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

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

<div><p>Background</p><p>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.</p><p>Results and Conclusions</p><p>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.</p></div

RNA-Binding Protein VICKZ Is Expressed in a Germinal Center Associated Pattern among Lymphoma Subtypes

Abstract Recent effort in the molecular characterization of diffuse large B-cell lymphoma (DLBCL) has led to the recognition that patients with DLBCL of germinal center origin exhibit a better overall survival. Thus, identification and characterization of markers of germinal center derivation are of importance in dissecting prognostic subclasses of DLBCL. The VICKZ (Vg1 RBP/Vera, IMP1, 2, 3, CRD-BP, KOC, ZBP-1) family members are RNA-binding proteins that recognize specific RNA targets and have been implicated in diverse cellular functions including cell polarity, migration, proliferation and tumorigenesis/metastasis. We generated an antibody that recognizes all three isoforms of VICKZ protein and characterized its expression in normal lymphoid tissue and in lymphoma subtypes. In normal tonsils, VICKZ protein showed a germinal center-specific pattern of expression with staining localized to the cytoplasm. Among 868 non-Hodgkin and Hodgkin lymphomas tested by immunohistochemistry on tissue microarrays, staining for VICKZ protein was present in 76% (126/165) of follicular lymphoma, 78% (155/200) of DLBCL, 90% (9/10) of mediastinal large B-cell lymphoma, and 100% (2/2) of Burkitt lymphoma. A subset of mantle cell lymphoma (11%, 2/19), extranodal (8%, 2/25), and nodal (20%, 1/5) marginal zone lymphoma and lymphoblastic lymphoma (25%, 4/13), showed VICKZ staining. The majority of lymphocyte predominant Hodgkin (92%, 12/13) and classical Hodgkin (94%, 101/108) lymphoma were found to be positive. Among T cell lymphoma, anaplastic large cell lymphoma were positive (75%, 6/9). Additional work is in progress to correlate VICKZ protein expression with other germinal center markers such as HGAL, BCL6 and CD10 as well as with prognostic subclasses of DLBCL. The differential expression pattern of VICKZ protein in lymphoma subtypes suggests a potential utility for VICKZ in the identification of subgroups of DLBCL associated with different prognoses

Overexpression of cVICKZ1 inhibits CNC emigration.

<p>2-4ss embryos were electroporated with either control GFP (A,C) or cVICKZ1-GFP (B,D), and fixed 20 hours post-electroporation. Embryos were stained for HNK-1 expression (red) and GFP (green) and photographed either as whole mount preparations (A,B) or after sectioning (C,D). The grey dotted line in (A) and (B) indicate the plane of section shown in (C) and (D), respectively. Almost no CNC delamination occurs in the cVICKZ1-GFP embryos, although GFP-negative, HNK-1-positive cells emigrate normally. (E) The percentage of the total number of GFP-positive cells that delaminated by 20 hours post-electroporation was calculated for each embryo and was graphed as the mean ± SEM (N = 5). Abbreviations: E, eye.</p

VICKZ1 mediates Integrin α6 expression and co-immunoprecipitates with Integrin α6 mRNA.

<p>2-4ss embryos were electroporated with control-GFP (A), full length VICKZ1- GFP (B), or Y396F-GFP (C), and then fixed and stained for Integrin α6 (red) and GFP (green) 10 hours later. In control-GFP transfected embryos, Integrin α6 is down-regulated in the dorsal aspect of the tube in the region where CNC delaminate, on both the transfected and non-transfected sides (A, inset 1 and 2). Overexpression of VICKZ1 maintains Integrin α6 expression even in the most dorsal regions of the tube, but only on the transfected side (B, inset 1), and not on the non-transfected side (B, inset 2). Y396F expression causes a precocious emigration of CNC and down-regulation of Integrin α6 in more lateral regions of the tube, only on the transfected side (C, inset 1) and not on the non-transected side (C, inset 2). All insets show only the red channel (ITGA6). Arrows in insets indicate areas of downregulation of ITGA6. (D) RNP complexes were prepared from 3d and 4d old chick embryos and immunoprecipitated with either pre-immune serum or pan-VICKZ antibody. Equal volumes of total lysates (Total) and immunoprecipitates (IP) were subjected to western blot analysis using the pan-VICKZ antibody. VICKZ protein is pulled down exclusively by the pan-VICKZ antibody. (E) Quantitative RT-PCR analysis was performed on cDNAs prepared from both pre-immune and pan-VICKZ immunoprecipitations and tested for the presence of ITGA6 mRNA. Values of pan-VICKZ IP mRNA were normalized to the amounts of total mRNA, and compared to the pre-immune normalized values (pan-VICKZ/pre-immune). A 15–16 fold enrichment of ITGA6 mRNA is observed in the pan-VICKZ IP, as compared to the pre-immune serum. A control RNA, 18S, shows only 1–2 fold enrichment when analysed in the same way. The data show the mean±SEM.</p

Down-regulation of VICKZ causes DM dissociation.

<p>(A-C) VICKZ is expressed in newly-formed somites in E2 embryos (A) particularly in the apical adherens junctions, and later in the early dermomyotome (B). In E3 embryos, VICKZ is down-regulated in the emerging dermis but maintained in the developing myofiber (C). (VICKZ is also down-regulated in the emigrating trunk NC cells (arrows in B)). (D-G) Embryos were electroporated with either control GFP (D, F) or Y396F-GFP (E, G) into the dorsal medial lip (DML) of early dissociating somites. Embryos were fixed either 12 (D,E) or 20 (F,G) hours post-electroporation and stained for the DM marker, Pax 7 (purple) and/or the myotome marker, Desmin (red). After 12 hours, control GFP-labeled DML cells retain their epithelial structure and are Pax7-positive (D) whereas many DML cells that received Y396F-GFP have begun rounding up and losing their epithelial pseudostratified morphology (E, arrowheads). By 20 hours post-electroporation, in the control-GFP embryos, GFP-positive, Desmin-positive myofibers derived from the DML occupy the medial-most aspect of the myotome (F, arrows). In Y396F-GFP treated embryos, however, the DM was completely dissociated, with GFP-positive, Pax7-negative cells having lost their epithelial morphology. None of the GFP-positive cells generated myofibers. Abbreviations: ES, epithelial somite; DM, dermomyotome; D, dermis; M, myotome.</p

Global reduction of VICKZ in Xenopus embryos inhibits CNC migration non-cell autonomously.

<p>(A) A scheme illustrating the position of blastomere a2 in a 32-cell stage embryo that gives rise to most of the CNC cells in the embryo. (B,C) 32 cell-stage embryos were injected in a2 with Alexa-red and either control morpholino (CMO) (B) or xVICKZ antisense morpholino (AMO) (C) and allowed to develop to tailbud stage. Both AMO-and CMO-injected cells reach their homing sites in the branchial, hyoid, and mandibular arches (b.a., h, m). (D-F) CNC orthotopic transplantations. Wild–type CNC grafts (in green) exhibited normal migration in control embryos (D), with grafts taken from a rostral position in the neural folds migrating along the mandibular migration pathway (D1) whereas grafts from more caudal position migrating to the branchial arches (D2). Grafts taken from xVICKZ-depleted embryos in orange, were able to migrate properly in WT embryos (E). However, wild-type CNC (green) were unable to migrate in xVICKZ AMO-injected embryos (orange). Abbreviations: m, mandibular; h, hyoid; b.a, branchial arches.</p