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The interaction of β-actin mRNA with zipcode-binding protein 1 (ZBP1) is necessary for its localization to the lamellipod of fibroblasts and plays a crucial role in cell polarity and motility. Recently, we have shown that low ZBP1 levels correlate with tumor-cell invasion and metastasis. In order to establish a cause and effect relationship, we expressed ZBP1 in a metastatic rat mammary adenocarcinoma cell line (MTLn3) that has low endogenous ZBP1 levels and delocalized β-actin mRNA. This leads to localization of β-actin mRNA, and eventually reduces the chemotactic potential of the cells as well as their ability to move and orient towards vessels in tumors. To determine how ZBP1 leads to these two apparently contradictory aspects of cell behavior -- increased cell motility but decreased chemotaxis -- we examined cell motility in detail, both in cell culture and in vivo in tumors. We found that ZBP1 expression resulted in tumor cells with a stable polarized phenotype, and reduced their ability to move in response to a gradient in culture. To connect these results on cultured cells to the reduced metastatic ability of these cells, we used multiphoton imaging in vivo to examine tumor cell behavior in primary tumors. We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion. [ABSTRACT FROM AUTHOR]

Summary Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to β-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with β-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding. [ABSTRACT FROM AUTHOR]

In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted β-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP3-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization. [ABSTRACT FROM AUTHOR]

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the β-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 5' element (CGGAC) followed by a variable 3' element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34. [ABSTRACT FROM AUTHOR]