The 3' Untranslated Region of Localized Maternal Messages Contains A

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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7164-7168, August 1992 Cell Biology The 3' untranslated region of localized maternal messages contains a conserved motif involved in mRNA localization ELLEN GOTTLIEB Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England Communicated by Aaron KMug, February 19, 1992 (receivedfor review December 2, 1991)

ABSTRACT Messenger RNA (mRNA) localization is transcripts residing at distinct oocyte locations while most emerg as a means ofr ig gene expression. This process messages are uniformly distributed. Both bicoid and Vgl is operational in fly and fog development, where a subset of mRNAs obtain their positions posttranscriptionally via mul- maternally inherited RNAs are asymmetrically distributed and tistep, dynamic processes (11, 13, 14). Cytoskeletal compo- thought to impart axial polarity to the embryo. Since most nents are implicated in localization: for Vgl, microtubules maternal mRNAs are unioly distributed, an a l must participate in translocation while microfilaments and inter- exist to recognize and specifically transport these rare loalized mediate filaments may facilitate cortical anchoring (14, 15). species. Here I report the Identificaton of a nine-nudeotde Localized mRNAs probably exist as mRNA-protein com- motif, YUGUUYCUG, common to the 3' unta regions of plexes (mRNPs). Some mRNP components could be required four sequenced of this class: Drosophia bicold and for localization by interacting with the localization machinery nanos mRNAs and Xenopus An2 and Vgl mRNAs. To test the or with a receptor at the localization site. Implicit in this role of this nner sequence in the l tio process, a model are the existence of cis-acting localization signals and Drosophila transient assay has been established. The assay trans-acting localization factors that could account for local- reveals that bicoid mRNA fically lacking this noner is ization specificity and selectivity. Signals for localization partal~ly mislocalized. In contrast, namer deletion is incon- reside in the RNA: synthetic Vgl transcripts can be localized sequential to message stabilt. The existence of specific and following injection into Xenopus oocytes (16), and a trans- genera IIRNA loction igal isproposed and itis s t genic fly analysis revealed that bicoid RNA localization that this conserved motif belongs to the latter category. requires signals harbored within 625 nucleotides (nt) of the bicoid 3' untranslated region (UTR) (17). Spatial organization of cells and subcellular regions arises in In an in-depth search for a defined localization signal, I part from the sorting and subsequent localization of proteins compared the available sequences of localized maternal and RNA. Although the study of RNA trafficking is in its RNAs. Here I report the finding of a 9-nt motif common to infancy, this process has already been documented in several the 3' UTR of several of these mRNA species. With a organisms (reviewed in ref. 1). Present indications are that transient localization assay, it is demonstrated that the se- RNA localization is likely to take its place alongside splicing, quence does indeed participate in the localization ofsynthetic polyadenylylation, stabilization, and translation as a major bicoid RNA but is inconsequential to message stability. regulatory step in the expression of some mRNAs and that localizing an RNA may have profound implications for the other processes that affect a transcript during its lifetime. MATERIALS AND METHODS Striking examples of localized messages can be found Computer Analysis. Sequence comparisons were per- among the maternal mRNAs of flies and frogs, where local- formed with DIAGON and ANALYSEQ (R. Staden). ization has been implicated in the establishment of axial Preparation ofRNA Substrates. m7GpppG-capped injection polarity. In Drosophila, the primary determinants of the substrates were synthesized using T7 RNA polymerase (18), embryonic anterior-posterior axis are