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The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants [1]. In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis. Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays [2]. The Drosophila family member, encoded by the mini spindles ( msps ) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles [3–5]. Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid ( bcd ) mRNA in the oocyte is a microtubule-mediated event [6, 7]. We show that bcd RNA localization is defective in msps mutants. We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers. Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.

Subcellular localization of mRNAs within the Drosophila oocyte is an essential step in body patterning. Yps, a Drosophila Y-box protein, is a component of an ovarian ribonucleoprotein complex that also contains Exu, a protein that plays an essential role in mRNA localization. Y-box proteins are known translational regulators, suggesting that this complex might regulate translation as well as mRNA localization. Here we examine the role of the yps gene in these events. We show that yps interacts genetically with orb, a positive regulator of oskar mRNA localization and translation. The nature of the genetic interaction indicates that yps acts antagonistically to orb. We demonstrate that Orb protein is physically associated with both the Yps and Exu proteins, and that this interaction is mediated by RNA. We propose a model wherein Yps and Orb bind competitively to oskar mRNA with opposite effects on translation and RNA localization.

We analyzed the acquisition of fluorescence of a GFP fusion protein expressed from gene constructs designed to regulate either the time of translation of the protein or the time of gene transcription. Both gene constructs expressed a fusion protein with wild-type GFP appended to the N-terminus of the Bicoid (Bcd) transcription factor. One gene construct, P[gfp-bcd] expressed an mRNA that was initially translated at the time of egg deposition. GFP–Bcd fluorescence could first be detected in anterior nuclei in the embryo at syncytial blastoderm stage, within 1.5–2.5 h after translation commenced. The second gene construct, P[hs-gfp-bcd], utilized thehsp70promoter to regulate the induction of gene expression. In larval salivary glands, GFP–Bcd fluorescence could first be detected in nuclei after a 40-min recovery period following a 10-min heat shock induction of gene expression. These estimates of the time required for the acquisition of a fluorescent GFP product inDrosophilatissues are less than suggested by previously published reports.

The Drosophila exuperantia gene (exu) functions in both oogenesis and spermatogenesis. Alternative RNA processing and promoter usage generates sex-specific transcripts which differ in their 5' and 3' untranslated regions, but encode the same predicted protein. We have sequenced the breakpoints of an exu allele which is defective in spermatogenesis but functions normally in oogenesis. This allele deletes most of the sequence specific to the male 3'-UTR, together with some flanking DNA, and causes a reduction in steady-state level of exu mRNA in the testis. In addition, we find that a smaller deletion which removes only sequence within the male 3'-UTR reduces the steady-state level of the mRNA and prevents an exu transgene from rescuing male sterility. Males carrying multiple copies of this transgene are fertile, suggesting that the male-specific 3'-UTR functions to maintain a proper level of exu product in the germline.

Subcellular RNA localization in different cell types leads to asymmetric distribution of proteins in these cells. The localization of bicoid (bcd) messenger RNA to the anterior pole of the developing Drosophila oocyte gives rise in embryogenesis to a steep concentration gradient of the bcd protein, a transcription factor that activates expression of zygotic genes needed for anterior development. The exuperantia (exu) gene is necessary for this localization of bcd mRNA. Here we express a chimaeric gene encoding a fusion between the Acquorea victoria green fluorescent protein (GFP) and the exu protein (Exu) in female germ cells, and find that the fusion protein fluoresces strongly in both live and fixed cells during Drosophila oogenesis. The fusion protein rescues an exu null allele, restoring full fertility to females, and is expressed and localized in a temporal and spatial pattern similar to native Exu. The high sensitivity of the GFP tag provides important new details on the subcellular localization of Exu. The fusion protein is found in particles concentrated at ring canals, where transport occurs between nurse cells and the oocyte. Drugs such as colchicine and taxol that affect microtubule stability alter localization of the particles. We propose that the particles are ribonucleoprotein complexes or vesicles which transport bcd mRNA along microtubules and target it to the anterior oocyte cortex.

RNAs are localized by microtubule-based pathways to both the anterior and posterior poles of the developing Drosophila oocyte. We describe a new gene, wispy, required for localization of mRNAs to both poles of the egg. Embryos from wispy mothers arrest development after abnormal oocyte meiosis and failure of pronuclei to fuse. Our analysis of spindle and chromosome movements during meiosis reveals defects in spindle structures correlated with very high frequencies of chromosome nondisjunction and loss. Spindle defects include abnormally shaped spindles, spindle spurs, and ectopic spindles associated with lost chromosomes, as well as mispositioning of the meiosis II spindles. The polar body nuclei do not associate with their normal monastral arrays of microtubules, the sperm aster is reduced in size, and the centrosomes often dissociate from a mitotic spindle that forms in association with the male pronucleus. We show that wispy is required to recruit or maintain known centrosomal proteins with two types of microtubule organizing centers (MTOCs): (1) the central MTOC that forms between the meiosis II tandem spindles and (2) the centrosomes of the mitotic spindle. We propose that the wispy gene product functions directly in several microtubule-based events in meiosis and early embryogenesis and speculate about its possible mode of action.

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu–Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.

The Drosophila exuperantia (exu) gene encodes overlapping sex-specific, germline-dependent mRNAs. In this work, the structural differences between these sex-specific exu mRNAs were determined by sequence analysis of 9 ovary and 10 testis cDNAs. The transformer 2 (tra-2) gene functions in sex determination of female somatic cells through its role in regulating female-specific splicing of doublesex (dsx) RNA. We report here that tra-2 is required in male germ cells for efficient male-specific processing of exu RNA; in the absence of tra-2, X/Y males produce a new mRNA which is processed at its 3' end so that it contains sequences normally specific to the female 3' untranslated region. Although the processing event that requires tra-2 occurs in an untranslated region of the exu transcript, the isolation and characterization of a male-specific exu allele which deletes male 3' untranslated sequence indicate that this processing is biologically significant.

The white gene of Drosophila is required for the pigmentation of the eyes, ocelli, Malpighian tubules and testis sheath. We have examined the regulation of white expression in germ line transformants that carry the mRNA-coding sequences of white flanked by varying amounts of the sequences normally located adjacent to them. A segment with as little as 1.9 kb of 5'- and 2.1 kb of 3'-flanking DNA was expressed to give essentially wild-type pigmentation in all of these tissues and interacted in trans with the zeste-1 allele. In contrast, transformants having segments with 1.1 kb of 5'-flanking DNA had reduced levels of pigment in their eyes, no detectable pigment in their testes and did not interact with zeste-1. The eyes, Malpighian tubules and ocelli of transformants with as little as 0.4 kb of 5'-flanking DNA were pigmented and the eye pigmentation exhibited dosage compensation. We conclude that there are sequence elements present in the region from 1.1 to 1.9 kb upstream from the 5' end of the white transcript which are required for expression in the testes and interaction of white and zeste-1. The same or other elements in this region appear to augment the pigmentation of the eye.

Nuclear history affects the fates of mRNAs in the cytoplasm of cells. Proteins loaded onto mRNAs in the nucleus mark RNAs for subsequent translational regulation, stability, degradation, and subcellular RNA localization. New results show that the Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) Hrp48 contributes to coordinated RNA localization and translational control in oocytes.