoplasmic protein factors (proteins) which can recognise the AU-rich elements in the 3'-UTR thus inducing mRNA masking, and also respond to a signal by releasing its masking activity.

Indeed, several groups reported on identification of a number of cytoplasmic RNA-binding proteins specifically recognising the AU-rich and U-rich sequences in the 3'-UTRs of cytokine, lymphokine and oncogene mRNAs. The proteins identified by different groups vary in their size from 15 to 70 kDa. It is not clear which of them are mRNA-destabilising factors and which can participate in mRNA masking. A remarkable observation is that some of the proteins earlier known as constituents of heterogeneous nuclear ribonucleoproteins (hnRNPs), namely proteins A1 and C, are found in the cytoplasm to be associated with the AUUUA sequences in the 3'-UTRs of mRNAs. In agreement with this, the proteins were shown to shuttle between the nucleus and the cytoplasm. Both proteins, A1 and C, possess typical RNA-binding domains (RBD) of the "RNP consensus sequence" (RNP-CS) type, with two conserved "RNP-1" and "RNP-2" sequence motifs. About ninety amino acid residues of the protein C RBD are arranged into a two-layer pappap structure where a four-stranded antiparallel b-sheet with the conserved RNP-1 and RNP-2 sequences is laid on two antiparallelely packed a-helices.

There are experimental indications that some special sequences in 3'-UTR may be required for unmasking of mRNA. Thus, it was reported that the removal of the 3'-proximal hundred nucleotide sequence of the tissue plasminogen activator (t-PA) mRNA 3'-UTR prevented both translational activation and destabilisation of the masked mRNA, as well as its polyadenylation. The existence of some protein factors recognising specific structures of the 3'-UTRs and thus inducing mRNA (mRNP) unmasking has been suspected. Such a trans-acting unmasking factor or activator relieving the masking effect of the TNF mRNA 3'-UTR has been detected in one of the human cell lines. The factor seems to abolish the effect of the 3'-UTR-binding masking factor which was discussed above and found to be more universal for different human and mammalian cell lines. It is not known if the unmasking factor directly or indirectly competes with the masking factor and displaces it from mRNA, or the unmasking effect overpowers the masking.

17.6.5.Models of mRNA Masking

Virtually in all the cases of mRNA masking the effect of the 3'-UTR and the 3'-UTR-binding protein(s) is displayed as the block of the initiation step of translation. At the same time the initiation takes place at the 5'-part of mRNA. The question arises: How can the 3'-located events affect the 5'-located processes? One hypothesis could be that the 3'-part and the 5'-part of mRNA are in a protein-mediated contact with each other providing some kind of a "cross-talk" or even a non-covalent "circularisation" (see Section 15.3.7 and Fig. 15.14).

An alternative model can be based on the fact that during all its life-time mRNA is complexed with a large amount of protein and thus organised in mRNP structures. Global structural reorganisations of mRNP (e.g., like condensation/decondensation of chromatin) could result in mRNA masking/unmasking. Interactions of the 3'-UTR with signal-regulatable proteins can be supposed to induce structural reorganisations of mRNP particles. Masked mRNA may be considered as a "condensed" form of mRNP (Fig. 17.13) where RNA is not available for the functional interactions with other macromolecules, including ribosomes and/or translational initiation factors, poly(A) polymerase, and ribonucleases. This "structural masking theory" does not necessarily exclude the idea of the 3'-part being in proximity to the 5'-part of masked mRNP.

In addition to the key role of 3'-UTR-binding proteins in mRNA masking, the core mRNP protein p50 (see above, Section 17.2.2) has been mentioned as a principal participant of masking processes (Sommerville, 1992; Sommerville & Ladomery, 1996; Ranjan et al., 1993; Tafuri et al., 1993; Bouvet & Wolffe, 1994; Wolffe, 1994; Matsumoto et al., 1996). Indeed, the extensive masking of mRNA during oogenesis and spermatogenesis is always accompanied by massive accumulation of p50 in the cell cytoplasm. Overproduction of p50 in the cell leads to enhanced masking of cytoplasmic mRNAs. Masked mRNAs of all types are found in association with large amounts of p50. It may be thought that, while a specific "masking protein" bound at the 3'-UTR in a single or few copies triggers the masking process, the sequence-nonspecific protein p50 loading the full sequence of mRNA completes the masking and forms a proper quaternary structure of the masked mRNP particle (Spirin, 1994). The proposed structural reorganisation of mRNP of the condensation type (Fig. 17.13) may be a function of p50. A co-operative interaction of multiple copies of this protein within mRNP may be induced by the specific 3'-UTR-bound "masking" protein.




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