encoded by the bicoid with reaction mixtures containing 0.12 mM UTP to produce and nanos mRNAs (reviewed in ref. 2). These messages are full-length transcripts and [a-[35S]thio]UTP (20 ,uCi; 400 localized to the anterior and posterior poles ofthe Drosophila Ci/mmol, Amersham; 1 Ci = 37 GBq) to generate localization egg, respectively (3-5). bicoid mRNA translation produces a substrates. Transcripts were poly(A)-tailed by poly(A) poly- transcription factor that specifies head and thoracic struc- merase (Escherichia coli, Pharmacia) in 50 mM TrisHCl, pH tures (6-9) whereas nanos mRNA translation is required to 7.4/10 mM MgCl2/2.5 mM MnCl2/250 mM NaCl/12.5 mM generate abdominal structures (5). Mutations at several loci ATP at 370C (25 min). Resulting tails were sized at 35-155 nt, disrupt localization of these messages, resulting in aberrant with the major species around 60 nt. embryos. This illustrates the importance of the localization The Assay. Injections and autoradiography. Canton-S fly process and argues that an apparatus exists to establish eggs were collected (15 min), prepared (19), and injected with and/or maintain RNA position. In Xenopus, searches for RNA in 5 mM KCI/0.1 mM sodium phosphate, pH 6.8. determinants of polarity uncovered a few localized maternal Recipients were incubated at 200C for 95 min to allow mRNAs in the oocyte and early egg. Of these, An2 RNA is transcript diffusion. This is roughly the longest time possible localized to the animal pole and its product may contribute to before endogenous bicoid RNA is degraded (13). Of 106 a respiration gradient along the animal-vegetal axis (10). Vgl sample embryos, 83% developed past the experiment's end, RNA is localized to the vegetal pole and its product has been and of those 47% hatched. Embryos were fixed in 2% implicated in mesoderm induction (11, 12). glutaraldehyde, devitellinized, washed with phosphate- Our understanding ofthe molecular mechanisms governing buffered saline/1% bovine serum albumin/0.1% Triton localization ofthe aforementioned mRNAs is minimal. RNA X-100, mounted on polylysine subbed slides, dipped in NTB2 sorting operates in Drosophila and Xenopus eggs, with rare emulsion (Kodak), and stored at 40C. Autoradiographs were developed according to ref. 20. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: mRNP, mRNA-protein complex; nt, nucleotide(s); in accordance with 18 U.S.C. §1734 solely to indicate this fact. UTR, untranslated region. 7164 Downloaded by guest on October 9, 2021 Cell Biology: Gottlieb Proc. Natl. Acad. Sci. USA 89 (1992) 7165 Transcripts were injected into the anterior of the embryo, Table 1. A conserved 9-nt motif, YUGUUYCUG, is found in the direction from which bicoid mRNA normally enters the the 3' UTR of four sequenced maternal messages known to be egg in vivo. Lateral injection resulted in preferential anterior localized posttranscriptionally concentration of the bicoid transcripts. However, the exper- Sequence imental time window is insufficient to chase most of the RNA Maternal RNA (5' -- 3') 3' UTR position* to this pole. Observation of sequence-dependent localization in this assay requires the presence of carrier RNA. Embryos Localized messages were injected with 0.1 mg of RNA per ml, 5% of which was Drosophila bicoid UUGUUCCUG 52/817 radiolabeled bicoid mRNA and 95% of which was yeast Drosophila nanos CUGUUUCUG 244/872 tRNA carrier. This is "'3 x 104 molecules of exogenous Xenopus An2 UUGUUCCUG 43/146 bicoid mRNA per embryo. Xenopus Vgl UUGUUCCUG 77/1268 Constructs. Constructs were derived from the bicoid Potential candidates cDNA (4) cloned into pGEM-1 (Promega). For the bicoid Xenopus cyclin b2 CUGUUCCUG 69/170 construct missing 45nt between the Hpa I and EcoRV sites, Xenopus D-7 CUGUUCCUG 35/188 the 5-kilobase Bgl II-EcoRV and 229-base Bgl II-Hpa I Consensus YUGUUYCUG fragments from the bicoid parent clone were ligated. For the Included are species localized to each of the two egg poles in both bicoid clone devoid ofthe nonamer, the 229-base Bgl II-Hpa frogs and flies; these encode disparate functions. A subset of cyclin I and 5-kilobase