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 (received for review December 2, 1991)

ABSTRACT Messenger RNA (mRNA) localization is emerg as a means of r ig gene expression. This process is operational in fly and fog development, where a subset of maternally inherited RNAs are asymmetrically distributed and thought to impart axial polarity to the embryo. Since most maternal mRNAs are unioly distributed, an a l must exist to recognize and specifically transport these rare loalized species. Here I report the Identificaton of a nine-nudeotde motif, YUGUUYCUG, common to the 3' unta regions of four sequenced of this class: Drosophia bicold and nanos mRNAs and Xenopus An2 and Vgl mRNAs. To test the role of this nner sequence in the l tio process, a Drosophila transient assay has been established. The assay reveals that bicoid mRNA fically lacking this noner is partal~ly mislocalized. In contrast, namer deletion is inconsequential to message stabilt. The existence of specific and genera IIRNA loction igal is proposed and itis s t that this conserved motif belongs to the latter category.

transcripts residing at distinct oocyte locations while most messages are uniformly distributed. Both bicoid and Vgl mRNAs obtain their positions posttranscriptionally via multistep, dynamic processes (11, 13, 14). Cytoskeletal components are implicated in localization: for Vgl, microtubules participate in translocation while microfilaments and intermediate filaments may facilitate cortical anchoring (14, 15). Localized mRNAs probably exist as mRNA-protein complexes (mRNPs). Some mRNP components could be required for localization by interacting with the localization machinery or with a receptor at the localization site. Implicit in this model are the existence of cis-acting localization signals and trans-acting localization factors that could account for localization specificity and selectivity. Signals for localization reside in the RNA: synthetic Vgl transcripts can be localized following injection into Xenopus oocytes (16), and a transgenic fly analysis revealed that bicoid RNA localization requires signals harbored within 625 nucleotides (nt) of the bicoid 3' untranslated region (UTR) (17). In an in-depth search for a defined localization signal, I compared the available sequences of localized maternal RNAs. Here I report the finding of a 9-nt motif common to the 3' UTR of several of these mRNA species. With a transient localization assay, it is demonstrated that the sequence does indeed participate in the localization of synthetic bicoid RNA but is inconsequential to message stability.

Spatial organization of cells and subcellular regions arises in part from the sorting and subsequent localization of proteins and RNA. Although the study of RNA trafficking is in its infancy, this process has already been documented in several organisms (reviewed in ref. 1). Present indications are that RNA localization is likely to take its place alongside splicing, polyadenylylation, stabilization, and translation as a major 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. Striking examples of localized messages can be found among the maternal mRNAs of flies and frogs, where localization has been implicated in the establishment of axial polarity. In Drosophila, the primary determinants of the embryonic anterior-posterior axis are encoded by the bicoid and nanos mRNAs (reviewed in ref. 2). These messages are localized to the anterior and posterior poles of the Drosophila egg, respectively (3-5). bicoid mRNA translation produces a transcription factor that specifies head and thoracic structures (6-9) whereas nanos mRNA translation is required to generate abdominal structures (5). Mutations at several loci disrupt localization of these messages, resulting in aberrant embryos. This illustrates the importance of the localization process and argues that an apparatus exists to establish and/or maintain RNA position. In Xenopus, searches for determinants of polarity uncovered a few localized maternal mRNAs in the oocyte and early egg. Of these, An2 RNA is localized to the animal pole and its product may contribute to a respiration gradient along the animal-vegetal axis (10). Vgl RNA is localized to the vegetal pole and its product has been implicated in mesoderm induction (11, 12). Our understanding ofthe molecular mechanisms governing localization of the aforementioned mRNAs is minimal. RNA sorting operates in Drosophila and Xenopus eggs, with rare

MATERIALS AND METHODS Computer Analysis. Sequence comparisons were performed with DIAGON and ANALYSEQ (R. Staden). Preparation of RNA Substrates. m7GpppG-capped injection substrates were synthesized using T7 RNA polymerase (18), with reaction mixtures containing 0.12 mM UTP to produce full-length transcripts and [a-[35S]thio]UTP (20 ,uCi; 400 Ci/mmol, Amersham; 1 Ci = 37 GBq) to generate localization substrates. Transcripts were poly(A)-tailed by poly(A) polymerase (Escherichia coli, Pharmacia) in 50 mM TrisHCl, pH 7.4/10 mM MgCl2/2.5 mM MnCl2/250 mM NaCl/12.5 mM ATP at 370C (25 min). Resulting tails were sized at 35-155 nt, with the major species around 60 nt. The Assay. Injections and autoradiography. Canton-S fly eggs were collected (15 min), prepared (19), and injected with RNA in 5 mM KCI/0.1 mM sodium phosphate, pH 6.8. Recipients were incubated at 200C for 95 min to allow transcript diffusion. This is roughly the longest time possible before endogenous bicoid RNA is degraded (13). Of 106 sample embryos, 83% developed past the experiment's end, and of those 47% hatched. Embryos were fixed in 2% glutaraldehyde, devitellinized, washed with phosphatebuffered saline/1% bovine serum albumin/0.1% Triton X-100, mounted on polylysine subbed slides, dipped in NTB2 emulsion (Kodak), and stored at 40C. Autoradiographs were developed according to ref. 20.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: mRNP, mRNA-protein complex; nt, nucleotide(s); UTR, untranslated region.

7164

Cell

Proc. Natl. Acad. Sci. USA 89 (1992)

Biology: Gottlieb

Transcripts were injected into the anterior of the embryo, the direction from which bicoid mRNA normally enters the egg in vivo. Lateral injection resulted in preferential anterior concentration of the bicoid transcripts. However, the experimental time window is insufficient to chase most of the RNA to this pole. Observation of sequence-dependent localization in this assay requires the presence of carrier RNA. Embryos were injected with 0.1 mg of RNA per ml, 5% of which was radiolabeled bicoid mRNA and 95% of which was yeast tRNA carrier. This is "'3 x 104 molecules of exogenous bicoid mRNA per embryo. Constructs. Constructs were derived from the bicoid cDNA (4) cloned into pGEM-1 (Promega). For the bicoid construct missing 45nt between the Hpa I and EcoRV sites, the 5-kilobase Bgl II-EcoRV and 229-base Bgl II-Hpa I fragments from the bicoid parent clone were ligated. For the bicoid clone devoid of the nonamer, the 229-base Bgl II-Hpa I and 5-kilobase Bgl II-Mlu I fragments from the parent clone and a double-stranded DNA fragment (5'-AACCACTGATTGTACAAATACCAAGTGATTGTAGATATCTA-3') were ligated. Constructs were screened by restriction analysis and confirmed by sequencing (21). Controls included hunchback, generated from a 2.4-kilobase Xba I fragment inserted into Bluescribe plasmid (Stratagene); tramtrack lacking the first 330 nt and cloned into pET 11a; and scute inserted into Bluescribe. Before transcription, plasmids were linearized with Sac II (bicoid/Sac II), Stu I (bicoid/Stu I), HindIII (other bicoid derivatives, hunchback, and scute), or EcoRI (tramtrack). Transcript stability. 32P-radiolabeled transcripts were injected as above. Following incubation, embryos were homogenized in extraction buffer (50 mM Tris-HCl, pH 7.5/50 mM NaCl/10 mM EDTA/0.5% SDS), treated with proteinase K, and extracted with phenol. Scintillation counting revealed that 79-94% of the counts were retrieved in each case. Samples were fractionated by polyacrylamide gel electrophoresis under denaturing conditions. The gel was backed with DEAE paper to minimize the loss of small RNA fragments, dried, and exposed to Fuji film at -70°C.

RESULTS Identification of a 9-nt Motif in the 3' UTR of Localized Maternal RNAs. In an attempt to identify cis-acting localization signals, the available sequenced localized maternal mRNAs were examined, revealing a 9-nt motif, YUGUUYCUG, in the 3' UTR of four localized species: Drosophila bicoid and nanos mRNAs and Xenopus An2 and Vgl mRNAs (Table 1). The calculated frequency of this motif is -1 in 66,000 bases. It is the longest contiguous stretch of sequence shared among these message trailers, the next longest common motif being a heptamer. The nonamer is absent from nonlocalized maternal RNAs and is distinct from other motifs in the 3' UTR that direct RNA-level events such as polyadenylylation (26), mRNA destabilization (27), and poly(A)-tail elongation during Xenopus oocyte maturation (28, 29). Although the data set of localized RNAs is presently small, functional importance of the nonamer is underscored by its conservation in the bicoid 3' UTR from all eight Drosophila species examined, diverging up to 60 million years (see refs. 30 and 31 for sequences). These data suggest that the motif YUGUUYCUG is a reasonable candidate RNA localization signal. Transient Localization Assay. To ask whether the conserved motif is required for localization, the Drosophila bicoid mRNA (Fig. 1) was selected as a prototypical localized transcript and a Drosophila transient assay was established. [a-[35S]thio]UTP-radiolabeled, T7 RNA polymerase transcripts were generated and injected into syncytial Drosophila embryos. Following incubation to allow diffusion, their em-

bryonic position was assessed by autoradiography.

7165

Table 1. A conserved 9-nt motif, YUGUUYCUG, is found in the 3' UTR of four sequenced maternal messages known to be localized posttranscriptionally Sequence 3' UTR position* (5' -- 3') Maternal RNA Localized messages 52/817 UUGUUCCUG Drosophila bicoid 244/872 CUGUUUCUG Drosophila nanos 43/146 UUGUUCCUG Xenopus An2 77/1268 UUGUUCCUG Xenopus Vgl Potential candidates 69/170 CUGUUCCUG Xenopus cyclin b2 35/188 CUGUUCCUG Xenopus D-7 YUGUUYCUG Consensus Included are species localized to each of the two egg poles in both frogs and flies; these encode disparate functions. A subset of cyclin B transcripts are localized to the posterior pole of the Drosophila 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 one of two Xenopus cyclin B homologues. This and D-7 are listed as potential candidates because they are maternal messages for which subcellular localization data are not available but which harbor the 9-nt motif; this may indicate that they are localized. The localized Drosophila oskar mRNA has a partial match (UUGUCUUG; i.e., YUG-UYYUG). A related oskar sequence may provide nonamer function or the motif may govern a localization step not operational for oskar (see Discussion). Indeed, the localization ofoskar and other 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 oskar RNA localization is disrupted by mutations in the oskar coding region that affect oskar protein synthesis but not oskar RNA synthesis (24, 25). *First residue in the motif relative to the stop codon/3' UTR length in bases.

A fly with four bicoid genes can be constructed and the resulting RNA localized (9, 32), implying that wild-type embryos possess excess message localization capacity. Hence, they were used as injection hosts. Transcripts were 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 embryos. This is the direction from which bicoid mRNA normally enters the egg from the nurse cells in vivo. Due to the injection site, this is in essence a retention assay and constructs are scored with respect to whether they are retained at the anterior or how far they diffuse from it. Under the conditions employed, full-length bicoid transcripts are tightly localized to the embryo's anterior tip -60% of the time (Fig. 2 A and F) whereas several control Drosoph3, UTR

5,

5,

UTR

CODING REGION

UIR~~~~~~~~~~~~A A.. ....

a

Hrd-aI

'

n

3,

E 19 . ,;5r = EC RV

AACCACTrGUGrTCCTGATTGTACAAATACCAAGTGATrGTAGAT FIG. 1. Diagram of the 2.4-kilobase bicoid cDNA. The 817-nt 3' UTR contains the sequence of interest and the restriction sites used. All sites are unique except Hpa I (position 1723). Unless stated otherwise, synthetic bicoid transcripts ended at the HindIII site 23 nt 5' of the authentic polyadenylylation signal; the nonmutated form is denoted wild type. The 45-nt sequence between the Hpa I and EcoRV sites, which is deleted in bicoid/D45nt, is shown with the nonamer is (positions 52-60 of the 3' UTR) underlined. Only the nonamer deleted in bicoid/D9nt. The 625 nt between the Mlu I and Stu I sites harbors a previously described localization signal (17). This abuts the region described here since the EcoRV and Mlu I sites are S nt apart.

7166

Cell Biology: Gottlieb

Proc. NatL. Acad. Sci. USA 89 (1992)

ila transcripts (scute, tramtrack, hunchback; Fig. 2 B-D and F) fail to localize. The latter two control species correspond to

A

".

B

C ;p

a'...

i,

D A4:

iff i.

-!"., At".,. +. A'::..,..M .1

4"

..

"'

.

E -.N,

"I .,-

I

..

I

..-*Ii .,

%Partaily % _% Loc Misloc Unloc Total

RA Species

btcoZd (wild type) 57 bkoid /StUI scute tramtrack hunchback

62 0 0 4 bicold /SacII 7 bicoid /HpaI-StuI 10

6 14 0 0 0 1.4 0

24 19

95 95 88 74 75

51 48 37 42 48 70 40

FIG. 2. Whole mount autoradiographs of Drosophila embryos injected with radiolabeled T7 polymerase-generated transcripts. Constructs are wild-type bicoid (A) or synthetic mRNAs from the Drosophila genes scute (B), tramtrack (C), hunchback (D), or bicoid without the 3' UTR (E). Embryos were exposed simultaneously and are arranged with anterior (in vivo bicoid mRNA localization site) to the left. RNA is seen as dark grains. Some embryos have grains encircling their periphery; this may result from emulsion stress, has been noted before (20), and can be seen around uninjected autoradiographed embryos. Since the assay is basically cortical, one focuses on several planes while scoring. Photographs are at the plane

of maximum grain density; at the focal plane used to photograph a nonlocalized embryo, localized radiolabeled RNA could be easily detected. The frequency of injected embryos exhibiting localization with each species is shown (F) where localized (Loc) is the phenotype in A, unlocalized (Unloc) is in B-E, and partially mislocalized (Partially Misloc) is in Fig. 3 B and C. A representative experiment where all embryos were autoradiographed together is shown. Total is the number of embryos scored; percentages do not tally to 100, because some embryos (but