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IGF2BP1 is a protein that controls the stability, localization, and translation of various mRNA targets. Poor clinical outcomes in numerous cancer types have been associated with its overexpression. As it has been demonstrated to impede tumor growth and metastasis in animal models, inhibiting IGF2BP1 function is a promising strategy for combating cancer. A lead chemical, 7773, which specifically decreased IGF2BP1 RNA binding and cellular activities, was previously identified in a high-throughput screen for effective IGF2BP1 inhibitors. Additional optimization of 7773 described in this manuscript led to the discovery of six compounds that performed equally well or better than 7773. In cell lines with high levels of endogenous IGF2BP1, one of 7773 derivatives, AVJ16, was found to be most efficient at preventing cell migration. Further, AVJ16 was found to be IGF2BP1-specific because it had no effect on cell lines that expressed little or no IGF2BP1 protein. The direct binding of AVJ16 to IGF2BP1 was validated by binding tests, with a 12-fold increase in binding efficiency over the lead compound. AVJ16 was shown to bind to a hydrophobic region at the protein's KH34 di-domain interface between the KH3 and KH4 domains. Overall, the findings imply that AVJ16 is a potent and specific inhibitor of IGF2BP1 activity., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or personal relationships that could inappropriately influence the reported work., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Research over the last 10 to 15 years has revealed that intracellular RNA localization is a widespread phenomenon found in a large range of different cell types in an equally impressive number of different organisms (Bashirullah et al., 1998; St. Johnston, 1995). Efforts have focused both on the molecular mechanisms involved in localizing RNAs to particular intracellular targets and on the functional importance (to the cell) of placing certain RNAs at particular cellular sites. In many cases, an understanding of the role of RNA localization seems to be predicated on a careful analysis of how a particular RNA achieves its characteristic distribution. A generalized model of RNA localization usually invokes cellular factors recognizing RNA target sequences. This review will focus on several systems in which cis-acting elements and trans-acting factors recognizing these elements are involved in RNA localization: how they have been defined, how they relate to each other, and how they interact and function to help achieve defined intracellular localization. Conservation of both RNA elements and protein factors across species suggests that RNA localization is probably a fundamental cellular process.

The 3'-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3'-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3'-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3'-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell.

We have analyzed the expression and intracellular distribution, during oogenesis and embryogenesis, of Vg1 RBP, a protein implicated in the intracellular localization of Vg1 mRNA to the vegetal cortex of Xenopus oocytes. Vg1 RBP (protein) colocalizes with Vg1 RNA at all stages of oogenesis. Vg1 RBP RNA, however, localizes to the animal pole during late oogenesis, and remains in the animal blastomeres and ectodermal precursors until its zygotic transcription is activated, around stage 12. Vg1 RBP mRNA then becomes expressed throughout the neural epithelium. Vg1 RBP mRNA expression is also detected in what appears to be neural crest cells undergoing delamination and lateral migration. By tailbud stages, Vg1 RBP expression is present in the branchial arches, otic vesicle, pronephros, and along the neural tube. To examine the expression pattern in different species, we cloned the zebrafish homolog of Vg1 RBP by using a highly homologous EST clone to screen an embryonic cDNA library. In situ hybridization reveals that Vg1 RBP RNA localizes early in oogenesis to the animal pole. Although Vg1 RBP RNA is detected in all blastomeres of the early embryo, the expression pattern in the one day old zebrafish embryo is almost identical to that of the equivalent stage Xenopus embryo. These results indicate that the zygotic expression pattern is similar in frogs and fish, and that there is a conserved zygotic expression of Vg1 RBP distinct from its expression in the oocyte.

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

The multistep pathway leading to intracellular RNA localization is known to involve cis-acting signals in targeted mRNAs, which are presumably recognized by specific RNA-binding proteins and interact with a functional cytoskeleton. Tau RNA is localized to the proximal hillock of rat axons, and this movement requires intact microtubules. Because Xenopus oocytes demonstrate a clear polarity involving microtubule-mediated RNA localization, we have studied the distribution of tau RNA injected into oocytes. We find that a fragment from the 3'-untranslated region of tau RNA is localized to the vegetal cortex of stage III/IV oocytes in a distribution indistinguishable from Vg1 RNA, a vegetally localized oocyte mRNA. A fragment from the tau RNA coding region, however, is homogeneously distributed in oocytes. Tau RNA contains a functional binding site for Vg1 RBP, a Xenopus microtubule-associated protein that binds vegetally localized oocyte RNAs with high affinity, and this binding correlates with vegetal localization ability. The present studies demonstrate, for the first time, localization of heterologous RNA in oocytes. Given the role of Vg1 RBP as a mediator of specific RNA-microtubule interactions, these results are strong evidence that Vg1 RBP is involved in the vegetal localization of RNAs in oocytes and raise the intriguing possibility of the existence of proteins with similar function in neurons.