Bgl II-Mlu I fragments from the parent clone B transcripts are localized to the posterior pole of the Drosophila and a double-stranded DNA fragment (5'-AACCACTGAT- embryo (22, 23). While the 9-nt motif is not contained in the one available Drosophila cyclin B sequence, it occurs in the 3' UTR of TGTACAAATACCAAGTGATTGTAGATATCTA-3') one of two Xenopus cyclin B homologues. This and D-7 are listed as were ligated. Constructs were screened by restriction anal- potential candidates because they are maternal messages for which ysis and confirmed by sequencing (21). Controls included subcellular localization data are not available but which harbor the hunchback, generated from a 2.4-kilobase Xba I fragment 9-nt motif; this may indicate that they are localized. The localized inserted into Bluescribe plasmid (Stratagene); tramtrack Drosophila oskar mRNA has a partial match (UUGUCUUG; i.e., lacking the first 330 nt and cloned into pET 11a; and scute YUG-UYYUG). A related oskar sequence may provide nonamer inserted into Bluescribe. Before transcription, plasmids were function or the motif may govern a localization step not operational linearized with Sac II (bicoid/Sac II), Stu I (bicoid/Stu I), for oskar (see Discussion). Indeed, the localization ofoskar and other HindIII (other bicoid derivatives, hunchback, and scute), or RNAs may differ; bicoid and Vgl mRNAs are localized at the RNA level in a process dictated by RNA signals in their 3' UTRs, whereas EcoRI (tramtrack). oskar RNA localization is disrupted by mutations in the oskar coding Transcript stability. 32P-radiolabeled transcripts were in- region that affect oskar protein synthesis but not oskar RNA syn- jected as above. Following incubation, embryos were ho- thesis (24, 25). mogenized in extraction buffer (50 mM Tris-HCl, pH 7.5/50 *First residue in the motif relative to the stop codon/3' UTR length mM NaCl/10 mM EDTA/0.5% SDS), treated with proteinase in bases. K, and extracted with phenol. Scintillation counting revealed that 79-94% of the counts were retrieved in each case. A fly with four bicoid genes can be constructed and the Samples were fractionated by polyacrylamide gel electro- resulting RNA localized (9, 32), implying that wild-type phoresis under denaturing conditions. The gel was backed embryos possess excess message localization capacity. with DEAE paper to minimize the loss of small RNA frag- Hence, they were used as injection hosts. Transcripts were ments, dried, and exposed to Fuji film at -70°C. mixed with carrier RNA to enhance discrimination between nonspecific RNA-binding protein(s) and those that facilitate localization. They were then injected into the anterior of RESULTS embryos. This is the direction from which bicoid mRNA Identification of a 9-nt Motif in the 3' UTR of Localized normally enters the egg from the nurse cells in vivo. Due to Maternal RNAs. In an attempt to identify cis-acting localiza- the injection site, this is in essence a retention assay and tion signals, the available sequenced localized maternal constructs are scored with respect to whether they are mRNAs were examined, revealing a 9-nt motif, YUGUUY- retained at the anterior or how far they diffuse from it. CUG, in the 3' UTR of four localized species: Drosophila Under the conditions employed, full-length bicoid tran- bicoid and nanos mRNAs and Xenopus An2 and Vgl mRNAs scripts are tightly localized to the embryo's anterior tip -60% (Table 1). The calculated frequency of this motif is -1 in of the time (Fig. 2 A and F) whereas several control Drosoph- 66,000 bases. It is the longest contiguous stretch of sequence 5, 3, shared among these message trailers, the next longest common UTR CODING REGION UTR 5, .... 