Munro TP, Kwon S, Schnapp BJ, St Johnston D.

Wellcome Trust

	Figure 1. IMP localization during oogenesis. (A–G) Localization of IMP during oogenesis visualized using G080, a GFP protein trap line (A, B, and E–G), or a GFP–IMP fusion construct specifically expressed in the germline (C and D). (A) IMP localizes to future oocyte in the germarium, accumulates in the oocyte through stage 7–8, appears enriched at both the anterior and posterior of the oocyte at stage 8, and is restricted to a crescent at the oocyte posterior pole by stage 9–10. These localizations are similar to those of Staufen protein, which marks the localization of osk mRNA. Within nurse cells, IMP is primarily cytoplasmic, but also rims the nucleus (inset). (B–G) IMP localization (green) and actin (red) in oocytes at stage 9 (B) or 10 (C) in WT and mutant backgrounds (D–G). (D) IMP is absent from the posterior crescent in a stau-null mutant that fails to localize osk RNA. (E) Like osk RNA (not depicted), IMP localizes ectopically in a transheterozygous par-1 allele combination that disrupts the polarity of the egg chamber. (F) IMP localizes normally in a vasa mutant that interferes with pole plasm formation. (G) IMP localizes to the posterior at stage 9 in an osk nonsense mutant (osk54/Df(3R)pXT103) egg chamber, indicating its localization depends on osk RNA, not protein. (H) IMP contains four KH-type RNA-binding domains and a glutamine-rich COOH terminus. Numbers indicate the percentage of amino acid identity between IMP's KH domains and those of its homologues. Bars, 25 μm.
	Figure 2. Characterization of the IBE UUUAY. (A and B) Representative RNA sequences selected after 12 rounds of SELEX against IMP's KH3 (A) and KH4 (B) domains. IBEs are in red. (C and D) Filter-binding assays between three tandem repeats of the winner sequence 4-12-13 (or the same RNA with IBEs mutated to UUgAU or gggcg) and either IMP KH3 (C) or the entire protein (D). (E) Electrophoretic mobility shift assay between IMP and three tandem copies of the winner sequence 4-12-13 containing either WT or mutant IBE motifs. Only RNAs with the WT (UUUAU) motifs induced a band shift.
	Figure 3. The osk 3′UTR contains 13 IBEs and UV cross-links to IMP. (A) Positions of the 13 IBEs in the osk 3′UTR. (B) A 65-kD protein (IMP) in D. melanogaster ovary extracts specifically cross-links the 1,120-nt osk 3′UTR, but not the 817-nt bcd 3′UTR, which contains only two UUUAY motifs. (C and D) Anti-IMP immunoblot of ovary extract (C) labels the same band that UV cross-links to the 32P-osk 3′UTR (D). (E) 32P-osk 3′UTR cross-links to an ∼95-kD polypeptide (GFP–IMP) in embryo extracts of the protein trap line G080. (F) Cross-linking reactions between the 32P-osk 3′UTR and oocyte extracts in the presence of increasing concentrations of cold, competitor RNAs, including WT and mutant osk 3′UTRs and the bicoid 3′UTR.
	Figure 4. osk mRNA, IMP, and Stau distributions in osk13 TTgAY flies. (A) Fluorescent in situ hybridizations comparing WT and mutant transgenic osk RNAs in flies that otherwise lack endogenous osk RNA. Mutant osk RNA localizes normally through stage 9 (middle), but by stage 10 (bottom), is evident as diffuse fluorescence fanning out from the posterior pole. (B) IMP (green) and Stau (blue) proteins in WT and mutant osk oocytes before stage 9 (top), at stage 9 (middle), and at stage 10 (bottom). Actin is visualized with rhodamine-phalloidin (red). At stage 9, Stau is localized normally at the posterior pole in oocytes that express the mutant osk transgene, whereas IMP is diffuse and not concentrated at the posterior. Both Stau and IMP are missing from the posterior pole in stage 10 oocytes that express the mutant osk RNA. Bars, 25 μm.
