The Destinies and Destinations Meeting Review of Rnas

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Cell, Vol. 95, 451–460, November 13, 1998, Copyright 1998 by Cell Press The Destinies and Destinations Meeting Review of RNAs

Tulle Hazelrigg GMC. Since Prospero is independently localized to the Department of Biological Sciences GMC, is prospero mRNA localization gratuitous? The Columbia University answer is no. While staufen mutants alone are not defec- New York, New York 10027 tive in GMC differentation, staufen is important for GMC fate, since staufen mutations enhance defects in GMC fate caused by hypomorphic prospero alleles. Thus, this The third biennial FASEB Summer Research Confer- binary cell fate decision appears to be controlled redun- ence, “Intracellular RNA Sorting, Transport, and Local- dantly by localization of both prospero mRNA and Pros- ization,” was held June 6–11 in Snowmass, Colorado. pero to the GMC daughter cell. Topics included the biological functions of localized Early Embryonic Development RNAs, the nature of nuclear–cytoplasmic RNA transport, Several mRNAs are localized to the animal or vegetal the role of signaling pathways in RNA localization, the poles of the Xenopus oocyte, and some are implicated nature of cis-acting localization elements within RNAs, in axial patterning of the embryo (reviewed in Schnapp the proteins that bind these elements, and the cellular et al., 1997). Mary Lou King (University of Miami Medical mechanisms that achieve cytoplasmic transport and an- School) presented definitive evidence for an essential choring of RNAs to specific domains within cells. role of one vegetally localized mRNA, VegT mRNA, in early embryogenesis. VegT encodes a T box transcription factor (Zhang and King, 1996). Since VegT is ex- Biological Functions of RNA Localization pressed both maternally and zygotically, the contribu- Subcellular RNA localization is a means that cells use tions of maternal and zygotic transcripts had to be to achieve high local concentrations of protein products separated to determine the role of the vegetally localized (reviewed in St. Johnston, 1995; Bashirullah et al., 1998). oocyte mRNA. This was accomplished by injecting anti- RNAs are localized in many different types of cells (Fig- sense oligonucleotides into the vegetal pole of oocytes. ure 1). Localization of mRNA is considered a more effi- Depletion of maternal VegT had global effects on cell cient way to localize protein products than targeting the fates in all regions of the embryo (Zhang et al., 1998). proteins themselves, since a single mRNA can give rise The nature of the developmental defects indicated that to many protein molecules, provided that it is associated maternal VegT is required to establish the three primary with components of the translation machinery. Indeed, germ layers of the embryo. in many cases mRNAs are localized as RNP complexes Ascidian eggs contain localized determinants that are along with translation components, ensuring that local- important for the early embryonic development of these ized translation can occur efficiently. At this meeting, simple chordates. Billie Swalla (Pennsylvania State Uni- abundant examples were reported that demonstrate the versity) reported on the characterization of some of biological roles of localized RNAs. these localized maternal factors, which include both Binary Cell Fates RNAs and proteins (see Swalla and Jeffery, 1996). RNAs A striking example of mRNA localization regulating bi- localized in distinct regions of the ascidian egg include nary cell fate choices is ASH1 mRNA localization in bud- ScYC (which is likely to be the ascidian mitochondrial ding cells of the yeast Saccharomyces cerevisae, where 16S rRNA) and L5 (which encodes a ribosomal protein), localization to the daughter cell is required for repres- both localized in the myoplasm, and PCNA RNA, local- sion of mating type switching in this cell exclusively ized to the ectoplasm. Swalla described a localized pro- (Long et al., 1997; Takizawa et al., 1997; discussed below tein, p58, and experiments using closely related species in The Role of the Cytoskeleton, Motor Proteins, and that suggest that p58 is required for establishing the Cellular Organelles in RNA Transport Pathways). RNA dorsal/ventral embryonic axis. localization also underlies a binary cell fate choice that Germ Cell Determination and Function occurs in the Drosophila embryonic nervous system, The establishment of germ cells is one of the earliest described by Daniel St. Johnston (Wellcome/CRC Insti- cell fate decisions made in organisms with distinct soma tute) and Chris Doe (University of Illinois) (reviewed in and germlines. Germ cell fate often correlates with the Fuerstenberg et al., 1998; Oleynikov and Singer, 1998). inheritance of specialized maternal egg cytoplasm, the During the generation of the Drosophila embryonic CNS, germ plasm, enriched for mitochondria and polar gran- neuroblasts undergo an asymmetric cell division that ules, organelles containing proteins and RNAs (reviewed produces another neuroblast, and a ganglion mother in Ikenishi, 1998). One RNA localized to Drosophila germ cell (GMC), which gives rise to neurons and glia. Local- plasm is the mitochondrial encoded large ribosomal ization of Prospero, a transcription factor, to the GMC RNA, mtlrRNA. Because of its mitochondrial source, daughter is a key event in this cell fate choice. Both classical genetic approaches were not useful to deter- Prospero and prospero mRNA are partitioned to the mine the functional significance of its localization. Sa- GMC. Staufen, a dsRNA-binding protein required for toru Kobayashi (University of Tsukuba, Japan) showed, localizing maternal RNAs in the oocyte (reviewed in St. by inactivating mtlrRNA with injected targeted ribo- Johnston, 1995), binds to prospero mRNA and is re- zymes, that it is required for the formation of pole cells quired for its localization to the GMC (Broadus et al., (Iida and Kobayashi, 1998). Ultrastructural analysis re- 1998; Schuldt et al., 1998). Another protein, Miranda, is vealed that mtlrRNA moves from its site of synthesis in required to localize both Prospero and Staufen to the mitochondria to the surface of nearby polar granules. Cell 452

