VICKZ proteins mediate cell migration via their RNA binding activity

The highly conserved, RNA binding VICKZ proteins help regulate RNA localization, stability, and translation in many eukaryotes. These proteins are also required for cell migration in embryos and cultured cells. In adults, many tumors overexpress VICKZ homologs, and it has been hypothesized that the proteins can mediate cell motility and invasion. How these proteins facilitate cell movement and, in particular, whether their ability to bind RNA plays a role in their function remain unclear. Using HPLC and mass spectrometry to identify a region of Xenopus Vg1 RBP (xVICKZ3) that binds the vegetal localization element of Vg1 RNA, we generated a deletion construct that functions in a dominant-negative manner. The construct associates with full-length xVICKZ3 and severely reduces binding to target RNAs. This dominant-negative construct phenocopies the effect of down-regulating xVICKZ3 in Xenopus embryos. A corresponding deletion in the human homolog hVICKZ1 similarly functions in a dominant-negative fashion to reduce the ability of full-length hVICKZ protein to bind RNA. Expression of the dominant-negative construct in human carcinoma cells inhibits cell movement by several criteria. We conclude that the ability of VICKZ proteins to mediate cell migration, in vitro and in vivo, requires their RNA binding activity.     

Classifying RNA-binding proteins based on electrostatic properties

Protein structure can provide new insight into the biological function of a protein and can enable the design of better experiments to learn its biological roles. Moreover, deciphering the interactions of a protein with other molecules can contribute to the understanding of the protein's function within cellular processes. In this study, we apply a machine learning approach for classifying RNA-binding proteins based on their three-dimensional structures. The method is based on characterizing unique properties of electrostatic patches on the protein surface. Using an ensemble of general protein features and specific properties extracted from the electrostatic patches, we have trained a support vector machine (SVM) to distinguish RNA-binding proteins from other positively charged proteins that do not bind nucleic acids. Specifically, the method was applied on proteins possessing the RNA recognition motif (RRM) and successfully classified RNA-binding proteins from RRM domains involved in protein-protein interactions. Overall the method achieves 88% accuracy in classifying RNA-binding proteins, yet it cannot distinguish RNA from DNA binding proteins. Nevertheless, by applying a multiclass SVM approach we were able to classify the RNA-binding proteins based on their RNA targets, specifically, whether they bind a ribosomal RNA (rRNA), a transfer RNA (tRNA), or messenger RNA (mRNA). Finally, we present here an innovative approach that does not rely on sequence or structural homology and could be applied to identify novel RNA-binding proteins with unique folds and/or binding motifs.     

The RNA-binding protein Vg1 RBP is required for cell migration during early neural development

After mid-blastula transition, populations of cells within the Xenopus embryo become motile. Using antisense morpholino oligonucleotides, we find that Vg1 RBP, an RNA-binding protein implicated in RNA localization in oocytes, is required for the migration of cells forming the roof plate of the neural tube and, subsequently, for neural crest migration. These cells are properly determined but remain at their site of origin. Consistent with a possible role in cell movement, Vg1 RBP asymmetrically localizes to extended processes in migrating neural crest cells. Given that Vg1 RBP is a member of the conserved VICKZ family of proteins, expressed in embryonic and neoplastic cells, these data shed light on the likely role of these RNA-binding proteins in regulating cell movements during both development and metastasis.     

RNA-protein interactions in vivo: global gets specific

RNA-binding proteins (RBPs) impact every process in the cell; they act as splicing and polyadenylation factors, transport and localization factors, stabilizers and destabilizers, modifiers, and chaperones. RNA-binding capacity can be attributed to numerous protein domains that bind a limited repertoire of short RNA sequences. How is specificity achieved in cells? Here we focus on recent advances in determining the RNA-binding properties of proteins in vivo and compare these to in vitro determinations, highlighting insights into how endogenous RNA molecules are recognized and regulated. We also discuss the crucial contribution of structural determinations for understanding RNA-binding specificity and mechanisms.     

A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis

Background

VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos.

Results and Conclusions

Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.     

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.     

Identification and characterization of proteins binding A + U-rich elements

A + U-Rich elements (AREs) have been extensively investigated as cis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to the study of any protein displaying sequence-specific RNA binding activity. The techniques described here include the purification of native AUF1 from cultured cells as well as the preparation of recombinant AUF1 proteins using a bacterial expression system. Analyses of RNA-protein interactions are also described, including the use of gel mobility shift assays with synthetic RNA probes to monitor specific RNA binding activity in cell extracts or with recombinant proteins. Variations of this technique are also described to evaluate the RNA binding affinity of recombinant proteins and the use of specific RNA competitors to assess RNA determinants of protein binding specificity. Other techniques presented include the identification of specific proteins in RNA:protein complexes using antibody supershifts and the estimation of molecular weights of RNA-binding proteins by UV crosslinking. Results of individual experiments are presented as examples of some techniques. Throughout the article, suggestions are included to avoid commonly encountered problems and to assist in the optimization of these techniques for the study of other RNA-binding proteins.