A role for VICKZ proteins in the progression of colorectal carcinomas: regulating lamellipodia formation.

VICKZ proteins are a highly conserved family of RNA binding proteins, implicated in RNA regulatory processes such as intracellular RNA localization, RNA stability, and translational control. During embryogenesis, VICKZ proteins are required for neural crest migration and in adults, the proteins are overexpressed primarily in different cancers. We hypothesized that VICKZ proteins may play a role in cancer cell migration. In patients, VICKZ expression varies with tumour type, with over 60% of colon, lung, and ovarian tumours showing strong expression. In colorectal carcinomas (CRCs), expression is detected at early stages, and the frequency and intensity of staining increase with progression of the disease to lymph node metastases, of which 97% express the protein at high levels. Indeed, in stage II CRC, the level of VICKZ expression in the primary lesion correlates with the degree of lymph node metastasis. In culture, VICKZ proteins rapidly accumulate in processes at the leading edge of PMA-stimulated SW480 CRC cells, where they co-localize with beta-actin mRNA. Two distinct cocktails of shRNAs, each targeting all three VICKZ paralogues, cause a dramatic drop in lamellipodia and ruffle formation in stimulated cells. Thus, VICKZ proteins help to facilitate the dynamic cell surface morphology required for cell motility. We propose that these proteins play an important role in CRC metastasis by shuttling requisite RNAs to the lamellipodia of migrating cells.

Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development.

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.

RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.

Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3' UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of beta actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.

A Xenopus nonmuscle myosin heavy chain isoform is phosphorylated by cyclin-p34cdc2 kinase during meiosis.

There are two vertebrate nonmuscle myosin heavy chain (MHC) genes that encode two separate isoforms of the heavy chain, MHC-A and MHC-B. Recent work has identified additional, alternatively spliced isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides (chicken and human) or 48 nucleotides (Xenopus) at a site corresponding to the ATP binding region in the MHC protein (Takahashi, M., Kawamoto, S., and Adelstein, R.S. (1992) J. Biol. Chem. 267, 17864-17871) and Bhatia-Dey, N., Adelstein, R.S., and Dawid, I.B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2856-2859). The deduced amino acid sequence of these inserts contains a consensus sequence for phosphorylation by cyclin-p34cdc2 (cdc2) kinase. In cultured Xenopus XTC cells, we have identified two inserted MHC-B isoforms and a non-inserted MHC-A isoform by immunoblotting of cell extracts. When myosin was immunoprecipitated from XTC cells and phosphorylated in vitro with cdc2 kinase, the kinase catalyzed the phosphorylation of both inserted MHC-B isoforms but not MHC-A. Isoelectric focusing of tryptic peptides generated from MHC-B phosphorylated with cdc2 kinase revealed one major phosphopeptide that was purified by reverse-phase high performance liquid chromatography and sequenced. The phosphorylated residue was Ser-214, the cdc2 kinase consensus site within the insert near the ATP binding region. The same site was phosphorylated in intact XTC cells during log phase of growth and in cell-free lysates of Xenopus eggs stabilized in second meiotic metaphase but not interphase. Moreover, Ser-214 phosphorylation was detected during maturation of Xenopus oocytes when the cdc2 kinase-containing maturation-promoting factor was activated, but not in G2 interphase-arrested oocytes. These results demonstrate that MHC-B phosphorylation is tightly regulated by cdc2 kinase during meiotic cell cycles. Furthermore, MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve different functions in these cells.

A 69-kDa RNA-binding protein from Xenopus oocytes recognizes a common motif in two vegetally localized maternal mRNAs.

Vg1 mRNA, a maternal message encoding a member of the transforming growth factor beta superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nucleotide sequence has been identified in Vg1 RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, 991-994]. To understand how cis- and trans-acting factors are involved in Vg1 mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vg1 mRNA. S100 extracts of late-stage oocytes contain a protein-binding activity that protects specific regions of labeled Vg1 mRNA from degradation by RNase T1. The use of different regions of Vg1 RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vg1 message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the down-stream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor beta 5 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.

Characterization of the early steps of herpes simplex virus replication in interferon-treated human cells.

Interferon (IFN) was shown to inhibit herpes simplex virus type 1 (HSV-1) replication at the transcription of the immediate early (alpha) genes. This apparent inhibition could be due to a direct effect on viral transcription or on one of the preceding steps of infection, i.e., penetration and uncoating. In the present study, we analyze the effects of IFN on the very early steps of HSV-1 infection in HEp-2 human cells. Analysis of the parental HSV-1 viral DNA accumulated in the infected cell nuclei indicated that viral DNA penetration, migration to the nuclei, and stability were not affected by IFN treatment. To analyze the effect of IFN on the uncoating of the parental HSV-1 DNA following infection, we developed a technique based on DNase I sensitivity of the infecting viral DNA genome. Within 1 h of infection, the parental viral DNA became sensitive to DNase I digestion. HSV-1 DNA in the nuclei of both control and IFN-treated cells was equally sensitive to DNase I digestion, suggesting that IFN's mode of action was unrelated to the uncoating of HSV-1 virions. Because IFN does not affect the events in the HSV-1 lytic cycle prior to the onset of the immediate-early gene transcription, it appears that IFN exerts a direct effect on this process.

Inhibition of transcription of herpes simplex virus immediate early genes in interferon-treated human cells.

The effect of interferon (IFN) treatment on the early stages of herpes simplex virus type 1 (HSV-1) replication in three types of human cells was investigated. Interferon pretreatment was shown to reduce the steady state levels of both total and polysomebound HSV-1 immediate early alpha mRNAs. Using the nuclear run-off transcription assay, we showed that IFN selectively inhibited transcription of the HSV-1 genes, with no effect on transcription of total cellular RNA or that of the beta-tubulin RNA. Thus, IFN appears to inhibit the initiation of HSV-1 alpha gene transcription rather than affect the stability of the respective mRNAs. IFN did not prevent the HSV-1-induced early shut-off of host cellular protein synthesis caused by a structural protein of infecting virus. This observation indicated that the IFN-mediated inhibition of HSV-1 replication is at a stage beyond viral penetration into the cytoplasm. These results suggested that IFN blocked HSV-1 replication primarily at a very early stage, during the onset of alpha mRNA transcription.

Isolation and characterization of an interferon-resistant cell line deficient in the induction of (2'-5')oligoadenylate synthetase activity.

To screen for cells with different sensitivities to interferon (IFN), NIH 3T3 mouse fibroblasts were subcloned and examined for their response to IFN treatment. Of 30 clones tested, 2 appeared to be relatively resistant to IFN, since the replication of both vesicular stomatitis virus and mengovirus was not inhibited, even in the presence of 1,000 U of IFN per ml. One resistant (A10) and one sensitive (A5) clone were further analyzed. In both clones, murine leukemia virus replication was equally inhibited by IFN, indicating the presence of functional receptors for IFN in the resistant clone. Using the (2'-5')oligoadenylate (2-5A) radiobinding assay, we could demonstrate that both clones contained the RNase L protein. Furthermore, this enzyme appears to be active, since a similar reduction in the rate of protein synthesis was evident after the introduction of exogenous 2-5A to the cells. We also analyzed the activity of another enzyme in the 2-5A pathway, namely, 2-5A synthetase. In the sensitive cells (A5), the induction of enzyme activity was proportional to the IFN concentration used, reaching a maximum of more than a 10-fold increase over the background of untreated cells. However, little if any induction over the basal activity was observed in the resistant cells (A10) when similar doses of IFN were used. It is thus probable that the lack of induction of 2-5A synthetase activity by IFN in A10 cells is at least partly responsible for their relative resistance to IFN treatment.