Figure 1. Examples of Localized RNAs (a) Xenopus Vg1 mRNA localized to the vegetal pole of a stage 6 oocyte (courtesy of Mal- gosia Kloc and Laurence Etkin). (b) Xenopus Xlsirts RNA localized to the vegetal pole of a stage 5 oocyte (courtesy of Mal- gosia Kloc and Laurence Etkin). Localization of Vg1 mRNA and Xlsirts occurs by two inde- pendent pathways, and each is implicated in early embryonic development (reviewed in Schnapp et al., 1997). (c) Drosophila bicoid mRNA localized at the anterior pole of a young embryo (provided by Ava Brent and Tulle Hazelrigg). (d) Drosophila nanos mRNA localized at the posterior pole of a young embryo (courtesy of Elizabeth Gavis). Several maternal mRNAs are localized to the anterior or posterior poles of the developing Drosophila oocyte, where their protein products are essential for early development (reviewed in St. Johnston, 1995). In (a)–(d), in situ hybridizations were performed with probes detected by enzy- matic methods, and visualized by light microscopy. (e) ␤-actin mRNA (red) localized at the leading edge of chicken embryo fibroblasts. Localiza- tion is required for fibroblast morphology (Kislauskis et al., 1994). The mRNA was detected by in situ hybridization with a fluorescent probe, F-actin was stained with fluores- cein-phalloidin (green), and nuclei with DAPI (blue). (Courtesy of Julie Zhu and Edward Kis- lauskis.) (f) Drosophila pair-rule runt mRNA localized in an apical cap above an expressing syncytial blastoderm nucleus. The transcripts of several pair-rule segmentation genes are restricted to such apical caps (Davis and Ish- Horowicz, 1991). The RNA was detected by Cy3 tyramine signal amplification (Wilkie and Davis, 1998), and the nuclear envelopes are stained with FITC-coupled wheat germ ag- glutinin. (Courtesy of Ilan Davis and Gavin Wilkie.) (g) ␤-actin mRNA granules localized along microtubules in a cultured cortical neuron (Bassell et al., 1998). The ␤-actin RNA granules (red) were detected by in situ hybridization with a fluorescent probe, and are associated with microtubules (green), and extend into growth cones. The image was captured by digital imaging microscopy (DIM). (Courtesy of Maritza Martinez and Gary Bassell.) (h) ASH1 reporter mRNA particles visualized by green fluorescent protein (GFP) in live budding yeast cells (Bertrand et al., 1998). Localization of ASH1 mRNA to the daughter cell is required for repression of mating type switching (Long et al., 1997; Takizawa et al., 1997). The RNA particles are being transported to, or are localized at, the bud tip. The RNA is detected by a GFP-MS2 coat protein fusion, bound to MS2 RNA targets present in the ASH1 reporter RNA. (Courtesy of Shailesh Shenoy and Robert Singer).

Since mtlrRNA disappears when pole cells form, its func- leading to agametic gonads. Kobayashi and Miho Asa- tion, while necessary, is transitory. It is not immediately oka (University of Tsukuba) also presented results that apparent why it is associated with the polar granules, suggest that both Pumilio and Nanos act together to since mtlrRNA is normally a component of mitochondrial regulate gene expression in pole cells. Thus, Nanos, ribosomes. Ruth Lehmann (New York University Medical while not required for germ cell formation, is required School, Skirball Institute) reported on the germ cell func- for germ cell function (Kobayashi et al., 1996; Forbes tions of Drosophila Nanos, which is restricted to the and Lehmann, 1998). posterior pole by localization of its mRNA (reviewed in In nematodes, germ cell fate also correlates with the Gavis, 1997). In its role in abdomen formation, Nanos is inheritance of specialized cytoplasm containing elec- required, along with Pumilio, to negatively regulate the tron-dense granules called P granules. These granules, translation of maternal hunchback mRNA (reviewed in like their counterparts in flies and frogs, contain both Macdonald and Smibert, 1996). By genetic manipula- protein and RNA. Susan Strome (Indiana University) re- tions, embryos were produced that are missing the lethal ported on the role of a P granule–associated protein, abdominal defects normally associated with loss of ma- the product of the pgl-1 gene (Kawasaki et al., 1998). ternal nanos function. Pole cells, the germ cell precur- The Pgl-1 protein is predicted to be an RNA-binding sors, form in these embryos but do not migrate correctly, protein due to the presence of an RGG motif. She Meeting Review 453

showed that Pgl-1 is required for function of the germ- this meeting, localization-dependent translation of grk line, but not for its initial determination. P granules are mRNA was also shown to regulate its expression. In present in pgl-1 mutants but are defective. Pgl-1 could early oogenesis the oocyte nucleus is positioned at the function through its RNA binding capacity to localize posterior pole of the oocyte along with grk mRNA, and RNA to P granules, or possibly to regulate translation in mid-oogenesis both are repositioned to the anterior/ of RNAs associated with P granules. dorsal corner of the oocyte. Paul Lasko (McGill Univer- Coupled RNA Localization and sity) reported that Vasa, an RNA-binding protein with Translational Activation similarity to the DEAD box family of RNA helicases, is Translational regulation of localized mRNAs can further required for efficient translation of posteriorly localized refine the cellular distribution of their protein products. grk mRNA (Styhler et al., 1998; Tomancak et al., 1998). Several mRNAs localized in the Drosophila oocyte are Robert Cohen (University of Kansas) reported that trans- subject to localization-dependent translational control: lational repression also restricts the spatial distribution only localized transcripts are translated, while nonlocal- of Gurken. He showed that while some grk mRNA comi- ized transcripts are not (reviewed in Macdonald and grates with the oocyte nucleus as it moves from the Smibert, 1996). Elizabeth Gavis (Princeton University) posterior to the anterior of the oocyte, some grk mRNA presented surprising results that emphasize the biologi- is left behind at the posterior pole and subsequently cal importance of localization-dependent translation. moves independently to the anterior cortex, where it The levels of nanos (nos) mRNA and oskar (osk) mRNA localizes transiently to the ventral surface. This ventrally in bisected anterior and posterior halves of embryos positioned grk mRNA is translationally repressed. Co- were measured by Northern blots, revealing that much hen showed that repression requires the action of the more of these mRNAs are unlocalized than was sus- K10 gene, which probably acts indirectly since K10 pro- pected. Gavis estimated that 96% of nos mRNA, and tein is restricted to the oocyte nucleus. 