3, motif being a heptamer. The nonamer is absent from nonlo- UIR~~~~~~~~~~~~AA.. calized maternal RNAs and is distinct from other motifs in the n a ' E 3' UTR that direct RNA-level events such as polyadenylyla- 19 . tion (26), mRNA destabilization (27), and poly(A)-tail elonga- ,;5r = tion during Xenopus oocyte maturation (28, 29). Although the data set of localized RNAs is presently small, functional Hrd-aI EC RV importance ofthe nonamer is underscored by its conservation in the bicoid 3' UTR from all eight Drosophila species exam- AACCACTrGUGrTCCTGATTGTACAAATACCAAGTGATrGTAGAT ined, diverging up to 60 million years (see refs. 30 and 31 for sequences). These data suggest that the motifYUGUUYCUG FIG. 1. Diagram of the 2.4-kilobase bicoid cDNA. The 817-nt 3' is a reasonable candidate RNA localization signal. UTR contains the sequence of interest and the restriction sites used. Transient Localization Assay. To ask whether the con- All sites are unique except Hpa I (position 1723). Unless stated served motif is required for localization, the Drosophila otherwise, synthetic bicoid transcripts ended at the HindIII site 23 nt as a prototypical localized 5' of the authentic polyadenylylation signal; the nonmutated form is bicoid mRNA (Fig. 1) was selected denoted wild type. The 45-nt sequence between the Hpa I and EcoRV transcript and a Drosophila transient assay was established. sites, which is deleted in bicoid/D45nt, is shown with the nonamer [a-[35S]thio]UTP-radiolabeled, T7 RNA polymerase tran- (positions 52-60 of the 3' UTR) underlined. Only the nonamer is scripts were generated and injected into syncytial Drosophila deleted in bicoid/D9nt. The 625 nt between the Mlu I and Stu I sites embryos. Following incubation to allow diffusion, their em- harbors a previously described localization signal (17). This abuts the bryonic position was assessed by autoradiography. region described here since the EcoRV and Mlu I sites are S nt apart. Downloaded by guest on October 9, 2021 7166 Cell Biology: Gottlieb Proc. NatL. Acad. Sci. USA 89 (1992) A ila transcripts (scute, tramtrack, hunchback; Fig. 2 B-D and F) fail to localize. The latter two control species correspond to maternal messages that enter the egg in vivo at the same time and by the same route as bicoid mRNA but are not anteriorly localized. Further, they are approximately the same size as the ". synthetic bicoid transcript, indicating that bicoid's preferential anterior retention in this assay is not the result ofits size. The B transient assay mirrors the in vivo situation in an additional respect. A deletion encompassing 625 nt ofthe bicoid 3' UTR, which disrupts bicoid RNA localization in vivo (17), can disrupt exogenous bicoid RNA localization in the transient assay (Fig. 2 E and F). These data indicate that discrimination between different RNAs occurs and that exogenous transcripts can be localized in a sequence-dependent manner. C Therefore, it is reasonable to use this assay to assess the effects of RNA sequences on the localization process. Deletion of the 9-nt Motif Results In Altered bicoid RNA ;p a'... i, Localizatio. To determine whether the region containing the conserved 9-nt motif plays a role in bicoid RNA localization, a 45-nt-deletion mutant was created (see Fig. 1). This portion of the bicoid 3' UTR encompasses the 9-nt motif and sur- D rounding nucleotides that show extended similarities when the 3' UTRs are compared pairwise. It can be drawn as a computer-generated stem-oop structure (data not shown). Anal- .1 "' A4: i. iff +. A'::..,..M-!"., At".,. ysis of the corresponding mutant bicoid transcripts in the 4" .. . transient assay revealed that deletion ofthe region does indeed have a qualitative effect on localization. While the wild-type transcript is tightly capped at the anterior pole (Fig. 3A), E synthetic mutant mRNA is partially mislocalized in a comparable exposure: transcripts spread from the apex and are found -.N, I within the anterior 30%o or so of the embryo (Fig. "I 3B). - ..