A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates.

BACKGROUND: Cytoplasmic mRNA localization is a widespread mechanism for restricting the translation of specific mRNAs to distinct regions of eucaryotic cells. This process involves specific interactions between cellular factors and localization signals in the 3' untranslated regions of the localized mRNA. Because only a few of these cellular factors have been identified, it is not known whether common factors are utilized for the localization of different mRNAs. We recently discovered Vera, a protein that binds specifically to the Vg1 localization element and is involved in the localization of Vg1 mRNA in Xenopus oocytes. RESULTS: To characterize further the role of Vera in the localization of Vg1 mRNA, we have purified the Vera protein and cloned its gene. Vera is homologous to chicken zip-code-binding protein (ZBP), which binds to a short RNA sequence required for localization of beta-actin mRNA in chick embryo fibroblasts. The predicted amino-acid sequences of Vera and ZBP contain five RNA-binding domains and putative signals for nuclear localization and export. Like the binding of ZBP to beta-actin mRNA, Vera specifically binds to a repeated sequence motif in the Vg1 localization element that is required for Vg1 mRNA localization in Xenopus oocytes. CONCLUSIONS: Vera, a highly conserved component of the mRNA localization machinery, participates in localizing different mRNAs in different cell types. Thus, Vera appears to be a general factor for mRNA localization, and additional factors may be required to specify diverse patterns of RNA localization.

Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum.

In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.

The nucleolar architecture of polymerase I transcription and processing.

The nucleolus, the site of transcription and processing of the major ribosomal genes, generally reveals three distinct ultrastructural components in conventional thin-section electron micrographs (fibrillar centres, dense fibrillar component and granular component). We show here that different parts of the transcription and transcript processing pathway can be mapped to the different nucleolar components in pea root cells. This study shows the full three-dimensional arrangement of the different domains by in situ hybridization and confocal microscopy, and their correspondence with the major ultrastructural components of the nucleolus is revealed by parallel serial section electron microscopy. The active rDNA is widely dispersed in discrete foci, the larger of which, at least, correspond to well-defined fibrillar centres. A probe to the external transcribed spacer (ETS) sequence of the pre-rRNA transcripts labels clearly demarcated regions surrounding the foci of rDNA, and which we show correspond to the dense fibrillar component. Finally, a probe to the entire 45S transcript shows a higher concentration in regions corresponding to the granular component, surrounding the dense fibrillar component labelled by the ETS probe. The changes in structure that occur with heat shock show that nucleolar organization is dynamic and dependent upon transcriptional activity. These results show that the various RNA processing events are spatially highly organized and suggest a vectorial or radial model of transcription and transcript processing, where nascent and newly completed transcripts occupy zones surrounding the genes, which are in turn surrounded by regions containing the older more mature transcripts.

Localization of 5 S genes and transcripts in Pisum sativum nuclei.

We have investigated the position of the 5 S gene clusters in the nuclei of Pisum sativum root tip cells, and of their transcripts in the nucleolus, using in situ hybridization and confocal microscopy. Single-stranded RNA probes were produced by in vitro transcription, incorporating fluorescein or digoxygenin label. There are known to be 3 pairs of 5 S gene clusters in this species, which would be expected to give 6 sites of hybridization in G1 cells and 12 sites in G2 cells. In presumed G2 cells, many of the sites appeared as paired spots very close together, which we interpret as the sites on sister chromatids. A clear association of one or more clusters with the nucleolar periphery was observed, and it is possible that this has a functional significance. The transcript labelling within the nucleolus showed a reproducible and highly ordered pattern, consisting of cavities devoid of transcript labelling surrounded by sheets of bright labelling, making a characteristic and often very symmetrical cage-work structure. This labelling pattern may represent an underlying organization of transcript processing within the nucleolus, and is highly reminiscent of the distribution of a previously described nucleolar matrix protein.