Mark J. Snee

Live imaging of nuage and polar granules : evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components

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.

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

RNA trafficking and stabilization elements associate with multiple brain proteins

Two of the best understood somatic cell mRNA cytoplasmic trafficking elements are those governing localization of β-actin and myelin basic protein mRNAs. These cis-acting elements bind the trans-acting factors fibroblast ZBP-1 and hnRNP A2, respectively. It is not known whether these elements fulfil other roles in mRNA metabolism. To address this question we have used Edman sequencing and western blotting to identify six rat brain proteins that bind the β-actin element (zipcode). All are known RNA-binding proteins and differ from ZBP-1. Comparison with proteins that bind the hnRNP A2 and AU-rich response elements, A2RE/A2RE11 and AURE, showed that AURE and zipcode bind a similar set of proteins that does not overlap with those that bind A2RE11. The zipcode-binding protein, KSRP, and hnRNP A2 were selected for further study and were shown by confocal immunofluorescence microscopy to have similar distributions in the central nervous system, but they were found in largely separate locations in cell nuclei. In the cytoplasm of cultured oligodendrocytes they were segregated into separate populations of cytoplasmic granules. We conclude that not only may there be families of trans-acting factors for the same cis-acting element, which are presumably required at different stages of mRNA processing and metabolism, but independent factors may also target different and multiple RNAs in the same cell.

Dynamic organization and plasticity of sponge bodies

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.

Two distinct domains of Bruno bind specifically to the oskar mRNA.

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.

Bicaudal C and trailer hitch have similar roles in gurken mRNA localization and cytoskeletal organization.

Bicaudal C and trailer hitch are both required for dorsoventral patterning of the Drosophila oocyte. Each mutant produces ventralized eggs, a phenotype typically associated with failure of the oocyte to provide a dorsalization signal--the Gurken protein--to the follicle cells. Bicaudal C and trailer hitch are both implicated in post-transcriptional gene regulation. Bicaudal C acts in recruiting a deadenylase to specific mRNAs, leading to translational repression. The role of trailer hitch is less well defined, but mutants have defects in protein secretion, and show aberrant distribution of an endoplasmic reticulum exit site marker whose mRNA is associated with Trailer hitch protein. We show that Bicaudal C and trailer hitch interact genetically. Mutants of these two genes have shared defects in localization of gurken and other anteriorly-localized mRNAs, as well as altered microtubule organization which may underlie the mRNA localization defects. Bicaudal C and trailer hitch mutants also share a syndrome of actin-related abnormalities, including the formation of ectopic actin cages near the anterior of the oocyte. The cages sequester Gurken protein, blocking its secretion and thus interfering with signaling of the follicle cells to specify dorsal fate.

Recognition of the bcd mRNA localization signal in Drosophila embryos and ovaries.

ABSTRACT The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. We used variants of the bicoid mRNA localization signal to explore recognition requirements in the embryo. We found that bicoid stem-loop IV/V, which is sufficient for ovarian localization, was necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V did localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bound specifically to III/IV/V and fushi tarazu localization signals copurified through multiple fractionation steps, suggesting that they are related. Binding to these two signals was competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. We have proposed a model for a conserved mechanism of localization signal recognition in multiple contexts.

miRNA-Dependent Translational Repression in the Drosophila Ovary

Background The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. Methodology/Principal Findings Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. Conclusions/Significance Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

Multiple cis -acting signals, some weak by necessity, collectively direct robust transport of oskar mRNA to the oocyte

ABSTRACT Localization of mRNAs can involve multiple steps, each with its own cis-acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila oskar mRNA − transport from nurse cells to the oocyte − relies on multiple cis-acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. We propose that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization. Summary: Multiple cis-acting signals direct the first step in localization of oskar mRNA. These signals are collectively strong but individually weak, a property essential for later steps of localization.