Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5 untranslated regions (5 UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5 UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5 UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growthdependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 -luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5 UTR-binding proteins control IGF-II biosynthesis during late mammalian development.Specific RNA-binding proteins are emerging as regulators of cytoplasmic mRNA events such as translatability, stability, and localization. Several examples of these types of regulatory events have been reported in studies of invertebrate embryogenesis and amphibian oogenesis, in which the 3Ј untranslated region (3Ј UTR) has been identified as a repository of regulatory elements (reviewed in reference 35). It is anticipated that similar mechanisms operate during mammalian development, since important physiological roles for RNA-binding proteins have been discerned from deletions of the DAZ and RBM genes leading to azoospermia (9, 26) and from a point mutation in the FMR1 gene resulting in the fragile X mental retardation syndrome (7). RNA-binding proteins often contain one or more RNA-binding motif such as the RNA recognition motif (RRM) and the K homology (KH) domain (reviewed in reference 28), which may either ensure increased specificity towards a single RNA molecule or provide an ability to bind different molecules simultaneously. Moreover, solution structures of the N-terminal RRM domain of the human U1A protein in complex with its own pre-mRNA and of the first KH domain of FMR1 suggest that flexible loop regions provide discriminating binding surfaces for RNA recognition (1, 20).Insulin-like growth factor II (IGF-II) is a fetal growth factor with auto-and paracrine modes of action. In the mouse, lack of IGF-II results in a small but apparently normal progeny (3), whereas an increased IGF-II dose is more detrimental (13, 30). In humans, increased levels of IGF-II are associated with the Beckwith-Wiedemann syndrome, which is characterized by a disproportionate overgrowth of the fetus and malformations (32). IGF-II expression is controlled by parental imprinting, since only the paternal allele is expressed in most tissues (8). However,...

Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). To characterize the physiological role of IMP1, we generated IMP1-deficient mice carrying a gene trap insertion in the Imp1 gene. Imp1؊/؊ mice were on average 40% smaller than wild-type and heterozygous sex-matched littermates. Growth retardation was apparent from E17.5 and remained permanent into adult life. Moreover, Imp1؊/؊ mice exhibited high perinatal mortality, and only 50% were alive 3 days after birth. In contrast to most other organs, intestinal epithelial cells continue to express IMP1 postnatally, and Imp1 ؊/؊ mice exhibited impaired development of the intestine, with small and misshapen villi and twisted colon crypts. Analysis of target mRNAs and global expression profiling at E12.5 indicated that Igf2 translation was downregulated, whereas the postnatal intestine showed reduced expression of transcripts encoding extracellular matrix components, such as galectin-1, lumican, tenascin-C, procollagen transcripts, and the Hsp47 procollagen chaperone. Taken together, the results demonstrate that IMP1 is essential for normal growth and development. Moreover, IMP1 may facilitate intestinal morphogenesis via regulation of extracellular matrix formation.Insulin-like growth factor II (IGF-II) mRNA-binding protein 1 (IMP1) and the closely related IMP2 and IMP3 proteins belong to a conserved family of RNA-binding proteins consisting of two RNA recognition motifs (RRM) and four K-homology (KH) domains (for review, see references 36 and 56). The latter are phylogenetically conserved in Drosophila melanogaster and Caenorhabditis elegans (GenBank T23837) and constitute a functionally independent entity (37). IMP1 is orthologous to the chicken zipcode-binding protein 1 (ZBP1) and the mouse c-Myc coding region determinant-binding protein (CRD-BP) (10, 45), whereas IMP3 is orthologous to the Xenopus laevis Vg1 mRNA-binding protein (Vg1-RBP/Vera) (8, 16). IMP2 has diverged phylogenetically from IMP1 and IMP3 and has no known orthologues, but a splice variant p62 has been isolated from hepatic carcinoma (59).So far only a handful of mRNAs, including Igf2 leader 3, H19, c-myc, ␤-actin, and Vg1 mRNAs, have been identified as targets for the proteins (16,25,39,45,48). Binding sites are located in the 5Ј untranslated region (UTR), the coding region, and the 3Ј UTR of the mRNAs. Similar to other RNA-binding proteins such as Bruno, which mediates translational repression of oskar mRNA in D. melanogaster (24), multiple IMP molecules may associate with the RNA target. So far, there is no absolute consensus about the nature of the IMP attachment site. Therefore, it is not possible to predict putative target mRNAs solely on the basis of their sequences.The IMPs have been implicated in posttranscriptional processes such as mRNA localization,...

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3'-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a 'locked' stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.

No abstract

Insulin-like growth factor (IGF)-II is important for fetal growth and development. The human IGF-II gene generates multiple mature transcripts with different 5' untranslated regions (5'UTRs) but identical coding regions and 3'UTRs. We have previously shown that a minor 4.8-kilobase messenger RNA was engaged in the synthesis of preproIGF-II, and a major 6.0-kb mRNA was untranslated and stored in a 100S ribonucleoprotein particle. Here we demonstrate that the 6.0-kb mRNA is selectively mobilized and translated in dispersed exponentially growing cells. Translational activation is prevented by rapamycin and mimicked by anisomycin, which suggests that translation of the 6.0-kb mRNA is regulated by the p70S6k/85S6k kinase signalling pathway. Therefore, the minor 4.8-kb mRNA generates a constitutive production of prepro-IGF-II, whereas the major 6.0-kb mRNA provides a post-transcriptionally regulated species.

Mouse coding region determinant-binding (mCRD-BP)and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

The role of phosphatidylinositol 3-kinase and FKSO6-binding protein . rapamycin-associated protein (FRAP) in translational control has been examined by treating RD-rhabdomyosarcoma cells with wortmannin and rapamycin and studying the effects on cell-growth, translation initiation, and protein synthesis. Whereas wortmannin and rapamycin exhibit subtle effects on global translation, examination of individual mRNAs in sucrose gradients and of individual proteins in two-dimensional polyacrylamide gels reveals that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs. Wortmannin represses the synthesis of a third of cellular proteins, whereas rapamycin affects a subset of these proteins. Since ribosomal protein S6 was rapidly dephosphorylated following wortmannin and rapamycin treatment, and the phosphorylation status of the eukaryotic initiation factor 4E was unchanged, our data imply that the p70 signalling pathway has at least one branch-point upstream of FRAP leading to an additional route of translational control.Keywords : translation ; wortmannin ; rapamycin ; ~7 0 "~~ signaling pathway.Protein synthesis is a key regulatory point in the control of gene expression and cell growth. Translation is mainly controlled at the level of initiation, and several critical components of this process are controlled by distinct signalling pathways. Translation initiation is generally considered to occur by a capdependent scanning mechanism where the 43s ribosomal preinitiation complex initially is brought into contact with the 5' terminus of the mRNA by binding to the eukaryotic initiation factor (eIF) 4G in the cap-binding complex eIF-4F. The eIF-4F complex also contains the cap-binding protein eIF-4E that recognises the 7-methyl-guanosine (m'G) structure and eIF-4A that, in conjunction with eIF-4B, exhibits an ATP-dependent RNA helicase activity. Besides the general translation apparatus, specific trunsacting factors may be required for translation of individual mRNAs (for reviews, see [1,21).The p70 ribosomal protein S6 kinase (p70'") plays an important role in the progression of cells from G I to S phase of the cell cycle [3] and in translational activation by insulin and growth factors (reviewed in [4] ; eEF-la, elongation factor la; 5'TOP, mRNA with a 5'-terminal cytosine followed by a short stretch of pyrimidines, IGF-11, insulin-like growth factor 11; 5'-UTR, 5'hntranslated region; GraP-DH, glyceraldehyde-3-phosphate dehydrogenase ; DMEM, Dulbecco's modified Eagles medium; 2D, two-dimensional. try, Rigshospitalet, DK-2100 Copenhagen, DenmarkEnzyme. Phosphatidylinositol 3-kinase (EC 2.7.1.137).stream of both phosphatidylinositol 3-kinase and the phosphatidylinositol-3-kinase-activated protein kinase B [7]. Moreover, Cdc42 and Racl have recently been reported to associate with ~7 0 '~~ [8]. Studies of the p7Whk pathway have been facilitated by the availability of wortmannin and rapamycin that inhibit phosphatidylinositol 3-kinase and FRAP, respectively. Wortmannin is a fungal metabol...