-*Ii .,- ., I .. To ask whether this diffuse mislocalization phenotype is specifically attributable to the nonanucleotide motif, a bicoid construct missing just the 9 nt 5'-UUGUUCCUG-3' was created. The resulting mutant bicoid transcripts also exhibit the delocalized appearance in the transient assay (Fig. 3C) _% %Partaily % RA Species Loc Misloc Unloc compared with the more spatially restricted localization seen Total with the wild-type transcript (Fig. 3A). Conceivably, this btcoZd (wild type) 57 6 24 51 delocalized distribution could correspond to a wild-type bkoid /StUI 62 14 19 48 bicoid RNA localization intermediate. The partially mislo- scute 0 0 95 37 calized phenotype can be scored at low level in the population tramtrack 0 0 95 42 of embryos injected with wild-type bicoid transcripts (7%; hunchback 4 0 88 48 Fig. 3D). This contrasts, however, with the much bicold /SacII 7 1.4 74 70 higher bicoid 0 incidence of this phenotype when either the 45 nt or the 9nt /HpaI-StuI 10 75 40 are deleted (49% and 50%6, respectively). FIG. 2. Whole mount autoradiographs of Drosophila embryos It is unlikely that these observations result solely from injected with radiolabeled T7 polymerase-generated transcripts. increased transcript mobility due to decreased transcript Constructs are wild-type bicoid (A) or synthetic mRNAs from the size, since each mutant is missing <2% of the wild-type Drosophila genes scute (B), tramtrack (C), hunchback (D), or bicoid sequence. However, to test this possibility, I assessed the without the 3' UTR (E). Embryos were exposed simultaneously and localization ability of a construct containing a 47-nt deletion are arranged with anterior (in vivo bicoid mRNA localization site) to elsewhere in the bicoid 3' UTR. Most embryos injected with the left. RNA is seen as dark grains. Some embryos have grains the corresponding mutant RNA show proper wild-type lo- encircling their periphery; this may result from emulsion stress, has calization (Fig. 4B; see 2F, construct been noted before (20), and can be seen around uninjected autora- Fig. bicoid/Stu I). diographed embryos. Since the assay is basically cortical, one Embryos displaying the partially mislocalized phenotype do focuses on several planes while scoring. Photographs are at the plane occur in the population =2-fold over background. This is of maximum grain density; at the focal plane used to photograph a insufficient to explain the partial mislocalization observed nonlocalized embryo, localized radiolabeled RNA could be easily with the 45-nt and 9-nt deletions, each of which occurs at a detected. The frequency of injected embryos exhibiting localization frequency z7-fold over background (49o and 50%, respec- with each species is shown (F) where localized (Loc) is the pheno- tively; Fig. 3D). I therefore conclude that the altered bicoid type in A, unlocalized (Unloc) is in B-E, and partially mislocalized localization observed in Fig. 3 B and C is specifically attrib- (Partially Misloc) is in Fig. 3 B and C. A representative experiment utable to deletion of the 9-nt motif UUGUUCCUG and that where all embryos were autoradiographed together is shown. Total this is the number of embryos scored; percentages do not tally to 100, sequence is necessary for complete bicoid RNA local- because some embryos (but <15%) were not easily categorized. ization under the assay conditions employed. bicoid/one restriction site, run-off transcripts ending at that site; bicoid/two restriction sites, boundaries of an internal deletion with of all (bicoid/Sac II) or most (bicoid/Hpa I-Stu I) of the bicoid 3' transcripts ending at the HindIII site. Localization in the assay is not UTR interferes with localization. Both constructs are missing the 1000% efficient. Rather, anterior localization is seen in '600% of the 625nt designated to contain signals for anterior concentration in vivo embryos injected with wild-type bicoid RNA (57% in this experi- and the nonamer described here. Phenotypes in A-E (and in Figs. 3 ment). This contrasts with the frequency ofanterior localization with and 4) represent the major distinguishing localization pattern seen controls: scute (0%/6), tramtrack (4%), and hunchback (0%o). Deletion with each construct. Downloaded by guest on October 9, 2021 Cell Biology: Gottlieb Proc. Natl. Acad. Sci. USA 89 (1992) 7167

A A _*- e * - - (. _w.A* A*

B B