82% of osk mRNA, are unlocalized. The biological signif- In some cases, expression of a localized mRNA may icance of localization-dependent translation is under- be spatially restricted within its localized domain. Jo- scored by these results. seph Yost (University of Utah) reported that in the case How is posterior translational activation achieved? of Xenopus Xwnt11 expression, subcellular localization Anne Ephrussi (EMBL, Heidelberg) reported that transla- of the mRNA does not alone predict the final protein tional activation of osk mRNA requires interaction be- distribution. Although Xwnt11 mRNA is localized to the tween 5Ј and 3Ј elements in the mRNA. The Bruno trans- entire vegetal cortex of the oocyte, Xwnt11 protein is lational repressor binds specifically to osk 3Ј UTR RNA more abundant in prospective dorsal embryonic cells. sequences called BREs (Bruno response elements) Yost showed that this dorsal/ventral difference in protein (Kim-Ha et al., 1995). A derepressor/activator element levels correlated with increased polyadenylation and was identified between two translation start codons in polysome loading of Xwnt11 mRNA in dorsal cells. the osk 5Ј UTR, along with two proteins, p50 and p68, that bind this RNA sequence (Gunkel et al., 1998). This element functions only in the context of RNAs that un- cis-Acting RNA Localization Signals dergo BRE-mediated repression. p50 is able to bind Localized RNAs contain cis-acting signals that target both the 5Ј derepressor element as well as the BRE them to domains within cells. These signals have been elements in the 3Ј UTR and may function as a corepres- termed RNA “zipcodes” (Kislauskis and Singer, 1992), sor with Bruno when it is bound at the BRE. The function since they direct an RNA to its final address within a of the p68 protein is currently being investigated. cell. The following picture emerges from considerable Elizabeth Gavis (Princeton University) presented evidence that in the case of nos mRNA, activation of trans- genetic and molecular dissection of these RNA se- lation at the posterior pole does not require special local- quences. RNA localization signals usually lie in the 3Ј ized activation factors. Gavis previously identified a 90 UTRs of mRNAs, in regions predicted to be rich in sec- NT element in the nos 3Ј UTR, the translation control ondary structures. Genetic studies support the in vivo element (TCE), that is required for translational repres- existence of these secondary structures. In several in- sion of unlocalized nos mRNA (Gavis et al., 1996). At stances redundant localization elements are dispersed this meeting, Gavis showed that a region of the 3Ј UTR, throughout a large region of the 3Ј UTR. In some cases designated the “ϩ2” element, which contains the TCE localization elements are closely linked to cis-acting se- plus an adjacent 89 NT, can independently direct RNA quences that regulate translation. localization and can also interact with translational re- Paul Macdonald (Stanford University) presented repressors. She proposed that the ϩ2 element can bind sults from his extensive analysis of the cis-acting RNA to either translational repressors or localization factors, sequences required for bicoid (bcd) mRNA localization but not to both. Thus, when localization factors are to the anterior pole of the Drosophila oocyte. A large bound to nos mRNA at the posterior pole, translational segment of the bcd 3Ј UTR was originally shown to repressors cannot bind, and translation ensues. contain its localization signal (Macdonald and Struhl, Gurken, a TGF␣ homolog, is required for a set of pre- 1988). Recently Macdonald has shown that elements in cisely localized signaling events between the oocyte and the bcd 3Ј UTR mediate two RNA localization programs; overlying follicles cells that establish first the anterior- one is initiated by an RNA recognition event designated posterior and then the dorsal-ventral axes of the oocyte “event A,” and the other is initiated by an RNA recogni- (reviewed in Lehmann, 1995). Restriction of Gurken to tion event designated “event B” (Macdonald and Kerr, the sites in the oocyte where signaling occurs is ac- 1997). During oogenesis, event A– and event B–initiated complished by localization of gurken (grk) mRNA. At localization programs overlap (i.e., are redundant) from Cell 454

stage 6 onward, but only event A is active during stages form particles in association with bcd mRNA (Ferrandon 4–6. Macdonald described a detailed analysis of the et al., 1994), is known to function in RNA localization RNA sequence requirements for event A–mediated RNA pathways because of the effects of staufen mutations localization (Macdonald and Kerr, 1998). These se- (reviewed in St. Johnston, 1995; see also Broadus et quences lie within a stem/loop (SLV) of the proposed al., 1998; Schuldt et al., 1998). Biochemical approaches secondary structure of the bcd 3Ј UTR. The effects of have recently been used to identify components of RNA point mutations in this region identified two short nucle- transport particles by searching for proteins that bind otide bulges and adjacent nucleotides where identities cis-acting RNA localization elements. Exl protein, which are constrained, indicating that these sites constitute binds a bcd RNA localization element (MacDonald et al., recognition sites for an RNA-binding protein or proteins, 1995), was discovered in this manner. At this meeting, as well as an adjacent helical stem that may have a several RNA-binding proteins implicated in RNA local- structural role. The existence of redundancy in the localization were reported. A particularly interesting theme ization elements implies that proteins that bind RNA and that is emerging from these studies is that the same mediate localization could be functionally redundant. RNA-binding protein may mediate RNA localization in Macdonald has