hts mRNA expression and localization - Journal of Cell Science - The ...

3385

Journal of Cell Science 112, 3385-3398 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0591

Different 3′ untranslated regions target alternatively processed hu-li tai shao (hts) transcripts to distinct cytoplasmic locations during Drosophila oogenesis Kellie L. Whittaker1,3, Dali Ding3,‡, William W. Fisher1,3,§ and Howard D. Lipshitz1,2,3,* 1Program in Developmental Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 2Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada 3Division of Biology, California Institute of Technology, Pasadena, California, USA

*Author for correspondence at address 1 (e-mail: [email protected]) ‡Present address: Wilson, Sonsini, Goodrich and Rosati, 650 Page Mill Road, Palo Alto, CA 94304, USA §Present address: Exelixis Pharmaceuticals, Inc., 260 Littlefield Avenue, South San Francisco, CA 94401, USA

Accepted 15 July; published on WWW 22 September 1999

SUMMARY Cytoplasmic mRNA localization is one method by which protein production is restricted to a particular intracellular site. We report here a novel mechanism for localization of transcripts encoding distinct protein isoforms to different destinations. Alternative processing of transcripts produced in the Drosophila ovary by the hu-li tai shao (hts) locus introduces distinct 3′ untranslated regions (3′UTRs) that differentially localize the mRNAs. Three classes of hts mRNA (R2, N32 and N4) are synthesized in the germ line nurse cells and encode proteins with adducin-homologous amino-terminal regions but divergent carboxy-terminal domains. The R2 and N32 classes of mRNA remain in the nurse cells and are not transported into the oocyte. In contrast, the N4 class of transcripts is transported from the nurse cells into the oocyte starting at stage 1, is subsequently localized to the oocyte cortex at stage 8 and then to the anterior pole from stage 9 on. All aspects of N4 transcript transport and localization are directed by the

INTRODUCTION The targeting of specific mRNAs to distinct subcellular compartments is an important mechanism for generating asymmetry in many cell types, including fibroblasts, neurons and oocytes (reviewed in Bashirullah et al., 1998). Localization of RNA can be accomplished either by translocation of a specific RNA to a particular subcellular location, or by degrading a ubiquitous RNA everywhere except a particular region. Both of these cytoplasmic localization mechanisms require sequences within the RNA (cis-acting signals) as well as a trans-acting localization machinery. This machinery includes factors that are able to recognize the cis-acting elements, direct the RNA to its appropriate destination and tether it there, or protect the RNA from degradation in a specific cytoplasmic region while directing degradation

345-nucleotide(nt)-long 3′ untranslated region (3′UTR). The organization of localization elements in the N4 3′UTR is modular: a 150 nt core is sufficient to direct transport and localization throughout oogenesis. Additional 3′UTR elements function additively together with this core region at later stages of oogenesis to maintain or enhance anterior transcript anchoring. The swallow locus is required to maintain hts transcripts at the anterior pole of the oocyte and functions through the N4 3′UTR. In addition to the three classes of germ line-expressed hts transcripts, a fourth class (R1) is expressed in the somatic follicle cells that surround the germ line cells. This transcript class encodes the Drosophila orthologue of mammalian adducin.

Key words: Drosophila, Adducin, hu-li tai shao, Oocyte, swallow, Cytoskeleton, RNA localization, 3′ untranslated region (3′UTR), RNA splicing, RNA transport

elsewhere (Bashirullah et al., 1999). Cis-acting localization signals have generally been found to reside within the 3′ untranslated region (UTR) of mRNAs. In Drosophila, the oocyte itself can be thought of as a specific cytoplasmic compartment because it develops as part of a syncytium (King, 1970; Spradling, 1993). Each egg chamber contains a single oocyte plus 15 nurse cells, with the nurse celloocyte complex surrounded by a monolayer of somatically derived follicle cells. The oocyte is connected to the nurse cells by cytoplasmic bridges known as ring canals, through which RNAs and other synthetic products of the nurse cells are transported into the oocyte. During early to mid-oogenesis, only a subset of maternal mRNAs is strongly enriched within the oocyte relative to the nurse cells (reviewed in Bashirullah et al., 1998). Thus cis-acting elements may serve at least two functions in localizing mRNAs during Drosophila oogenesis:

3386 K. L. Whittaker and others first, to target a mRNA specifically to the developing oocyte within the nurse cell-oocyte complex and, second, to direct that mRNA to a specific destination within the oocyte itself. Much of the work on localized RNAs in Drosophila has focused on transport and localization of transcripts encoding pattern determinants in the oocyte and early embryo, but not all localized maternal mRNAs encode such pattern specifiers. Since one aspect of cell and oocyte asymmetry is the organization of the cytoskeleton, it might be expected that some localized RNAs would encode cytoskeletal proteins. Many of the localized RNAs identified in somatic cells encode cytoskeletal proteins and/or cytoskeleton-associated proteins such as actin, myosin heavy chain and MAP-2 (Cheng and Bjerknes, 1989; Garner et al., 1988; Lawrence and Singer, 1986; Pomeroy et al., 1991). hu-li tai shao (hts) is the only example to date of a transcript that is localized throughout Drosophila oogenesis and encodes a cytoskeleton-associated protein (Ding et al., 1993; Yue and Spradling, 1992). This locus has previously also been referred to as Adducin-like (Ding et al., 1993; Whittaker, 1997; Whittaker and Lipshitz, 1995; Zaccai and Lipshitz, 1996a,b), but will be referred to here exclusively as hts to avoid confusion. Mammalian adducin is a membrane-cytoskeletal protein that contributes to local cytoskeletal assembly, particularly at sites of cell-cell contact and communication (Kaiser et al., 1989; Seidel et al., 1995). Adducin binds spectrin-actin complexes, recruits spectrin to spectrin-actin-adducin complexes, bundles actin filaments, and is capable of regulating actin filament length by capping the barbed end of actin (Bennett et al., 1988; Gardner and Bennett, 1987; Kuhlman et al., 1996; Mische et al., 1987). Three mammalian adducin genes encode α, β and γ adducin subunits (Joshi et al., 1991; Katagiri et al., 1996), and the subunits associate as αβ heterodimers or heterotetramers (Hughes and Bennett, 1995; Joshi et al., 1991) or αγ heterodimers (Dong et al., 1995). The Drosophila hts gene has been reported to encode an 1156-amino-acid protein with substantial homology to the N-terminal 590 or so amino acids of mammalian adducin (Ding et al., 1993; Yue and Spradling, 1992). We show here that the hts locus generates a family of transcripts through alternative mRNA splicing and polyadenylation. The hts mRNA splice variants share aminoterminal open reading frames (ORF) but have variant carboxyterminal ORF sequences and 3′UTRs. One of these transcript classes, the R1 class, is expressed in the follicle cells and encodes an adducin-related protein isoform very similar in size and overall structure to the mammalian adducins, and thus represents the Drosophila orthologue of mammalian adducin. Three hts transcript classes are expressed in the germ line nurse cells during oogenesis but only one of these, the N4 class, is localized specifically to, and within, the developing Drosophila oocyte. Cis-acting signals necessary and sufficient for N4 oocyte localization are contained within its 3′UTR. We have mapped these signals and have identified a 150 nucleotide (nt) core region capable of directing transport of RNA into the oocyte as well as subsequent cortical and anterior transcript localization. Additional 3′UTR elements act in concert with this element to enhance or maintain anterior anchoring of transcripts. Alternative processing of 3′UTRs represents a novel mechanism for differential intracellular targeting of transcripts encoding distinct protein isoforms.

MATERIALS AND METHODS cDNA isolation and DNA sequence analysis An ovarian cDNA library (the RAT1 library; Ding and Lipshitz, 1993) and a 0- to 4-hour embryonic cDNA library (pNB library; Brown and Kafatos, 1988) were screened with a 2.2 kb EcoRI-PstI genomic fragment as previously described (Ding et al., 1993). Eight cDNA clones were isolated, which were grouped into four classes. These cDNA classes have been designated as N4, R2, N32 and R1, with the letters N or R referring to the cDNA library (Nick Brown or λRAT1) from which the clones were recovered. Three clones each were recovered for the N4 class (N4, R8, N1) and the R2 class (R2, R10, R16) (Whittaker, 1997). The N32 and R1 classes are each represented by a single clone (Whittaker, 1997). GenBank accession numbers for the cDNAs are: AF151705 (R1), AF151706 (R2), AF151707 (N32) and L07617 (N4). Sequence analysis of the hts cDNA clones was done using the GELASSEMBLE, FASTA and BLAST programs of the Genetics Computer Group package (GCG). RNA folding was modeled using the GCG MFOLD program. Genomic sequence was obtained from GenBank #AC004299 (P1 clone DS07982, submitted by the Berkeley Drosophila Genome Project). In situ hybridization to whole-mount ovaries Standard methods (Ding et al., 1993; Tautz and Pfeifle, 1989) were followed for whole-mount in situ hybridization to ovaries and embryos (for details see Whittaker, 1997). Probes were generated as follows: Digoxigenin-labeled antisense-β-galactosidase RNA probe (corresponding to the entire 3.4 kb lacZ sequence) and antisense N4 RNA probe (representing the full-length cDNA, transcribed from SalI-digested pNB40-N4 cDNA) were transcribed by T7 RNA polymerase using Ambion’s Megascript kit, resuspended in H2O, and hydrolyzed by treatment with 100 µl of 100 mM NaCO3 buffer, pH 10.2 for 1 hour at 60°C. Antisense R1 3′UTR digoxigenin-labeled RNA probe was transcribed using SP6 polymerase from BlpI-digested R1 cDNA. Transcription reactions were performed as for N4 except that SP6-specific transcription buffer (BRL) and SP6 polymerase (Gibco/BRL) were used. Digoxigenin-labeled DNA probes for each of the hts splice form 3′UTRs were made by random priming of gelpurified PCR products. For the N4 3′UTR, PCR primers were 5A1(5′TAC CCT AGG TAG ATA CAT ATT ATG-3′) and 3N1 (5′-ACG GCG GCC GCT AAT GAA AAC TAT ATT T-3′). The R1 3′UTR (plus part of the coding region unique to the R1 cDNA) was amplified using primers 972-84 (5′-AAT GGC GAT CAT TCG GAG GC-3′) and SP6-UBDT (5′-GCT GCG GCC GCA GAT TTA GGT G-3′). For the R2 3′UTR, PCR primers were Rat-Add 1 (5′-CCA CCA ACC TTA AGG AGA TTG-3′) and Rat-Add 2 (5′-GAT TTA TGC ACA TTA CAC3′). The N32 3′UTR was amplified with primers AddN32A (5′-GTGCCT CAT TTA GGG ATT TGC GCT G-3′) and T7 (5′-TAA TAC GAC TCA CTA TAG GG-3′). Additionally, the HindIII-NotI fragment of pNB40-N4 was gel-purified and labeled with digoxigenin by random priming, producing a full-length N4 DNA probe. Additional details can be found in Whittaker (1997). Construction of lacZ-Adducin-like transgenes for germline transformation For all constructs, the parent vector was pCaSpeR4β26. This vector contains the Hsp26-Sgs-3 promoter and 5′UTR, a cassette that promotes nurse cell transcription, and the entire 3.4 kb lacZ sequence. To construct pCaSpeR4β26, the hsp26-Sgs-3 cassette was excised from pGerm6 (a vector related to the published pGerm vectors, Serano et al., 1994) with EcoRI and NotI, end-filled and cloned into the StuI site of pCaSpeR4. The lacZ sequence was excised from pCaSpeRβgal (X+R+) (Thummel et al., 1988) using XbaI and cloned into the XbaI site of pCaSpeR4-26, to complete pCaSpeR4β26. In Drosophila, full-length lacZ ORF is often truncated during transcription (Kim-Ha et al., 1993); consequently a shortened lacZ tag was included in our reporter constructs. To shorten the lacZ tag, pCaSpeR4β26 was