Transient transfection into L8 myoblasts has been used to study the rat alpha-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the alpha-actin gene construct into the cell. The removal of the methyl moiety from the complementary strand takes place after a delay of at least 48 hr, and both events are actively carried out in the absence of DNA replication. By assaying gene activity during the course of the transfection, it was possible to demonstrate that demethylation of both strands at the critical CpG loci is essential to activate transcription. Genetic analysis revealed the existence of cis-acting elements required for demethylation. The recognition of these sites early in the differentiation process probably leads to the demodification events required to make the gene accessible to its transcription factors.

In an effort to understand how polarity is established in Xenopus oocytes, we have analyzed the process of localization of the maternal mRNA, Vg1. In fully grown oocytes, Vg1 mRNA is tightly localized at the vegetal cortex. Biochemical fractionation shows that the mRNA is preferentially associated with a detergent-insoluble subcellular fraction. The use of cytoskeletal inhibitors suggests that (1) microtubules are involved in the translocation of the message to the vegetal hemisphere and (2) microfilaments are important for the anchoring of the message at the cortex. Furthermore, immunohistochemistry reveals that a cytoplasmic microtubule array exists during translocation. These results suggest a role for the cytoskeleton in localizing information in the oocyte.

The animal and vegetal ends of Xenopus oocytes have distinctly different developmental fates. At the molecular level, several maternal mRNAs have been isolated that are localized to either the animal or vegetal hemisphere. One of these mRNAs, Vg1, is distributed homogeneously throughout the cytoplasm of early-stage oocytes and gets localized during oogenesis to a tight shell at the vegetal cortex of middle and late-stage oocytes. We have used an in vitro culture system to demonstrate that exogeneous Vg1 mRNA injected into middle-stage, but not late-stage, oocytes gets localized in a similar fashion to the endogenous message. Furthermore, translation of Vg1 mRNA is not required for the localization of the message itself. These results show that the information necessary to interpret the animal-vegetal polarity in oocytes is present in the naked mRNA transcript.

The maternal mRNA Vg1 is localized to the vegetal pole during oogenesis in Xenopus. We have cultured oocytes in vitro to begin to understand how this localization occurs. Endogenous Vg1 mRNA undergoes localization when oocytes are cultured in vitro, and synthetic Vg1 mRNA injected into such oocytes is localized in the same fashion. Vg1 mRNA is associated with a detergent-insoluble fraction from homogenized oocytes, suggesting a possible cytoskeletal association. The use of cytoskeletal inhibitors reveals a two-step process for localizing Vg1 mRNA. Microtubule inhibitors such as nocodazole and colchicine inhibit the localization of Vg1 mRNA in late stage III/early stage IV oocytes, but have no effect on Vg1 mRNA once it is localized. The microfilament inhibitor cytochalasin B, however, has little effect on the translocation of Vg1 mRNA in middle-stage oocytes but causes a release of the message in late-stage oocytes. We propose a model for the localization of Vg1 mRNA in which translocation of the message to the vegetal cortex is achieved via cytoplasmic microtubules and the anchoring of the message at the cortex involves cortical microfilaments.

This paper summarizes our recent work concerned with the developmental polarity of the frog egg and the patterning of the embryonic body plan. In two separate projects, we are studying genes involved in setting up basic embryonic axes. One of these genes, Vg1, codes for a maternal mRNA that is localized in the frog egg. The Vg1 gene is used in studies on the induction of mesoderm and as a probe to understand how the polarity of an egg is established. A second gene, Xhox3, contains a homeodomain and is differentially expressed in the axial mesoderm. Our studies suggest that this homeobox gene is critically involved in setting up different positional values along the anteroposterior axis.