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,...

Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular, we show that a 5.0 kb CD44 mRNA contained multiple IMP-binding sites in its 3 0 UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation.

SummaryClusters of regularly interspaced short palindromic repeats (CRISPRs) of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3Ј-end, within repeats, yielding heterogeneous 40-45 nt spacer RNAs. Processing of the pKEF9 leader transcript occurred partially in spacers, and was incomplete, probably reflecting defective repeat recognition by host enzymes. A similar level of transcripts was generated from complementary strands of each chromosomal repeat-cluster and they were processed to yield discrete~55 nt spacer RNAs. Analysis of the partially identical repeat-clusters of Sulfolobus solfataricus strains P1 and P2 revealed that spacer-repeat units are added upstream only when a leader and certain cas genes are linked. Downstream ends of the repeat-clusters are conserved such that deletions and recombination events occur internally.

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,...

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100 -300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly ( Moreover the exon junction complex, which is deposited during splicing, is removed during the so-called pioneering round of translation (for reviews, see Refs. 1 and 2). Finally a particular mRNA becomes embroidered with nuclear RNA-binding proteins, and the specific ensemble may determine cytoplasmic events such as RNA localization, translation, and stability (for a review, see Ref.3). Cytoplasmic mRNPs may become destined for local translation. In support of this possibility, RNAs have been found in large mRNP granules, which are transported along cytoskeletal structures and anchored at their final destination. Messenger RNA localization has mainly been examined in polarized oocytes and neurons, and it has been proposed that local postsynaptic protein synthesis is required for synaptic plasticity (4). Previous studies have identified neuronal Staufen (5) and FMRP granules (6, 7), containing mRNAs, small and large ribosomal subunits, translation initiation factors including eIF4E and eIF2␣, and RNA-binding proteins (Refs. 8 -11; for a review, see Ref. 12). The protein composition of neuronal mRNP granules is to some degree overlapping with stress granules and processing bodies (P-bodies). The hallmark of stress granules is the presence of stalled 48 S initiation complexes and stress-dependent RNA-binding factors such as G3BP (13,14), whereas P-bodies contain components of the 5Ј-3Ј mRNA decay machinery and factors involved in nonsense-mediated decay (15).The zip code-binding proteins IMP1, -2, and -3 (human), ZBP1 (chicken), Vg1-RBP/Vera (Xenopus), and coding region determinant-binding protein (mouse) are members of the From the ‡Department

Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2 and T2D.

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.

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