FIG. 4. Whole mount autoradiographs of Drosophila embryos injected with radiolabeled wild-type bicoid transcripts (A) or bicoid transcripts ending at the Stu I site and hence lacking the 47nt between C the Stu I and HindIII sites (B; bicoid/Stu I). The localization frequency of these constructs is comparable (see Fig. 2F). DISCUSSION The most important finding to emerge from this analysis is the identification ofa 9-nt motif, YUGUUYCUG, common to the 3' UTRs of localized maternal mRNAs in frogs and flies. Deletion ofthe sequence from one ofthese messages, bicoid, D % Partially causes partial mislocalization of bicoid mRNA in a transient RNA Species Mislocalized Total assay but is inconsequential to message stability. I conclude that the sequence participates in the bicoid mRNA localiza- bod (wild type) 7 70 tion process and infer that it may be instrumental in the bicoid /D 45 nt 49 77 bicoid /D9 nt 50 82 localization ofother mRNAs as well. It is unclear whether the 9-nt motif is a general feature of localized maternal mRNAs or FIG. 3. Whole mount autoradiographs of Drosophila embryos is limited to a subset that are localized via overlapping injected with radiolabeled wild-type bicoid (A), bicoid without the mechanisms. Nonamer occurrence in more than one orga- 45-nt Hpa I-EcoRV fragment containing the nonamer (B; bicoid/ nism already indicates that- different systems employ com- D45nt), or bicoid without the nonamer (C; bicoid/D9nt). Anterior is mon RNA localization machinery. to the left, RNA appears as dark grains, and comparable exposures are Existence of the motif may have predictive value in iden- shown. The percentage of embryos exhibiting the partially mislocal- tifying additional localized messages. A search of the EMBL ized phenotype portrayed in B and C for each construct is shown (D); data base (October 1991) and the literature revealed the motif total is the number of embryos scored in a representative experiment in the 3' UTRs of two other Xenopus transcripts of potential with all autoradiographed simultaneously. Since, on injection of wild-type bicoid RNA, only '60%o of embryos showed localization interest: D-7 and cyclin B2 (Table 1). While both are maternal (Fig. 2F), one cannot expect more efficient appearance ofthe partially mRNAs, no subcellular localization data are available. It is mislocalized phenotype when mutant RNA is injected. Thus, it is intriguing, however, that a subset of cyclin B mRNAs are significant that 49% and 50% of embryos injected with constructs devoid of the nonamer showed the partially mislocalized phenotype. Uninjected Retrieved This phenotype correlates with nonamer removal (B and C) and differs from the disruption of bicoid localization following deletion of the entire 3' UTR (Fig. 2E). In this experiment, only 1 of44 embryos (2%) injected with the bicoid/Sac II construct exhibited partial mislocal- -w _- ization. The low level of partial mislocalization seen with the bicoid wild-type construct is reproducible (6%, Fig. 2F; 7%, Fig. 3D). The 9-nt Sequence Does Not Appear to Affect Transcript Stability. Upon gastrulation, bicoid RNA becomes destabi- lized and disappears. Conceivably, removal of the motif could prematurely destabilize the mutant bicoid transcripts, increasing RNA mobility and resulting in apparent mislocalization. Indeed, this signal could play a role in the ultimate destruction of these mRNAs. To assess this possibility, [a-32P]UTP-radiolabeled mutant and wild-type synthetic bi- 1 2 3 4 1 2 3 4 coid transcripts were injected into early embryos and incu- FIG. 5. Stability of radiolabeled bicoid transcripts following bated as before. Embryos were then homogenized and the incubation in Drosophila embryos under localization conditions. retrieved transcripts were fractionated by gel electrophore- Shown are gel profiles ofuninjected (Left) and retrieved (Right) RNA sis. To gel resolution, intact RNA was recovered in all cases from a single exposure of a 4% polyacrylamide gel. The dark (Fig. 5 Right) compared with the uninjected material (Fig. 5 autoradiograph reveals the amount ofdegraded material. Each RNA Left). Significant amounts of degraded RNA were not ob- aliquot was from a pool of >120 injected embryos. Retrieved samples served in any case, even on prolonged are "smeary," probably due to the large amount of endogenous autoradiographic RNA. Lanes: 1, wild-type bicoid; 2, bicoid without the 3' UTR; 3, exposure. This suggests that alterations in transcript local- bicoid without the 45 nt including the nonamer; 4, bicoid without the ization during these experiments are not complicated by nonamer. Intact material was retrieved following injection of RNA transcript instability. Rather, they are consistent with se- missing the 3' UTR (Right, lane 2), demonstrating that the assay is quence-dependent effects on RNA localization. not complicated by transcript instability. Downloaded by guest on October 9, 2021 7168 Cell Biology: Gottlieb Proc. Natl. Acad. Sci. USA 89 (1992) localized to the Drosophila embryo's posterior pole (22, 23), have implicated staufen in the localization of maternal factors and these data suggest that a similar situation may exist in such as nanos to the posterior pole (13, 35, 36), and the Xenopus. staufen protein has been immunolocalized to both poles of The analysis described here tests the effect of an RNA the Drosophila embryo (37). sequence directly at the RNA level in an effort to better understand the molecular parameters dictating the localiza- I am deeply indebted to Hugh Pelham, Andrew Travers, and Aaron tion process. It does not address the question of whether Klug for support, encouragement, and stimulating discussions. This work profited from the superb technical assistance ofPaul Johnston. nonamer deletion from a localized message will ultimately Thanks also go to Mark Bretscher and Peter Lawrence for their contribute to pattern-formation defects in a complex orga- suggestions to refine the assay; Andy Newman, Steve Harrison, nism. Nonetheless, precedent for a localization profile such Maria Leptin, Mike Lewis, Sean Munro, and David Zarkower for as the one observed exists for two nonamer-containing their technical expertise; and Marcus N611, Louise Fairall, Kiyoshi RNAs. Mutation of the Drosophila staufen gene alters the Nagai, Carlos Cabrera, and Terry Smith for plasmids and oligonu- localization of endogenous bicoid RNA such that it spreads cleotides. This paper was improved by Andrew Travers, Hugh from the apex and resides within the anterior 30% or so ofthe Pelham, Andy Newman, Wes Sundquist, and Mark Bretscher. E.G. embryo (13). This indicates that it is possible to partially is a fellow of the Helen Hay Whitney Foundation. disrupt bicoid RNA localization in vivo. Similarly, a Vg1 1. Gottlieb, E. (1990) Curr. Opin. Cell Biol. 2, 1080-1086. RNA localization intermediate can be trapped by drug dis- 2. Nusslein-Volhard, C., Frohnh6fer, H.-G. & Lehmann, R. (1987) ruption of microfilaments (14). Under these conditions, Vgl Science 238, 1675-1681. RNA is confined to the egg's vegetal halfwhile being splayed 3. Frigerio, G., Burr, M., Bopp, D., Baungartner, S. & Noli, M. (1986) from its final localized position at the vegetal cortex. Cell 47, 735-746. 4. Berleth, T., Burri, M., Thoma, G. G., Bopp, D., Richstein, S., Although the 9-nt sequence is necessary for wild-type bicoid Frigerio, G., Noli, M. & Nfisslein-Volhard, C. (1988) EMBO J. 7, localization, it could not be sufficient: the motif resides in 1749-1756. RNAs that localize to different poles of the same cell and 5. Wang, C. & Lehmann, R. (1991) Cell 66, 637-647. consequently could not specify ultimate subcellular position. 6. Driever, W. & Nfisslein-Volhard, C. (1989) Nature (London) 337, 9 nt is 138-143. Indeed, a subset of the bicoid 3' UTR including these 7. Driever, W., Thoma, G. & Nusslein-Volhard, C. (1989) Nature unable to confer anterior retention to a non-bicoid message in (London) 340, 363-367. Drosophila embryos (17). Further, the motif identified here is 8. Struhl, G., Struhl, K. & Macdonald, P. M. (1989) Cell 57, 1259-1273. separable from a 625-nt region ofthe bicoid 3' UTR that is also 9. Driever, W. & Nuisslein-Volhard, C. (1988) Cell S4, 95-104. required for localization (17). This region was originally de- 10. Weeks, D. L. & Melton, D. A. (1987) Proc. NatI. Acad. Sci. USA scribed as necessary and sufficient for bicoid localization 84, 2798-2802. 11. Melton, D. A. (1987) Nature (London) 328, 80-82. following a study that compared the relative amount of trans- 12. Weeks, D. L. & Melton, D. A. (1987) Cell S1, 861-867. genic RNA in the anterior and posterior halves of bisected 13. Johnston, D., Driever, W., Berleth, T., Richstein, S. & Nfsslein- embryos. That study showed that the defined sequences are Volhard, C. (1989) Development (Cambridge, UK) 107, Suppl., necessary and sufficient to localize the bulk ofbicoid RNA to 13-19. the anterior halfofthe embryo but not necessarily sufficient to 14. Yisraeli, J. K., Sokol, S. & Melton, D. A. (1990) Development produce localization. With a construct devoid ofthe (Cambridge, UK) 108, 289-298. wild-type 15. Pondel, M. D. & King, M. L. (1988) Proc. Natl. Acad. Sci. USA 85, nonamer, I observe synthetic bicoid RNA spreading away 7612-7616. from the anterior tip of the embryo (Fig. 3 B and C). This is 16. Yisraeli, J. K. & Melton, D. A. (1988) Nature (London) 336, 592- confined to the anterior half and would have been scored as 595. "localized" in the previous analysis. As Fig. 2E shows, this 17. Macdonald, P. M. & Struhl, G. (1988) Nature (London) 336, 595- phenotype differs from the more drastic disruption of local- 598. are The 18. Black, D. L., Chabot, B. & Steitz, J. A. (1985) Cell 42, 737-750. ization that can be seen when both signals deleted. 19. Anderson, K. V. & Nfisslein-Volhard, C. (1984) Nature (London) bicoid 3' UTR including both these signals is sufficient for 311, 223-227. localization in the transient assay. 20. Baker, N. E. (1988) Development (Cambridge, UK) 103, 289-298. I suggest that RNA localization is dictated in a combinatorial 21. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nati. Acad. manner requiring multiple signals. Specificity signals would Sci. USA 74, 5463-5467. designate ultimate egg position whereas separate general sig- 22. Whitfield, W. G. F., GonzAlez, C., Sinchez-Herrero, E. & Glover, D. M. (1989) Nature (London) 338, 337-340. nals, which include the motif described here, could cause an 23. Lehner, C. F. & O'Farrell, P. H. (1990) Cell 61, 535-547. RNA to be packaged into an mRNP capable of full wild-type 24. Kim-Ha, J., Smith, J. L. & Macdonald, P. M. (1991) Cell 66, 23-35. localization or interact with the cellular localization machinery 25. Ephrussi, A., Dickinson, L. K. & Lehmann, R. (1991) Cell66, 37-50. (e.g., the cytoskeleton). These are designated "general" be- 26. Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London) 263, cause they can reside in RNAs destined for different cellular 211-214. locales. Their existence suggests overlap between the mech- 27. Shaw, G. & Kamen, R. (1986) Cell 46, 659-667. 28. McGrew, L. L., Dworkin-Rastl, E., Dworkin, M. B. & Richter, anisms that localize RNA to different poles in developing cells. J. D. (1989) Genes Dev. 3, 803-815. Since bicoid and Vgl RNA localization arises via complex, 29. Fox, C. A., Sheets, M. D. & Wickens, M. P. (1989) Genes Dev. 3, multistep processes (13, 14), it is not surprising that multiple 2151-2161. signals may be involved. 30. Seeger, MA. & Kaufmann, T. C. (1990) EMBO J. 9, 2977-2987. How could the nonamer function in the localization of 31. Macdonald, P. M. (1990) Development (Cambridge, UK) 110, 161- RNAs to different cellular positions? Presumably, it is the 171. 32. Frohnh6fer, H.-G. & Nusslein-Volhard, C. (1986) Nature (London) target (or core of a target) for a protein or small ribonucle- 324, 120-125. oprotein particle located at both egg poles. Mutations in three 33. Frohnh6fer, H.-G. & Nflsslein-Volhard, C. (1987) Genes Dev. 1, genes disrupt bicoid RNA localization, pinpointing potential 880-890. localization factors (4, 13, 33, 34). Of these, the best candi- 34. Stephenson, E. C., Chao, Y. & Fackenthal, J. D. (1988) Genes Dev. date to interact directly or indirectly with the consensus 2, 1655-1665. 35. Schfpbach, T. & Wieschaus, E. (1986) Roux's Arch. Dev. Biol. 195, sequence is the staufen gene product. Endogenous bicoid 302-317. RNA is mislocalized within the anterior 30% of staufen 36. Lehmann, R. 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