created RNA reporters stripped of re- different cell types, and in different species, via path- dundancy, which undergo only event A– or event B– ways with different cytoskeletal requirements (Oleyni- initiated localization (Macdonald and Kerr, 1997). These kov and Singer, 1998). should provide important tools for both the biochemical ZBP-1 and Its Homologs and genetic analysis of RNA-binding proteins that func- Robert Singer (Albert Einstein College of Medicine) de- tion in these redundant RNA localization pathways. scribed the characterization of zipcode-binding protein The Xenopus Vg1 mRNA localization signal also con- 1 (ZBP-1), a protein isolated from chick fibroblasts (Ross sists of redundant elements spread out over a large et al., 1997). ZBP-1 binds specifically to the proximal segment of the 3Ј UTR termed the Vg1 localization ele- portion of the 54 NT ␤-actin RNA localization element ment (LE) (Mowry and Melton, 1992). Several labora- (the “zipcode”), which targets the RNA to the leading tories have contributed to a detailed analysis of the edge of motile fibroblasts (Kislauskis et al., 1994). ZBP-1 Vg1 LE, using complementary approaches: Kim Mowry contains five RNA-binding domains: an RRM-type RNA- (Brown University), Bruce Schnapp (Harvard University), binding domain and four KH domains, as well as nuclear and Joel Yisraeli (Hebrew University Medical School). import and export signals. Singer presented recent re- Both the 5Ј and 3Ј ends of the Vg1 LE are required for sults of Yuri Oleynikov that show that ZBP-1 colocalizes localization, and several short repeated motifs, termed with ␤-actin mRNA, and that overexpression of ZBP-1 E1–E4, and VM1, contribute to RNA localization in quan- causes increased localization of ␤-actin mRNA. To iden- titative and redundant fashions (Deshler et al., 1997 and tify RNA-binding proteins required for localization of Vg1 1998; Gautreau et al., 1997; Havin et al., 1998). The E2 mRNA in Xenopus oocytes, three labs working indepen- element is reported to be the binding site for Vera (see below; Deshler et al., 1998), and Mowry reported that dently used biochemical approaches to identify several the VM1 element is required for VgRBP60 binding (see proteins that bind specifically to the Vg1 RNA localiza- below). Mary Lou King (University of Miami Medical tion element, Vg1 LE (Schwartz et al., 1992; Mowry, 1996; School) defined further the sequence requirements for Deshler et al., 1997). Bruce Schnapp (Harvard University) localizing Xenopus Xcat 2 and Veg T mRNAs. The Xcat and Joel Yisraeli (Hebrew University Medical School) 2 localization signal, located in the 3Ј UTR, is relatively reported that proteins their labs had identified as Vera short, only 57 NT long, and contains E1 and E2-like (Schnapp) or Vg1 RBP (Yisraeli) are in fact the same repeats, the significance of which is being tested. In protein, and that this protein is the Xenopus homolog contrast, the VegT 3Ј UTR localization signal is more of chicken ZBP-1 (Deshler et al., 1998; Havin et al., 1998). similar to the Vg1 LE, in that it is long and bipartite, with There is also a human homolog of unknown function, sequence requirements at both ends which contain E2 KOC (KH-domain protein overexpressed in cancer cells). repeats. A role for these proteins in localizing ␤-actin and Vg1 Robert Cohen (University of Kansas) reported an ex- mRNAs is strongly suggested by the fact that mutant ception to the rule that localization signals lie in the 3Ј versions of the ␤-actin or Vg1 mRNA localization ele- UTR: he mapped the Drosophila gurken mRNA localiza- ments that perturb RNA localization also fail to bind tion signal to the first 100 NT of the transcript. John ZBP-1 or Vera/Vg1 RBP, respectively. In addition, Yis- Carson (University of Connecticut Health Sciences Cen- raeli showed that the distribution of Vg1 RBP in the ter) described two elements within the 3Ј UTR of myelin cytoplasm precisely mirrors that of Vg1 mRNA. This un- basic protein (MBP) mRNA, required for distinct steps expected result, that the homologous protein in Xeno- in its localization to the processes of oligodendrocytes: pus and chicken is implicated in RNA localization, sug- the 21 nucleotide RNA transport sequence (RTS) and a gests that RNA localization pathways in vertebrates may longer RNA localization region (RLR) (Ainger et al., 1997). share common, essential features. Carson showed that the RTS sequence is bifunctional, A Second Zipcode-Binding Protein conferring both transport and translation enhancement While several mRNAs selectively localize to dendrites to heterologous reporter RNAs. in adult neurons, but are excluded from axons, the situa- tion is different in cultured developing neurons, where Proteins that Bind RNA Localization Elements some mRNAs are targeted to the growth cones of both In many cases, localized RNAs are transported as large immature dendrites and axons (reviewed in Steward, RNA/protein complexes that can be seen by light mi- 1997). Gary Bassell (Albert Einstein College of Medicine) croscopy. Staufen, a ds RNA-binding protein that can described the case of ␤-actin mRNA, which is localized Meeting Review 455

to granules that are sorted to dendritic and axonal both Vg1RBP60 and Vera/Vg1RBP may both bind to Vg1 growth cones (Bassell et al., 1998). ␤-actin RNA granules mRNA in the nucleus. Vera/Vg1RBP contains a nuclear contain translational components and are associated localization sequence, and Yisraeli reported that the pro- with microtubules in neuronal processes. Microtubule- tein is present in the germinal vesicle of stage 1 oocytes. destabilizing drugs, but not Cytochalasin, delocalize Other RNA-Binding Proteins ␤-actin RNA granules from growth cones. This is in con- While many RNAs in mature neurons are localized to trast to ␤-actin mRNA localization in fibroblasts, where dendrites, tau mRNA is an exception, being localized actin-based localization occurs (Sundell and Singer, to the proximal portion of axons (reviewed in Steward, 1991). Bassell proposed that in the two cell types, 1997). Irith Ginzburg (Weizmann Institute of Science) ␤-actin mRNA associates with different proteins, which reported that HuD, a member of the Elav family of neu- in turn mediate its association with different cytoskeletal ronal RNA-binding proteins, binds to a cis-acting zip- elements. To test this, Bassell and Singer, and Wei Gu code in the 3Ј UTR of tau mRNA. Injection into cultured in the Singer lab, undertook to biochemically identify neurons of HuD antisense oligonucleotides decreased proteins in neuronal extracts that bind the ␤-actin 54-NT HuD mRNA and tau mRNA and inhibited the outgrowth zipcode, and they identified a second zipcode-binding of processes. While HuD protein colocalizes with tau protein, ZBP-2, that colocalizes with ␤-actin mRNA in mRNA in the cell body and proximal portions of axons, neuronal processes. ZBP-2, a 95 kDa protein, is also HuD is also nuclear, indicating that HuD can be added present in fibroblasts and is both nuclear and cyto- to a growing list of proteins implicated in RNA stabiliza- plasmic. Singer suggested that ZBP-2 might be involved tion and hence localization that may cycle between the in both nuclear export and localization of RNAs. nucleus and the cytoplasm. hnRNP Proteins that Bind to Zipcodes A striking example of RNA localization in the central nervous system is localization of myelin basic protein The Role of the Cytoskeleton, Motor Proteins, (MBP) mRNA in the processes of oligodendrocytes. and Cellular Organelles in RNA MBP forms part of the myelin compartment of these Transport Pathways cells and is essential for the myelination of the nervous Both the microtubule and actin cytoskeletons play im- system. MBP RNA forms granules that also contain com- portant roles in RNA localization (reviewed in Wilhelm ponents of the translation machinery and are trans- and Vale, 1993; Bassell and Singer, 1997; Oleynikov and ported along microtubules to the cell processes, in a Singer, 1998). Cytoskeletal elements contribute to both kinesin-dependent manner (Ainger et al., 1993, 1997; the transport of RNAs through the cytoplasm, as well Barbarese et al., 1995; Carson et al., 1997). John Carson as the anchoring of RNAs at their final destinations in (University of Connecticut Health Sciences Center) de- cells. At this meeting, novel approaches to studying RNA scribed the identification of six proteins, in rat brain transport in living cells were described. These methods, extracts, that bind specifically to the RTS RNA localiza- as well as biochemical and genetic approaches, have tion element (Hoek et al., 1998). The predominant protein revealed the functions of factors, including motor pro- is heterogeneous nuclear ribonucleoprotein (hnRNP) A2. teins, in RNA localization pathways. While this protein is primarily nuclear, it is also cyto- Roy Long (Medical College of Wisconsin) and Ralf plasmic, where it localizes to granules. Carson used Jansen (ZMBH, Heidelberg) reported on ASH1 mRNA green fluorescent protein (GFP) to tag segments of localization in budding yeast cells (Long et al., 1997; hnRNP A2, allowing the identification of domains re- Takizawa et al., 1997). ASH1 mRNA localization to the quired for nuclear trafficking, RTS binding, and granule daughter cell is regulated by the SHE genes, including assembly. He presented a model about the biogenesis SHE1, which encodes a type V myosin. Long reported and function of the MBP RNA granules, suggesting that the results of an exciting set of experiments done in the the first step of their assembly may normally occur in laboratory of Robert Singer (Albert Einstein College of the nucleus, where hnRNP A2 is bound, and proposed Medicine) to study ASH1 mRNA localization in living that these granules coordinate transport and translation yeast cells, using a GFP-based detection system (Ber- of MBP mRNA. trand et al., 1998). ASH1 reporter RNAs were con- Kim Mowry (Brown University) characterized a second structed with the ASH1 localization signal linked to the protein previously identified as a Vg1 LE-binding protein target RNA sequence that is bound by the phage MS2 (Mowry, 1996). This 60 kDa protein, VgRBP60, belongs coat protein. This reporter RNA was visualized by co- to the hnRNP 1 protein family. Mowry showed that RNAs transforming yeast with a gene expressing a GFP-MS2 carrying mutations in the VM1 repeat of the Vg LE fail coat protein fusion. The ASH1 reporter RNA assembles to localize and also fail to bind VgRBP60, implicating into particles, typically one per budding yeast cell. These VgRBP60 in Vg1 mRNA localization. Mowry pointed out RNP particles are transported from mother to daughter that the typical experiment to test for localization of Vg1 cell, and particle movement requires the SHE1 myosin RNA segments in Xenopus oocytes utilizes injection of motor. Other SHE gene products appear to be structural exogenous RNA, and that it takes an unusually long time, components of this particle or to affect particle assem- typically several days, for exogenous RNAs to become bly. The advantages of this methodology are far reach- localized. She suggested that injected RNAs may first ing, since it should be applicable to many different types have to bind to proteins in the cytoplasm that they nor- of cells. mally bind in the nucleus. Thus, the initial steps in RNA Localization of bcd mRNA to the anterior pole of devel- localization pathways may first occur in the nucleus, oping Drosophila oocytes requires microtubules, as well with the assembly of RNA transport complexes. Indeed, as the products of several maternal genes, including Cell 456

exuperantia (exu) (reviewed in St. Johnston, 1995). GFP- Xlsirts, in stage 1 and 2 oocytes, whereas Vg1 and VegT tagged Exu forms particles in the nurse cells of devel- mRNAs are localized by the late pathway, which is initi- oping egg chambers, where bcd mRNA is synthesized ated in late stage 2 oocytes and completed by stage 4 before being transported to the oocyte (Wang and Ha- (Forristall et al., 1995; Kloc and Etkin, 1995). Laurence zelrigg, 1994). These particles are likely to contain bcd Etkin (University of Texas M. D. Anderson Cancer Cen- RNA and function as RNA transport particles. Bill Theur- ter) reported results that suggest these two pathways kauf (University of Massachussetts at Worcester) de- are linked (Kloc and Etkin, 1998). Early pathway RNAs scribed work done in collaboration with Tulle Hazelrigg are localized to a region of the mitochondiral cloud (Columbia University) using time-lapse video confocal called the METRO, which is transported from a site near microscopy to analyze the transport of Exu particles in the germinal vesicle (GV), to the vegetal cortex. Vg1 live egg chambers (Theurkauf and Hazelrigg, 1998). They mRNA is first localized to a wedge-shaped area lying found that particle transport in the nurse cell cytoplasm between the GV and the vegetal cortex. The wedge is is microtubule dependent, and they identified dynamic enriched for a specialized compartment of the endoplas- populations of microtubules likely to mediate this trans- mic reticulum (ER). The first physical manifestation of port. Variability in rates and directionality of movement this ER compartment is an accumulation of ER material of these particles were observed, suggesting that multi- in a cap on the trailing edge of the METRO, as it migrates ple motors may mediate movement, providing functional to the vegetal pole. The identification of a ␥-tubulin- redundancy to transport. containing body, possibly a centrosomal remnant, in the Kathryn Miller (Washington University, St. Louis) pre- METRO, suggests that it could provide the microtubule- sented results supporting the idea that coordinated ac- nucleating activity for microtubules that form in the tivity of the microtubule and actin cytoskeletons may wedge. Kloc and Etkin proposed that linking of the early underly the localization of some RNAs. Previous work and late pathways occurs as a set of sequential steps: of Miller’s demonstrated that an unconventional myosin, (1) elaboration of the ER-containing wedge by the Drosophila 95F Myosin, functions in actin-based trans- METRO, (2) association of Vg1 mRNA with ER vesicles port of organelles (Mermall et al., 1994). Miller identified in the wedge, and (3) nucleation of microtubules in the a 95F Myosin-binding protein, D-CLIP-190, the Dro- wedge, which then mediate Vg1 mRNA transport to the sophila homolog of the vertebrate cytoplasmic linker vegetal cortex. What role might the ER compartment protein, CLIP-170, which mediates linkage of vesicles play in Vg1 mRNA transport through the wedge to the to microtubules (Lantz and Miller, 1998). Both Myosin vegetal cortex? Two models have been proposed. Vera/ 95F and D-CLIP-190 are enriched at the posterior pole Vg1 RBP protein, which binds the Vg1 RNA localization of young embryos. Treatment of embryos with Cytocha- signal (see above) associates with both ER and microtu- lasin D caused polar plasm components, osk and nos bule fractions of Xenopus oocyte extracts (Elisha et al, mRNAs, and D-CLIP-190 and 95F Myosin, to be released 1995; Deshler et al., 1997). This suggests that Vera/ from the posterior pole. She proposed that D-CLIP-190 Vg1 RBP could link Vg1 mRNA to ER vesicles that are and 95F Myosin may coordinate the microtubule-based transport and actin-based anchoring of posteriorly lo- transported along microtubules to the vegetal cortex calized RNAs. (Etkin, 1997; Deshler et al., 1998). An alternative model Genetic approaches have also identified components is that the ER plays a structural role in forming the micro- of microtubule and actin-based mRNA localization path- tubule architecture of the wedge (Etkin, 1997). ways in Drosophila. The actin cytoskeleton is implicated in osk mRNA localization, since mutations in a cyto- Localization of RNA by Degradation plasmic Tropomyosin (TmII) disrupt localization of osk RNA localization can arise from generalized degradation mRNA at the oocyte posterior pole (Erdelyi et al., 1995). coupled with protection in specialized regions of the Miklos Erdelyi (Hungarian Academy of Sciences, Insti- cytoplasm (reviewed in Cooperstock and Lipshitz, 1997). tute of Genetics) isolated several mutations that en- This is true for the Drosophila maternally expressed hance specific TmII alleles. Further study of these genes Hsp83 mRNA, which is initially uniformly distributed should lead to a better understanding of the mecha- throughout the early embryo and subsequently de- nisms by which TmII and actin filaments contribute to graded, except at the posterior pole, where it is re- osk RNA localization. Microtubules are required for lo- stricted ultimately to the pole cells. Howard Lipshitz calizing RNAs to both poles of the Drosophila oocyte (Research Institute, Hospital for Sick Children and Uni- (Pokrywka and Stephenson, 1995). Tulle Hazelrigg (Co- versity of Toronto) reported on the nature of cis-acting lumbia University) described maternal effect lethal mu- RNA elements that mediate these coupled events. These tations that perturb RNA localization in the oocyte and elements lie in the Hsp83 3Ј UTR and consist of a 97 also disrupt female meiotic spindle functions. The mei- NT element called the “HDE” (Hsp83 degradation otic defects of these mutants include failure to maintain element) and an adjacent element called the “HPE” metaphase I arrest, and frequent loss and nondisjunc- (Hsp83 protection element). Interestingly, a link between tion of chromosomes. These phenotypes suggest that Hsp83 and nos mRNA degradation is indicated, since these genes encode factors shared by the RNA localiza- the HDE and nos translational control element (TCE) tion machinery and microtubule spindle functions. (Dahanukar and Wharton, 1996; Gavis et al., 1996) are A Role for the Endoplasmic Reticulum interchangeable. Lipshitz has shown that maternal in RNA Localization? factors for RNA degradation require egg activation to There are two pathways for localizing RNAs to the vege- function and cause the degradation of several mRNAs, tal pole of the Xenopus oocyte: the early pathway local- including Hsp83, nos, and string, and also noncoding izes Xwnt11 and XCat2 mRNAs, and the noncoding RNAs, including Pgc RNA. Meeting Review 457

Localization of RNAs in Response dendritic domains. Previous work identified mRNAs that to Extracellular Signals are expressed rapidly in response to electric shock, Growth factors stimulate the rapid recruitment of ␤-actin including the immediate early gene Arc (activity regu- mRNA to the leading edge of cultured fibroblasts (La- lated cytoskeletal protein) (Link et al., 1995; Lyford et tham et al., 1994). Several results reported at this meet- al., 1995). Arc mRNA, which is expressed in response ing affirm that mRNA localization is regulated by extra- to both electric shock and new behavior, is sorted to cellular signals, including growth factors, and signals dendrites soon after its expression. In the recent experi- derived from the interactions of cells with other cells ments, high-frequency localized stimulation of a specific and their environments. region of the hippocampus resulted in specific accumu- Signaling between cells and the extracellular matrix lation of Arc transcripts in the segments of dendrites (ECM) occurs when integrin receptors bind the ECM, synaptically activated in this region of the brain (Steward leading to the formation of focal adhesion complexes et al., 1998). These results are direct proof that neurons (FACs). Robert Singer (Albert Einstein College of Medi- localize mRNAs to specific subdendritic domains in re- cine) reported that this signaling causes the recruitment sponse to synaptic signals. of mRNA and ribosomes to FACs (Chicurel et al., 1998). Microbeads were coated with ECM material and allowed to bind to cultured cells; binding induced the formation Nuclear/Cytoplasmic RNA Transport of FACs at the sites where beads were bound. High- Transport in and out of the nucleus occurs through the (MDa 120ف) resolution in situ hybridization determined that poly(A)ϩ nuclear pore complexes (NPCs), very large and ribosomal RNAs were recruited to these newly in- macromolecular complexes that span the nuclear enve- -proteins called nucleo 40ف duced FACs. They showed that this effect was not the lope and are composed of result of induced gene transcription but was due to the porins (reviewed in Ohno et al., 1998). NPCs mediate recruitment of cytoplasmic mRNA to the FACs, and that transport in combination with receptors that bind car- mechanical tension on the actin cytoskeleton influences goes in the nucleus or cytoplasm, adaptor proteins that RNA recruitment. This latter effect is intriguing and may in some cases link receptors to their cargoes, and Ran, be related to the geometry of mechanically stressed a small GTPase that imparts directionality to transport. actin filaments, since previous work from the Singer lab Results reported at this meeting included the character- showed that mRNAs and translation factors accumulate ization of receptors and their interactions with cargo at the verteces of overlapping actin filaments (Bassell and the NPC, and the identification of RNA processing et al., 1994). and proofreading events essential to export. Kenneth Kosik (Harvard Medical School) described Iain Mattaj (EMBL-Heidelberg) described two recep- the sorting of RNA granules to dendritic domains in tors that function in nuclear export of RNAs: the tRNA cultured neurons. These RNA granules were identified receptor Exportin-t, which binds directly to the RNA, with fluorescent dyes and contain poly(A)ϩ RNA as well and the U snRNA receptor CRM1, which binds indirectly as translation components (Knowles et al., 1996). Micro- via interaction with adaptor proteins that bind the RNA tubules are required for their transport into dendrites, (Fornerod et al., 1997; Arts et al., 1998). He showed and the rates are typical of fast transport. An attractive that in both cases binding of the receptors (or receptor/ hypothesis is that sorting of these RNA granules to den- adaptors) to the RNA in the nucleus requires GTP-Ran, drites serves to target both mRNAs and their translation which binds cooperatively to the receptor in the pres- machinery to specific postsynaptic domains within neu- ence of the RNA or RNA/adaptor complex. Release of rons, and that neuronal plasticity may involve the redis- the cargo RNAs in the cytoplasm occurs upon hydrolysis tribution of these granules in response to extracellular of the Ran-bound GTP. Elsebet Lund (University of Wis- signals. In support of this idea, Kosik and his coworkers consin) reported on the role of nuclear proofreading in have shown that RNA granule translocation to dendrites the export of tRNAs from the nucleus. She and James can be stimulated by the neurotrophic factor NT-3 Dahlberg found that in Xenopus oocytes only mature (Knowles and Kosik, 1997). tRNAs with properly processed 5Ј and 3Ј ends are trans- Henri Tiedge (State University of New York at Brook- ported to the cytoplasm, and that aminoacylation of lyn) reported that expression and localization of BC1 tRNAs occurs in the nucleus and is important for efficient RNA, a noncoding polIII transcript that is localized to nuclear export (Lund and Dahlberg, 1998). These results neuronal cell bodies and dendrites, is responsive to cell– show that before export tRNAs are monitored within cell interactions. BC1 RNA is found only in cultured hip- nuclei, ensuring that only those with structural and func- pocampal neurons that have made synaptic connec- tional integrity are transported efficiently to the cyto- tions with other cells, and its presence can be reversibly plasm. reduced by treatment with tetrodotoxin, which blocks Charles Cole (Dartmouth Medical School) described neuronal activity (Muslimov et al., 1998). Tiedge pro- an RNA helicase that is required for nuclear export of posed that synaptic stimulation induces gene expres- mRNA in Saccharomyces cerevisiae. A genetic screen sion of BC1, followed by active transport of BC1 RNA for temperature-sensitive mutants that accumulate mRNA to dendrites, where the RNA may be selectively targeted in the nucleus identified 10 RAT (ribonucleic acid traf- to a subset of dendritic spines, and/or contribute to ficking) genes. Several encode nucleoporins, and one, selective regional translation within dendrites. RAT8/DBP5, encodes a DEAD box protein closely re- Oswald Steward (University of Virginia Medical School) lated to eIF4A, an RNA helicase required for translation reported that synaptic activation causes the selective initiation (Snay-Hodge et al., 1998). In yeast cells Rat8p accumulation of Arc mRNA at specific postsynaptic is punctate in the cytoplasm, accumulates at perinuclear Cell 458

sites, and associates with NPCs. A related helicase ex- key components have been identified, including pro- ists in diverse species, including Schizosaccharomyces teins that bind cis-acting RNA localization elements, pombe, Dictyostelium, flies, frogs, mice, and humans, and components of the translational machinery. RNA- suggesting conservation of this factor for mRNA export. binding proteins identified biochemically by their bind- Pamela Silver (Dana Farber Cancer Institute, Harvard ing to cis-acting RNA localization elements are impli- Medical School) reported recent work on the yeast NPL3 cated in RNA localization quite literally due to “guilt by gene, which encodes an hnRNP required for nuclear association”: since mutant forms of the cis-acting RNA mRNA export. Silver showed that Npl3p shuttles rapidly elements fail to localize correctly, the proteins that asso- in and out of the nucleus. Npl3p export occurs only when ciate with these elements are implicated in RNA localiza- mRNA is exported; Npl3p does not leave the nucleus tion. Although the implication is strong, a localization in PolII mutants or when transcriptional inhibitors are role for these RNA-binding proteins has not yet been added. Methylation of hnRNPs on arginine residues oc- proven. Genetic analysis, or other methods to produce curs in all eukaryotes. Silver reported the identification loss-of-function phenotypes, will provide the functional of an enzyme required for methylation of hnRNPs, analysis that is needed. Hmt1p, and showed that it regulates export of Npl3p How are RNAs started on their localization pathways, from the nucleus (Shen et al., 1998). She proposed that and how are they anchored at their final destinations in arginine methylation facilitates assembly of RNA/protein cells? The first and the last steps of RNA localization pathways are in many ways the least understood. Some complexes destined for nuclear export. proteins that bind RNA localization elements appear to Ilan Davis (University of Edinburgh) described im- shuttle between the nucleus and the cytoplasm, sug- proved methods that he developed, with Gavin Wilkie gesting that the first steps in RNA localization pathways (University of Edinburgh), for sensitive, high-resolution could occur within nuclei, where RNA transport com- in situ hybridization to detect transcripts in the cyto- plexes may be assembled. The recently developed plasm and nuclei of Drosophila tissues (Wilkie and Davis, methods (Femino et al., 1998) to visualize single RNA 1998). They used this method to screen temperature- molecules within nuclei will be valuable in the analysis sensitive lethal mutations to identify genes required for of these early assembly steps. The relationship between nuclear mRNA transport. Davis reported that one muta- the assembly of complexes, and their nuclear export, tion, fun[ts], causes mRNA to be retained within the will be important areas of enquiry. nuclei of blastoderm embryos, where it accumulates in Recent advances in RNA localization research include patches. Injection of a dominant-negative form of human the recognition that signaling pathways regulate mRNA Importin-␤ caused a similar phenotype. This phenotype localization in several types of cells, including neurons. may represent an intermediate in mRNA trafficking in Understanding how signaling pathways impinge on RNA the nucleus. The study of this gene, and others isolated localization is an important goal. by their screens, holds the promise of providing insights Answers will be forthcoming about how movement of into nuclear/cytoplasmic trafficking of mRNA during de- RNA particles on microtubules and actin filaments is velopment (see Davis, 1997). achieved. Already two motors have been identified that The Drosophila pair-rule genes are expressed in seven are required for RNA localization: the SHE1 myosin is stripes in syncytial blastoderm embryos, and their mRNAs required for ASH1 mRNA localization in budding yeast are localized in the cytoplasm apically of each express- cells (Long et al., 1997; Takizawa et al., 1997; Bertrand ing nucleus (Davis and Ish-Horowicz, 1991). David Ish- et al., 1998), and kinesin is required for localization of Horowicz (Imperial Cancer Research Fund) discussed MBP mRNA in oligodendrocytes (Carson et al., 1997). the proposition that vectorial nuclear export underlies The in vivo GFP-based methods to visualize RNA local- apical localization. Certain aneuploid embryos are de- ization pathways in living cells (Bertrand et al., 1998; fective in maintaining nuclei at the periphery of the syn- Theurkauf and Hazelrigg, 1998) will no doubt be applied cytial blastoderm embryo, so that some nuclei form an to a wide variety of cell types. Real-time analysis of internal layer. Pair-rule transcripts associated with these the RNA localization pathways in living cells, using this internal nuclei are still localized in apical caps (Francis- noninvasive methodology, will allow visualization of dy- Lang et al., 1996). These internal nuclei do not have the namic events that have not been accessible from the apical microtubule baskets or actin arrays typical of study of fixed material. superficial nuclei, suggesting that these cytoskeletal el- Acknowledgments ements do not localize dispersed pair-rule transcripts. Ish-Horowicz also reported that when fluorescently la- Thanks to Larry Etkin and Howard Lipshitz for organizing a very beled pair-rule transcripts are injected into the embryo informative and rewarding meeting. Larry Etkin, Howard Lipshitz, cytoplasm, the RNA is not apically localized, suggesting and Rob Singer provided helpful comments on the entire manu- that a nuclear history is indeed essential for apical local- script, and several of the meeting participants provided helpful infor- ization. mation for specific sections. Thanks to all of them, to Ava Brent for her help in preparing Figure 1, and to the people who contributed images for this figure, including Malgosia Kloc and Larry Etkin, Eliza- Future Directions beth Gavis, Shailesh Shenoy and Rob Singer, Julie Zhu and Edward This field is progressing rapidly, and developments are Kislauskis, Gavin Wilkie and Ilan Davis, and Maritza Martinez and expected on several fronts. In most cases, localized Gary Bassell. RNAs are transported as large complexes, visible by References light microscopy as particles. Understanding the nature of these particles, and how they regulate RNA localiza- Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, tion, is an important immediate goal. Already several E., and Carson, J.H. (1993). Transport and localization of exogenous Meeting Review 459

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