The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1 B , hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41-and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.Establishment of asymmetry in cells requires selective localization of proteins. This may be accomplished by directed protein transport, a well established pathway for plasma membrane and secreted proteins, or by trafficking and subsequent localization of mRNA. Localization of RNA has been intensively studied in Drosophila and Xenopus oocytes (for reviews see Refs. 1-6) and more recently in mammalian somatic cells (7-12).In 1982 Subsequent experiments demonstrated that MBP mRNA is translated close to myelin and the protein rapidly incorporated into the nascent membrane (14 -16) and lead to a model in which MBP mRNA is recruited into RNA transport granules in the perikaryon and then transported, by indirect attachment to the microtubule-bound motor protein kinesin, to the myelin compartment at the cell periphery (10, 17-21). The granules are localized in the myelin compartment, and the RNA cargo is translated, with the MBP being incorporated into the myelin membrane. Deletion studies led to the conclusion that a small element in the 3Ј-untranslated region of the MBP mRNA, the RNA transport sequence (RTS), is sufficient and necessary for this cytoplasmic RNA transport in oligodendrocytes (17). Cytoplasmic trafficking of RNA encoding ␤-actin is also dependent on inclusion in transport granules that are attached to the cytoskeleton. In fibroblasts ␤-actin mRNA transport is microfilament-dependent (9, 22), whereas microtubules are implicated in transport of this mRNA in neurons (23,24).trans-Acting factors have been isolated in pull-down experiments with RTS-labeled magnetic particles. The predominant RTS-binding protein from a number of rat tissues is heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (25), a constituent of...

Summary Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the oskar mRNA 3′ UTR. Here we show some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3′ cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an oskar mRNA with wild type BREs but itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread.

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. We used GFP-tagged versions of Vasa and Aubergine to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. We found that perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. We also find that Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes.

Sponge bodies, cytoplasmic structures containing post-transcriptional regulatory factors, are distributed throughout the nurse cells and oocytes of the Drosophila ovary and share components with P bodies of yeast and mammalian cells. We show that sponge body composition differs between nurse cells and the oocyte, and that the sponge bodies change composition rapidly after entry into the oocyte. We identify conditions that affect sponge body organization. At one extreme, components are distributed relatively uniformly or in small dispersed bodies. At the other extreme, components are present in large reticulated bodies. Both types of sponge bodies allow normal development, but show substantial differences in distribution of Staufen protein and oskar mRNA, whose localization within the oocyte is essential for axial patterning. Based on these and other results we propose a model for the relationship between P bodies and the various cytoplasmic bodies containing P body proteins in the Drosophila ovary. Developmental Dynamics 238: 918 -930, 2009.

Selective deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of oskar mRNA, combined with translational regulation to ensure that only the localized mRNA produces protein. The Bruno protein binds to Bruno Response Elements (BREs) in the oskar mRNA, and prevents translation of unlocalized oskar mRNA. Bruno contains three copies of the RNA Recognition Motif (RRM), a protein motif that often binds directly to RNA. Either of two nonoverlapping parts of Bruno-RRMs 1 and 2, and RRM 3 and 42 flanking amino acids-can bind specifically to BRE-containing RNA, but both domains are required for maximal binding. When expressed in Drosophila ovaries, Bruno proteins with a single RNA binding domain mutated have reduced repressive activity, while mutation of both binding domains largely eliminates this activity. Notably, the same proteins expressed as fusions to GFP accumulate in nuclei, with the most severe mislocalization occurring when both RNA binding domains are mutated. A similar mislocalization of endogenous Bruno occurs when mRNA export is blocked. Thus, Bruno shuttles between the nucleus and cytoplasm, and may first bind oskar mRNA in the nucleus.