hts mRNA expression and localization 3387 digested with HpaI and NotI. A 600-bp fragment of lacZ was amplified by PCR, such that the 3′ primer contained a NotI site. This fragment was digested with EcoRV and AvrII, and the resulting 500 bp fragment was then gel-purified. The hts N4 3′UTR was PCR amplified from pNB40-N4 using primers with AvrII and NotI sites, and digested with AvrII and NotI. A triple ligation was performed, which joined the pCaSpeR4β26 vector to the truncated lacZ sequence and the hts 3′UTR sequence, producing pAdd3′UTR. PCR primers used to amplify the full-length hts N4 3′UTR were 5A1 (see above), which includes an engineered AvrII restriction site, and 3N1 (see above), which includes an engineered NotI restriction site. For 5′ deletion constructs (p5∆-1 to p5∆-6), pAdd3′UTR was digested with AvrII and NotI to remove the full-length hts 3′UTR sequence. Using primers with engineered AvrII and NotI sites, the desired subset of the 3′UTR was amplified and then subcloned into the vector. Primers for the p5∆-1 insert were 5A2AB (5′-ATA CCT AGG GGA TCC ATA TTT GAA ATC GAC-3′) and 3N1; for the p5∆-2 insert, 5A3 (5′-ACG TAC CCT AGG GTT TAA TGA TGA TAA TTT GG-3′) and 3N1; for the p5∆-3 insert, 5A4AB (5′-ATA CCT AGG GGA TCC ACT TCT TTG TTC TC-3′) and 3N1; for the p5∆-4 insert, 5A5 (5′-ACG TAC CCT AGG ATG GAA ATG CAA AG-3′) and 3N1; for the p5∆-5 insert, 5A6AB (5′-GCG CCT AGG GGA TCC ACA TCT GTG AAA TTG-3′) and 3N1; for the p5∆-6 insert, 5A7AB (5′GCG CCT AGG GGA TCC TCA TAT TTT TAT AAT TAA AAT AT3′) and 3N1. For 3′ deletion constructs (p3∆-1 to p3∆-6), pAdd3′UTR was digested with AvrII and NotI as above. Using primers with engineered AvrII and BamHI sites, the desired subset of the hts N4 3′UTR was amplified and then subcloned into the vector. Since the 3′ deletion constructs are missing the predicted hts N4 polyadenylation signal, the α-tubulin 3′UTR was appended. The α-tubulin 3′UTR was amplified from p701, a gift from Paul Macdonald. Triple ligations were performed, joining the pAdd3′UTR vector to the desired segment of the hts 3′UTR and the α-tubulin 3′UTR. PCR primers for the p3∆-1 insert were 5A1 and 3B1 (5′-CCG GGA TCC AAC GAA AAT CAA GAT TCG AC-3′); for the p3∆-2 insert, 5A1 and 3B2 (5′-CAT GGA TCC TTA AAT GTA CAA TGG TTG GC-3′); for the p3∆-3 insert, 5A1 and 3B3 (5′-ACG TGG ATC CAT TGT GCT TAA GTT ACA G3′); for the p3∆-4 insert, 5A1 and 3B4 (5′-ACG TGG ATC CAA AAA ATG CAT TAG TTC G-3′); for the p3D∆-5 insert, 5A1 and 3B5 (5′ACG TGG ATC CTT AAT CTA GTT GCT TTA TAC-3′); for the p3∆6 insert, 5A1 and 3B6 (5′-ACG TGG ATC CCA CAA CAA AAG CGA AG-3′). For internal deletion constructs (p∆50-1 to p∆50-5 and p∆100-1 to p∆100-3), triple ligations were performed, joining AvrIIBamHI digested PCR-amplified fragments of the hts N4 3′UTR to BamHI-NotI PCR-amplified pieces of the 3′UTR, and to the AvrII-Not I digested pAdd3′UTR vector. For construction of p100-250, primers 5A3B (5′-ACG TAC GGA TCC GTT TAA TGA TGA TAA TTT GG3′) and 3B2 were used to amplify a 150-bp fragment of the N4 3′UTR; the digested PCR product was ligated into AvrII-NotI digested p3∆-1. pAddtag3′UTR was constructed by digesting pAdd3′UTR with NheI and ligating it to a PCR product consisting of the complete hts N4 5′UTR and open reading frame (ORF), with NheI-compatible AvrII ends engineered into the primers Add-Avr-5 and Add-Avr-3. pORFtagtub was constructed by digesting pAddtag3′UTR with AvrII, end-filling with the Klenow fragment of DNA polymerase, followed by digestion with NotI. p3∆-1 was digested with BamHI, end-filled, then cut with NotI and the fragment containing the tubulin 3′UTR was purified. The NotI digested pAddtag vector was then ligated to the NotI tubulin 3′UTR fragment. Germline transformation Germline transformation was carried out using standard procedures (Rubin and Spradling, 1982; Spradling and Rubin, 1982). w1118 embryos were co-injected with 500 µg/ml of pAdd3′UTR or the desired deletion construct and 100 µg/ml of pHSπ helper plasmid (Steller and Pirrotta, 1985). All transgenic lines were homozygosed or balanced over CyO or TM3.

Northern blot analysis Ovarian RNA was prepared and poly(A) RNA was selected as previously described (Ding et al., 1993) and electrophoresed in a lowpercentage formaldehyde gel (Tsang et al., 1993). Standard procedures were followed for transfer to solid support, hybridization and washing (Ausubel et al., 1994). PCR amplification of the 3′UTR from each of the five splice forms of hts mRNA was followed by purification and labeling with digoxigenin-dUTP using Boehringer Mannheim’s High Prime kit. Chemiluminescent detection using CSPD as a substrate was according to the protocol of Boehringer Mannheim. RT-PCR analysis To verify that full-length reporter RNA was present but delocalized (as opposed to prematurely truncated and therefore undetectable, or unstable due to misfolding/deletion of an element conferring stability), total RNA was prepared from whole female flies (pGerm6 confers germ line-specific expression; Serano et al., 1994) homozygous for selected transgenes (pAdd3′UTR, 5′∆2, 5′∆4, 5′∆6) and used as a substrate for RT-PCR. Total RNA was prepared from 25 female flies of each transgenic line, using an SDS lysis buffer (Andres and Thummel, 1994). RNase-free DNase I (BRL, amplification grade) was used to remove contaminating genomic DNA. First-strand cDNA synthesis was primed from 3 µg of total RNA with oligo-dT12-18, using the Superscript II Preamplification kit (Gibco/BRL). RNase H was then used to remove RNA from the RNA/DNA hybrids followed by PCR amplification. For amplification of the transgene from the beginning of the lacZ tag sequence through the hts 3′UTR, primers TG1 and 3N1 were used, producing an 874 bp product for the full-length 3′UTR transgene and correspondingly shorter products for deletion transgenes. The TG1 sequence was 5′ATC CTG CTG ATG AAG CAG AAC AAC-3′. For RNA prepared from homozygous 5′∆2, 5′∆4 and 5′∆6 female flies, single PCR products of 774, 674 and 574 bp, respectively, were predicted. In each case, a single band of the expected size was detected (data not shown) indicating that the lack of in situ signal in 5′∆4 and 5′∆6 was due to the reporter RNA being present but unlocalized. We cannot exclude the possibility that there are stage-specific stability effects that are not revealed by this type of analysis since RNA from all stages was combined. Fly strains For transgenics, w1118 stocks were used as host (Lindsley and Zimm, 1992). In order to ascertain whether reporter transcripts bearing the hts N4 3′UTR are localized properly in a swallow (sww) mutant background, ovaries were collected from mothers that were sww1/sww1; P[Add3′UTR]/P[Add3′UTR] or sww1/sww6; P[Add3′UTR]/P[Add3′UTR]. sww alleles have previously been described; sww1 (also known as fs(1)A1497) is a strong allele while sww6 (fs(1)A384) is weaker (Hegdé and Stephenson, 1993). Flies were cultured at 25°C.

RESULTS The hts gene produces at least four classes of transcripts that encode three adducin-related proteins, one of which is orthologous to mammalian adducin The hts gene has previously been reported to encode an 1156amino-acid (aa) protein (Ding et al., 1993; Yue and Spradling, 1992). Here, we show that alternative use of promoters, differential exon splicing and use of alternative transcript termination sites result in at least four classes of transcripts with two different 5′ untranslated regions (UTRs) and four

3388 K. L. Whittaker and others 1 kb

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5 2

10 3

15

20

4 5 6 7,8,9,10

N4 (R8, N1)

30

11 AAAAA

1

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4 5 6 7,8,9,10

R1

12 AAAAA

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Fig. 1. Exon organization of hu-li tai shao (hts) cDNAs. The hts genomic region covers approximately 27 kb. Alternative splicing of exons found within this region yields at least four classes of transcripts (N4, R1, R2, N32). For each of the four known classes of hts cDNAs, coding sequences are shown in red, 5′ untranslated regions (5′UTRs) are shown in green and 3′UTRs are shown in blue. Introns are represented by thin black lines.

25

R2 (R10, R16)

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4 5 6A AAAAA

4A 5 6B N32

different 3′UTRs (Fig. 1). Correlation of cDNA and genomic sequences suggests that the hts gene may have two promoters, one at the 5′ end of exon 1 and one at the 5′ end of exon 4A. Similarly there are four transcription termination and polyadenylation sites, at the 3′ ends of exons 6A, 6B, 11 and 12. The resulting four classes of hts mRNA encode three variant adducin-related protein products (Fig. 2), one of which is the 1156-amino-acid protein previously described (Ding et al., 1993; Yue and Spradling, 1992) and two of which have not previously been reported. These three protein isoforms are identical over their N-terminal 472 amino acids (aa) (Fig. 2A). The N4 class of transcripts encodes the longest protein, referred to here as HTSN4, with a predicted length of 1156 aa and predicted molecular mass of 128 kDa (Ding et al., 1993; Yue and Spradling, 1992). The C-terminal 498 aa region of HTSN4 does not show homology with any known proteins (Ding et al., 1993; Yue and Spradling, 1992). The R2 and N32 classes of cDNA encode a protein, HTSR2/N32, with a predicted length of 495 aa and predicted molecular mass of 55 kDa; the C-terminal 23 aa region lacks homology with any known protein (Fig. 2A). The R1 cDNA encodes a predicted protein of 718 aa (Fig. 2A), HTSR1, with an expected molecular mass of 79 kDa, similar in size to human adducins; human αadducin is 737 aa long (Joshi et al., 1991), β-adducin is 726 aa long (Joshi et al., 1991) and γ-adducin is 674 aa long (Dong et al., 1995). The HTSR1 protein is equally homologous to all three human adducin subunits (Fig. 2B), showing 39-40% amino acid identity and 59-61% similarity. The R1 protein contains a motif near the C terminus that is highly conserved in α-, β- and γ- forms of human adducin (Fig. 2B), but which is absent from the Drosophila HTSN4 and HTSR2/N32 adducinrelated proteins. This motif is highly polybasic and shows myristoylated alanine-rich C-kinase substrate (MARCKS) homology (Harlan et al., 1991). The MARCKS-related sequence has been shown to be the major calmodulin-binding site and the major phosphorylation site for PKA and PKC in

AAAAA

both human α- and β-adducin (Matsuoka et al., 1996). Phosphorylation occurs within the MARCKS-related sequence at Ser-726 for human α-adducin and Ser-713 for human βadducin (Matsuoka et al., 1996). The corresponding residues in human γ-adducin (Ser-660) and Drosophila HTSR1 (Ser705) are conserved (Fig. 2B) but have not been tested for their ability to be phosphorylated.

hts transcripts bearing different classes of 3′′UTRs show distinct expression and localization patterns in the ovary We next asked whether all four classes of hts transcripts are expressed in ovaries and then analyzed their spatial and temporal expression patterns during oogenesis. Northern blots of ovarian poly(A+) RNA hybridized with DNA probes specific for each 3′UTR class confirmed that transcripts bearing each class of hts 3′UTR are present in ovarian RNA (data not shown). To assess the pattern of expression of each class during oogenesis, whole-mount in situ hybridizations were carried out using probes specific for each 3′UTR (Fig. 3). Our analyses focused on stages 1-14 of oogenesis since at these stages nurse cell to oocyte transport, and intracellular localization within the oocyte, can readily be assayed. Transcripts bearing the hts R1 3′UTR accumulate in the somatic component of egg chambers. In the germarium the resolution of our analysis does not enable us to determine whether R1 is expressed exclusively in the somatic cells. However, in stage 2-14 egg chambers, expression is restricted to the follicle cells and is absent from the germ line (Fig. 3A,B). Transcripts with the hts R2/N32 class of 3′UTR are expressed at low levels during oogenesis. As a consequence, during the early stages of oogenesis we were unable to determine whether expression is restricted to the germ line (Fig. 3C). At later stages, however, R2/N32 transcripts clearly accumulate in nurse cells (Fig. 3D) but are excluded from the oocyte until late oogenesis. At stage 11 these transcripts are

hts mRNA expression and localization 3389

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Fig. 2. Alignment of predicted HTS open reading frames. (A) Sequences of all three predicted HTS protein isoforms are identical over the first 472 residues. The HTSR2/N32 open reading frame (ORF) continues for an additional 23 codons (black). Identity between the HTSN4 and HTSR1 ORFs continues to codon 658, after which the sequences diverge, the HTSN4 ORF continuing for an additional 498 codons (blue) and the HTSR1 ORF for an additional 60 codons (orange). The red and orange regions of the predicted proteins are homologous to the human adducins. (B) Conservation of sequence between Drosophila HTSR1 protein and human adducins. Amino acids conserved between Drosophila HTSR1 and all three human adducins (α, β and γ) are highlighted in blue; residues conserved between HTSR1 and one or two of the human adducins are highlighted in green. Numbers at right and left refer to the amino acids within the specific predicted protein. The MARCKS (myristoylated alaninerich C-kinase substrate) homology region near the carboxy terminus is underlined. The serine residue known to be phosphorylated in human α-adducin and human β-adducin is also conserved in Drosophila HTSR1 and human γ-adducin (asterisk).

Drosophila HTSR1 Human α-adducin Human β-adducin Human γ-adducin

1 1 1 1

MTEVEQPPQN MNGDSRAAVV MSEETVPEAA MSSDASQGVI

GIDPTAGEDD TSPPPTTAPH SPPPPQGQPTTPPPPSMPH

DNS------KERYFDRVDE ---YFDRFSE KERYFDRINE

--------KA NNPEYLRERN DDPEYMRLRN NDPEYIRERN

RPADIEQDMR MAPDLRQDFN RAADLRQDFN MSPDLRQDSS

35 50 46 50


36 51 47 51

EMERRKRVEA MMEQKKRVSM LMEQKKRVTM MMEQRKRVTR

IMGSKLFREE ILQSPAFCEE ILQSPSFREE ILQSPAFRED

LERIVDSARD LESMIQEQFK LEGLIQEQMK LECLIQEQMK

GGAGASGI-KGKNPTGLLA KGNNSSNIWA KGHNPTGLLA

LQQLSDIVGV LQQIADFMTT LRQIADFMAS LQQIADYIMA

83 100 96 100

84 Drosophila HTSR1 Human α-adducin 101 97 Human β-adducin Human γ-adducin 101

PVSRVGSVFK NVPNVYPAAP TSHAVFPTSS NSFSGFSSPP

---------S QGGMAALNMS --------MN --------LS

SNCMVPINDI LGMVTPVNDL VSMMTPINDL LGMVTPINDL

RGVESMGYAK RGSDSIAYDK HTADSLNLAK PGADTSSYVK

GEKILRCKLA GEKLLRCKLA GERLMRCKIS GEKLTRCKLA

124 150 138 142


125 151 139 143

ATFRLLDLYG AFYRLADLFG SVYRLLDLYG SLYRLVDLFG

WTQGLGAQIT WSQLIYNHIT WAQLSDTYVT WAHLANTYIS

ARLKVDQEYF TRVNSEQEHF LRVSKEQDHF VRISKEQDHI

LVNPYGLLYH LIVPFGLLYS LISPKGVSCS IIIPRGLSFS

EITASALNKV EVTASSLVKI EVTASSLIKV EATASNLVKV

174 200 188 192


175 201 189 193

DMQGQIVEQG NLQGDIVDRG NILGEVVEKG NIIGEVVDQG

TTNFGGNKSH STNLGVNQAG SSCFPVDTTG STNLKIDHTG

FVLHSVVHAA FTLHSAIYAA FCLHSAIYAA FSPHAAIYST

RPDIRCAIYI RPDVKCVVHI RPDVRCIIHL RPDVKCVIHI

GCSPVVAISS HTPAGAAVSA HTPATAAVSA HTLATAAVSS

224 250 238 242


225 251 239 243

LKTGLLPLTK MKCGLLPISP MKWGLLPVSH MKCGILPISQ

DACVLGEITT EALSLGEVAY NALLVGDMAY ESLLLGDVAY

HAYTG-LFDE HDYHGILVDE YDFNGEMEQE YDYQGSLEEQ

EERNRLVRSL EEKVLIQKNL ADRINLQKCL EERIQLQKVL

GPNSKVILLT GPKSKVLILR GPTCKILVLR GPSCKVLVLR

273 300 288 292


274 301 289 293

NHGALCCGET NHGLVSVGES NHGVVALGDT NHGVVALGET

IEEAFFAACH VEEAFYYIHN VEEAFYKIFH LEEAFHYIFN

IVQACETQLK LVVACEIQVR LQAACEIQVS VQLACEIQVQ

LLPV--GLDN TLASAGGPDN ALSSAGGVEN ALAGAGGVDN

LVLIPEESRK LVLLNPEKYK LILLEQEKHR LHVLDFQKYK

321 350 338 342


322 351 339 343

AIYEQSRRPP A--------P--------A---------

EDLEKKFAAV ----KSRSPG ----HEVGSV ----FTYTVA

AAAEDGAATA SPVGEG---QWAGST---ASGGGG----

EKDAAEAVPK --------------------------VN

VGSPPKWRVG TGSPPKWQIG FGPMQKSRLG MGSHQKWKVG

371 373 361 367


372 374 362 368

GAEFEALMRM EQEFEALMRM EHEFEALMRM EIEFEGLMRT

LDNAGYRTGY LDNLGYRTGY LDNLGYRTGY LDNLGYRTGY

IYRHPLIKSD PYRYPALR-E TYRHPFVQ-E AYRHPLIR-E

PPKPKNDVEL KSKKYSDVEV KTKHKSEVEI KPRHKSDVEI

PPAVSSLGYL PASVT--GYS PATVT--AFV PATVT--AFS

421 420 408 414


422 421 409 415

LEEEELFRQG FASDGDSGTC FEEDG--APV FEDDT--VLL

IWKKGDIRKSPLRHSFQKQ PALRQHAQKQ SPLKYMAQRQ

GGDRSRWLNS QREKTRWLNS QKEKTRWLNT QREKTRWLNS

PNVYQKVEVL G----RGDEA PNTYLRVNVA PNTYMKVNVP

ETGTPD---P SEEGQNGSSP DEVQRSMGSP EESRNGETSP

467 466 456 462


468 467 457 463

KKITKWV-AE KSKTKWTKED RPKTTWMKAD RTKITWMKAE

GSPTHST--P GHRTSTSAVP EVEKSSSGMP DSSKVSGGTP

VRIEDPLQFV ------NLFV IRIENPNQFV IKIEDPNQFV

PAGTNPREFK PLNTNPKEVQ PLYTDPQEVL PLNTNP-EVL

RVQQLIKDNR EMRNKIREQN EMRNKIREQN EKRNKIREQN

514 510 506 511


515 511 507 512

RADKISAGPQ LQDIKTAGPQ RQDVKSAGPQ RYDLKTAGPQ

SHILEGVTWD SQVLCGV--SQLLASV--SQLLAGI---

EASRLKDATV ----VMDRSL ----IAEKSR ----VVDKPP

SQAGDHVVMM VQGE-----L SPS----TES STM-------

GAASKGIIQR VTASKAIIEK QLMSKGDEDT --------QF

564 548 545 539


565 549 546 540

GFQHNATVYK EYQPHVIV-KDDS-----EDDDHG----

APYAKNPFDN STTGPNPFTT EETVPNPFSQ PPAPPNPFSH

VTDDELNEYK LTDRELEEYR LTDQELEEYK LTEGELEEYK

RTVERKKKSV REVERKQKGC KEVERKKLEL RTIERKQQGL

HGEY-----EENLDEAREQ DGEKETAPEE EENHELFSKS

608 596 589 585


609 597 590 586

---------KEKSPPDQPA PGSPAKSAPA -----FISME

---------VPHPPPSTPI SPVQSPAKEA VPVM------

-TDTDFSESE KLEEDLVPEP ETKSPLVSPS ----------

AVLQAGTKKY TTGDDSDAAT KSLEEGTKKT ----------

PQ-----SEP FKPTLPDLSP ETSKAATTEP ----------

632 646 639 594


633 647 640 595

ETEHQVIEIQ DEPSEALGFP ETT-QPEGVV ---------V

TQQAPVPRQA MLEKEEEAHR VNGREEEQTA VNGKDDMHDV

EVVLSDGENV PPSPTEAPTE EEILSKGLSQ EDELAKRVSR

QNGDHSEAHL ASPEPAPDPA MTTSADTDVLSTSTTIENI

STFSQSSKEF PVAEEAAPSA ----DTSKDK EITIKSPEKI

682 696 683 635


683 697 684 636

QDV------S VEEGAAADPG TESVTSGPMS EEVL-----S

TDGSPKKD-K SDGSPGKSPS PEGSPSKSPS PEGSPSKSPS

KKKKGLRTPS KKKKKFRTPS KKKKKFRTPS KKKKKFRTPS

dumped into, but are not localized within, the oocyte (data not shown). We did not use the unique portion of the N32 3′UTR as probe and thus cannot address whether there are any differences in distribution of transcripts carrying the R2 versus the N32 classes of 3′UTR. Transcripts with the hts N4 3′UTR are expressed in the nurse cells, accumulate within the presumptive oocyte in region 2 of the germarium and are subsequently targeted specifically to the

* FLKKKKEKKK AEA FLKKSKKKSD S FLKKSKKKEK VES FLKKNKKKEK VEA

718 737 726 673

oocyte in early egg chambers (Fig. 3E). These transcripts are restricted to the oocyte cortex at stage 8 (data not shown), and further restricted to the anterior oocyte cortex from stages 914 (Fig. 3F). The in situ localization pattern visualized by the N4 3′UTR-specific probe corresponds to previous reports using N4 coding region probes (see Fig. 4A-E) (Ding et al., 1993; Zaccai and Lipshitz, 1996a). We conclude that different hts 3′UTRs are associated with

3390 K. L. Whittaker and others Fig. 3. Localization patterns of endogenous hts transcripts with different 3′UTRs. In situ hybridization of DNA probes specific for each hts 3′UTR (N4, R1, R2/N32) to ovarioles from wild-type females. (A) Transcripts with the R1 3′UTR are first detectable within the germarium. In early egg chambers, these transcripts accumulate in the monolayer of follicle cells surrounding the egg chamber. (B) Stage 10 egg chambers show high levels of transcripts bearing the R1 3′UTR in the follicle cells but not in the germ line. The posterior of the oocyte is in a slightly different focal plane from the anterior; R1 transcripts accumulate in the posterior follicle cells though this is not clearly visible in this image. (C) Transcripts carrying the R2 3′UTR are present at low levels in the germarium and in early egg chambers. (D) At stage 10, R2 transcripts accumulate in the nurse cells but are not transported into the oocyte. (E) During the early stages of oogenesis, transcripts with the N4 3′UTR are found in discrete locations within the germarium (arrowheads, most likely the presumptive oocyte). In young egg chambers, transcripts with the N4 3′ UTR are found in the most posterior cell (arrowheads, the presumptive oocyte). (F) In stage 10 egg chambers, transcripts with the N4 3′UTR are strongly expressed in nurse cells and are localized within the oocyte to the anterior cortex. For all egg chambers shown, anterior is to the left and the dorsal side of the oocyte is towards the top of the page.

distinct mRNA expression and localization patterns within the egg chamber. Transcripts with the R1 3′UTR are expressed only in the somatic follicle cells while those with the N4 or R2/N32 3′UTR are expressed only in the nurse cells of the germ line. Of the germ line-restricted transcripts, only those with the N4 3′UTR are transported into, and localized within, the oocyte. The remainder of our analyses were directed at defining the mechanism of N4 transcript transport and localization. The hts N4 3′′UTR is necessary and sufficient to direct RNA transport into and localization within the oocyte To map the region of the N4 transcript necessary and sufficient for transport into and localization within the oocyte, three

transgenic reporter constructs were made. All three constructs contain the hsp26-Sgs-3 regulatory cassette (Serano et al., 1994) whose hsp26 enhancer elements activate nurse cellspecific transcription during oogenesis (Cohen and Meselson, 1985). In addition they contain a 0.5 kb fragment of the E. coli lacZ gene as a reporter tag such that transgenic transcripts could be detected with an antisense β-galactosidase RNA probe in whole-mount ovaries prepared from transgenic flies. For each construct, ovaries from three independent lines homozygous for the transgene were analyzed and the results were the same for all three lines. The first reporter construct, pAddtag3′UTR, carries the fulllength hts N4 cDNA sequence (Ding et al., 1993) with the lacZ tag inserted between the ORF and the 3′UTR. The resulting reporter transcript is localized in the normal N4 pattern (data

Fig. 4. Localization patterns of endogenous hts transcripts versus transgenic transcripts containing the hts N4 5′UTR and coding sequence, or the hts N4 3′UTR alone. (A-E) In situ hybridizations of DNA probe for the N4 coding region to ovarioles from w1118 females. (A) Endogenous hts N4 transcripts accumulate in the presumptive oocyte from stages 1-7 (arrowheads). (B) At stage 8, hts N4 transcripts are restricted to the cortex of the oocyte. (C) hts N4 transcripts are further restricted to the anterior cortex of the oocyte at stage 9. Expression in the nurse cells continues and anterior localization in the oocyte is maintained from stage 10 (D) through the end of oogenesis (E). (F-J) In situ hybridizations of antisense β-galactosidase RNA probe to ovarioles from females homozygous for the pORFtagtub transgene. The pORFtagtub transgene produces reporter transcripts with the hts N4 5′UTR and coding region, a lacZ tag sequence and the α-tubulin 3′UTR. pORFtagtub transcripts are never localized within the oocyte (F-J), although they accumulate within the nurse cells in later oogenesis (I,J). (K-O) In situ hybridizations of antisense β-galactosidase RNA probe to ovarioles from females homozygous for the pAdd3′UTR transgene. The pAdd3′UTR transgene produces reporter transcripts with a lacZ tag sequence and the full-length hts N4 3′UTR. pAdd3′UTR reporter transcripts are localized in the same pattern as the endogenous hts N4 transcripts: first to the oocyte at stages 1-7 (K, arrowheads), then to the oocyte’s cortex at stage 8 (L), followed by anterior localization from stage 9 to 14 (M-O).


1

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N4 3'UTR scale 150 200

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stage of oogenesis 8 9-10a 10b-11 12-14

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∆5 0 - 1

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∆5 0 - 2

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∆1 0 0 - 1

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∆1 0 0 - 2

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∆1 0 0 - 3

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A+ *

100-250

Fig. 5. hts N4 3′UTR deletion transgenes and summary of results in stages 1-14 egg chambers. All transgenes used in the deletion analysis are shown as black bars, with the numbers along the thin scale bar referring to the nucleotide position within the N4 3′UTR. A summary of data is presented for each transgene and each stage of oogenesis. The numbers in bold-face type above the data summary refer to the stage of oogenesis for which the relevant data is being presented. T, transport from the nurse cells into the oocyte; C, localization to the cortex of the oocyte; A, localization to the anterior of the oocyte; ++, process is normal; +, anterior enrichment but some delocalization, or the signal is weak; +*, anterior enrichment but some delocalization and/or ectopic localization; −, process does not occur.

not shown). Thus insertion of the lacZ tag does not disrupt transcript transport and localization. To examine whether the N4 3′UTR is necessary to direct localization of the N4 class of transcripts, pORFtagtub transcripts were assayed. pORFtagtub carries the hts N4 5′UTR and coding region as well as the lacZ tag, but replaces the N4 3′UTR with the α-tubulin 3′UTR. The α-tubulin 3′UTR has been shown to promote RNA stabilization and polyadenylation but does not direct RNA transport into, and localization within, the oocyte at any stage (Macdonald and Struhl, 1988; Theurkauf et al., 1986). pORFtagtub transcripts are present in nurse cells but are not transported into the oocyte (Fig. 4F-J). We conclude that the N4 3′UTR is necessary for transport into and localization within the oocyte. pAdd3′UTR was used to ask whether the N4 3′UTR is sufficient to direct transcript transport and localization. pAdd3′UTR transcripts contain the 0.5 kb lacZ sequence followed by the 345 nt hts N4 3′UTR. Localization of pAdd3′UTR reporter transcripts in transgenic egg chambers is identical to that of endogenous hts transcripts (Fig. 4K-O; cf. