Joel K. Yisraeli

Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases.

Publisher Summary This chapter discusses the basic technique for synthesizing long, capped transcripts in vitro by SP6 and T7 RNA polymerases. The sequence to be transcribed is cloned into the appropriate vector. The transcription reaction consists of a linear DNA template, ribonucleotide triphosphates, RNA polymerase, and, generally, an RNase inhibitor in a relatively simple buffer. An increasing number of plasmid vectors have been constructed to facilitate the cloning of sequences to be transcribed in vitro . Plasmids containing the cloned DNA sequence can be grown and purified using many techniques. Because supercoiled plasraids containing a bacteriophage promoter are very efficiently transcribed, yielding large, often multimeric transcripts, it is very important to cleave the entire template to completion. RNA transcripts containing modified nucleotides have been used in a variety of ways. RNAs containing biotinylated nucleotides can be used as nonradioactive probes in any sort of hybridization assay in place of DNA probes.

VICKZ proteins: a multi-talented family of regulatory RNA-binding proteins

The highly conserved VICKZ (Vg1 RBP/Vera, IMP‐1,2,3, CRD‐BP, KOC, ZBP‐1) family of RNA‐binding proteins recognize specific cis‐acting elements in a variety of different RNAs and have been implicated in cell polarity and migration, cell proliferation, and cancer. In just the last two years, the use of transgenic mice, antisense RNA, and RNAi (RNA interference) techniques have contributed to our understanding of the roles of these proteins and how they interface with many diverse processes in cells. In this article, I will review the recent advances relating to VICKZ proteins and try to suggest a framework for understanding how, in conjunction with other RNA‐binding proteins, they participate in a combinatorial fashion to help determine the fate of their RNA targets and, ultimately, the function of cells in which they are expressed. Such a ‘ post‐transcriptional operon ’ model, as proposed by Keene and Tenenbaum [(2002) Mol. Cell 9, 1161‐1167], can explain the differential, integrated action of multiple RNA‐binding proteins on mRNA targets.

Dynamics of demethylation and activation of the α-actin gene in myoblasts

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.

The involvement of a conserved family of RNA binding proteins in embryonic development and carcinogenesis

Vg1 RBP is a member of a family of highly conserved proteins that appear to be involved in RNA localization, stability, and/or translational control in a wide variety of cell types and organisms. Over the last few years, the human homologs of these proteins have been found to be overexpressed in an increasing number of different kinds of cancers. Although the role of these proteins in neoplasia is not understood, results from several labs, including our own, are beginning to suggest that many of these proteins may be important in cell motility, a necessary requirement for metastasis. This paper will review these data and suggest a model for the role of Vg1 RBP and its homologs in embryonic development and carcinogenesis.

Vg1 RNA binding protein mediates the association of Vg1 RNA with microtubules in Xenopus oocytes.

Localized RNAs are found in a variety of somatic and developing cell types. In many cases, microtubules have been implicated as playing a role in facilitating transport of these RNAs. Here we report that Vg1 RNA, which is localized to the vegetal cortex of Xenopus laevis oocytes, is associated with microtubules in vivo. Because of the ubiquitous nature of tubulin, the association of specific RNAs with microtubules is likely to involve factors that recognize both RNA and microtubules. Vg1 RNA binding protein (Vg1 RBP), previously shown to bind with high affinity to the vegetal localization site in Vg1 RNA, appears to function in this capacity. Vg1 RBP is associated with microtubules: it is enriched in microtubule extracts of oocytes and is also co‐precipitated by heterologous, polymerized tubulin. Furthermore, Vg1 RBP binding activity is required for the specific association of Vg1 RNA to microtubules in vitro. These data suggest a general model for how specific RNAs can be localized to particular sites via common cytoskeletal elements.

Muscle-specific activation of a methylated chimeric actin gene

To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.

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.

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.

Nested expression and sequential downregulation of the Xenopus caudal genes along the anterior-posterior axis.

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.

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 β‐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. Copyright