Nuclear RNP complex assembly initiates cytoplasmic RNA localization

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA-protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.     

Vg1 RNA localization in oocytes in the absence of xVICKZ3 RNA-binding activity

Vg 1 RNA becomes localized at the vegetal cortex of Xenopus oocytes in a process requiring both intact microtubules (MT) and microfilaments. This localization occurs during a narrow window of oogenesis, when a number of RNA-binding proteins associate with the RNA. xVICKZ3 (Vg1 RBP/Vera), the first Vg1 RNA-binding protein identified, helps mediate the association of Vg1 RNA with MT and is co-localized with the RNA at the vegetal cortex. Given the complexity of the Vg1 RNA ribonucleoprotein (RNP) complex, it has remained unclear how xVICKZ3 functions in Vg1 RNA localization. Here, we have taken a closer look at the process of xVICKZ3 localization in oocytes. We have made use of deletion constructs to perform a structure-function analysis of xVICKZ3. The ability of xVICKZ3-GFP constructs to vegetally localize correlates with their association to MT but not with Vg1 RNA-binding ability. We find that when the ability of xVICKZ3 to bind Vg1 RNA is inhibited by the injection of a construct that dominantly inhibits RNA binding, both the construct and Vg1 RNA still localize, apparently through their continued association with a Vg1 RNA-containing RNP complex. These results emphasize the importance of protein-protein interactions in both xVICKZ3 and Vg1 RNA localization.     

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination

The 3′ untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.     

Evidence for common machinery utilized by the early and late RNA localization pathways in Xenopus oocytes

In Xenopus, an early and a late pathway exist for the selective localization of RNAs to the vegetal cortex during oogenesis. Previous work has suggested that distinct cellular mechanisms mediate localization during these pathways. Here, we provide several independent lines of evidence supporting the existence of common machinery for RNA localization during the early and late pathways. Data from RNA microinjection assays show that early and late pathway RNAs compete for common localization factors in vivo, and that the same short RNA sequence motifs are required for localization during both pathways. In addition, quantitative filter binding assays demonstrate that the late localization factor Vg RBP/Vera binds specifically to several early pathway RNA localization elements. Finally, confocal imaging shows that early pathway RNAs associate with a perinuclear microtubule network that connects to the mitochondrial cloud of stage I oocytes suggesting that motor driven transport plays a role during the early pathway as it does during the late pathway. Taken together, our data indicate that common machinery functions during the early and late pathways. Thus, RNA localization to the vegetal cortex may be a regulated process such that differential interactions with basal factors determine when distinct RNAs are localized during oogenesis.     

Polarizing genetic information in the egg: RNA localization in the frog oocyte

RNA localization is a powerful strategy used by cells to localize proteins to subcellular domains and to control protein synthesis regionally. In germ cells, RNA targeting has profound implications for development, setting up polarities in genetic information that drive cell fate during embryogenesis. The frog oocyte offers a useful system for studying the mechanism of RNA localization. Here, we discuss critically the process of RNA localization during frog oogenesis. Three major pathways have been identified that are temporally and spatially separated in oogenesis. Each pathway uses a different mechanism to effect RNA localization. In some cases, localization elements within the 3' untranslated region have been identified and have provided unique insights into the localization process. This important field is still in its infancy, however, and much remains to be learned. BioEssays 21:546–557, 1999. © 1999 John Wiley & Sons, Inc.     

mRNA localization: motile RNA, asymmetric anchors

Techniques to label mRNA with green fluorescent protein (GFP) have provided the first real-time images of RNA motility in live yeast cells. Genetic screens for factors responsible for mRNA asymmetry (e. g. SHE genes) in yeast identified type V myosin among other proteins. Analysis of mRNA movement in various she mutants revealed the role of motor proteins in long-range transport, factors for particle formation, and cortical anchors for docking the mRNA.     

An Accurate Model for the Expression of the Antisense RNA Block Gene:the Firefly Luciferase Gene-Xenopus Oocyte System

The plasmid p~(SV-Luc20)or the mRNA of luciferase gene transcribed from p~(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.     

A novel procedure for the localization of viral RNAs in protoplasts and whole plants

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.     

The right place at the right time: chaperoning core histone variants

Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.     

Xvelo1 uses a novel 75-nucleotide signal sequence that drives vegetal localization along the late pathway in Xenopus oocytes

Vegetally localized RNAs in Xenopus laevis oocytes are involved in the patterning of the early embryo as well as in cell fate specification. Here we report on the isolation and characterization of a novel, vegetally localized RNA in Xenopus oocytes termed Xvelo1. It encodes a protein of unknown biological function and it represents an antisense RNA for XPc1 over a length of more than 1.8 kb. Xvelo1 exhibits a localization pattern reminiscent of the late pathway RNAs Vg1 and VegT; it contains RNA localization elements (LE) which do not match with the consensus structural features as deduced from Vg1 and VegT LEs. Nevertheless, the protein binding pattern as observed for Xvelo1-LE in UV cross-linking experiments and coimmunoprecipitation assays is largely overlapping with the one obtained for Vg1-LE. These observations suggest that the structural features recognized by the protein machinery that drives localization of maternal mRNAs along the late pathway in Xenopus oocytes must be redefined.     

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of Os Tudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. Os Tudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that Os Tudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that Os Tudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of Os Tudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of Os Tudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged Os Tudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of Os Tudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that Os Tudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.