	Figure 5. IBE mutations abolish osk RNA translational activation. (A and B) Anti-Osk immunostaining of osk87/Df(3R)pXT103 egg chambers expressing a WT osk transgene, showing a crescent of Osk protein (A). Osk protein is missing in egg chambers from oskTTgAY flies (B). (C and D) Cuticle preparations of larvae from osk87/Df(3R)pXT103 that express the WT (C) or mutant oskTTgAY (D) transgene. (E) Western blot of ovarian protein extracts probed with anti-Osk antibody, followed by an anti-tubulin antibody. Extracts were from WT flies with no osk transgene (WT) or from osk87/Df(3R)pXT103 flies expressing either a WT or the IBE mutant oskTTgAY osk transgene, or the nonsense mutant osk54 transgene. Neither long nor short Osk is present in the oskTTgAY or osk54 mutants. Bars, 25 μm.
	Figure 6. Mutations to nonoverlapping subsets of osk's IBEs and their affects on osk RNA and protein distributions in the oocyte. (A) Four subsets of IBEs in the osk 3′UTR: A, B, C, and D. (B–E) osk in situ hybridizations at stages 9 (B and C) and 10 (D and E). (F and G) Osk protein detected by immunofluorescence at stage 10. Oocytes that express osk RNA with mutations to IBE subset B display normal localization of osk mRNA and protein. Mutations to subset D, or to subsets A and C (not depicted) cause osk RNA delocalization from the posterior pole at stage 10; Osk protein is absent in these oocytes. Bars, 25 μm.
	Figure 7. Analysis of oocytes that lack IMP protein. (A) The imp gene is flanked by sesB, Ant2, and sbr. Diagram shows alternatively spliced isoforms (exons in blue), the location of GFP in protein trap line G080, and P element insertion EP(X)760 used to create three mutant alleles of IMP. The positions of the two alternate ATGs are shown in green. (B) Immunoblots of preblastoderm embryos laid by mothers with IMP mutant germline clones. IMP2 may produce a truncated protein, whereas IMP7 and IMP8 are protein nulls. (C, top) A germline clone (center egg chamber) marked by the absence of GFP. (bottom) IMP is absent only from the mutant clone. (D) The distributions of osk mRNA and Osk protein in IMP-null egg chambers (middle and right) are indistinguishable from WT (left) in IMP mutant germline clones generated using the FLP/FRT OvoD1 DFS technique. Bars, 25 μm.

Bergsten, Role for mRNA localization in translational activation but not spatial restriction of nanos RNA. 1999, Pubmed

Bergsten, Role for mRNA localization in translational activation but not spatial restriction of nanos RNA. 1999, Pubmed
Böhl, She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p. 2000, Pubmed
Brand, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. 1993, Pubmed
Breitwieser, Oskar protein interaction with Vasa represents an essential step in polar granule assembly. 1996, Pubmed
Bubunenko, A consensus RNA signal that directs germ layer determinants to the vegetal cortex of Xenopus oocytes. 2002, Pubmed , Xenbase
Castagnetti, Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte. 2003, Pubmed
Chang, The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes. 1999, Pubmed , Xenbase
Cook, The Drosophila SDE3 homolog armitage is required for oskar mRNA silencing and embryonic axis specification. 2004, Pubmed
Cote, A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization. 1999, Pubmed , Xenbase
Czaplinski, Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-beta-related mRNA during oogenesis. 2005, Pubmed , Xenbase
Deshler, Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. 1997, Pubmed , Xenbase
Deshler, A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates. 1998, Pubmed , Xenbase
Eom, Localization of a beta-actin messenger ribonucleoprotein complex with zipcode-binding protein modulates the density of dendritic filopodia and filopodial synapses. 2003, Pubmed
Ephrussi, Induction of germ cell formation by oskar. 1992, Pubmed
Ephrussi, Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. 1991, Pubmed
Farina, Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment. 2003, Pubmed
Gavis, Localization of nanos RNA controls embryonic polarity. 1992, Pubmed
Git, The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association. 2002, Pubmed , Xenbase
Gonzalez, ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation. 1999, Pubmed
Gunkel, Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA. 1998, Pubmed
Hachet, Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. 2004, Pubmed
Havin, RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization. 1998, Pubmed , Xenbase
Hay, Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity. 1990, Pubmed
Hoek, hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA. 1998, Pubmed , Xenbase
Huynh, The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization. 2004, Pubmed
Huynh, PAR-1 is required for the maintenance of oocyte fate in Drosophila. 2001, Pubmed
Jensen, The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain. 2000, Pubmed
Kim-Ha, Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. 1995, Pubmed
Kim-Ha, Multiple RNA regulatory elements mediate distinct steps in localization of oskar mRNA. 1993, Pubmed
Kim-Ha, oskar mRNA is localized to the posterior pole of the Drosophila oocyte. 1991, Pubmed
Kislauskis, Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. 1994, Pubmed
Kislauskis, Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. 1993, Pubmed
Kroll, A homolog of FBP2/KSRP binds to localized mRNAs in Xenopus oocytes. 2002, Pubmed , Xenbase
Kwon, UUCAC- and vera-dependent localization of VegT RNA in Xenopus oocytes. 2002, Pubmed , Xenbase
Lasko, Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. 1990, Pubmed
Lasko, The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. 1988, Pubmed
Lehmann, Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila. 1986, Pubmed
Lewis, Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome. 2000, Pubmed
Lewis, Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains. 1999, Pubmed
Lewis, Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes. 2004, Pubmed , Xenbase
Long, She2p is a novel RNA-binding protein that recruits the Myo4p-She3p complex to ASH1 mRNA. 2000, Pubmed
Markussen, Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly. 1995, Pubmed
Martin, The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen. 2003, Pubmed
Mathews, Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. 1999, Pubmed
Melton, Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes. , Pubmed , Xenbase
Micklem, The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. 1997, Pubmed
Micklem, Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation. 2000, Pubmed
Morin, A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. 2001, Pubmed
Nakamura, Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. 2004, Pubmed
Neuman-Silberberg, The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. 1993, Pubmed
Nielsen, The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila. 2000, Pubmed , Xenbase
Norvell, Specific isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis. 1999, Pubmed
Oleynikov, Real-time visualization of ZBP1 association with beta-actin mRNA during transcription and localization. 2003, Pubmed
Ramos, RNA recognition by a Staufen double-stranded RNA-binding domain. 2000, Pubmed
Rongo, Localization of oskar RNA regulates oskar translation and requires Oskar protein. 1995, Pubmed
Rongo, Regulated synthesis, transport and assembly of the Drosophila germ plasm. 1996, Pubmed , Xenbase
Rørth, Gal4 in the Drosophila female germline. 1998, Pubmed
Ross, Characterization of a beta-actin mRNA zipcode-binding protein. 1997, Pubmed
Shulman, The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole. 2000, Pubmed
Smith, Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos. 1992, Pubmed
St Johnston, Staufen, a gene required to localize maternal RNAs in the Drosophila egg. 1991, Pubmed
St Johnston, Moving messages: the intracellular localization of mRNAs. 2005, Pubmed
St Johnston, Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. 1989, Pubmed
Takizawa, The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. 2000, Pubmed
Tiruchinapalli, Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons. 2003, Pubmed
van Eeden, Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. 2001, Pubmed
Vanzo, Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte. 2002, Pubmed
Wilhelm, Mechanisms of translational regulation in Drosophila. 2005, Pubmed
Wilhelm, Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. 2003, Pubmed
Wilson, aubergine enhances oskar translation in the Drosophila ovary. 1996, Pubmed
Yano, Hrp48, a Drosophila hnRNPA/B homolog, binds and regulates translation of oskar mRNA. 2004, Pubmed
Zhang, Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning. 1996, Pubmed , Xenbase
Zhang, Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility. 2001, Pubmed
Zhao, A proline-rich protein binds to the localization element of Xenopus Vg1 mRNA and to ligands involved in actin polymerization. 2001, Pubmed , Xenbase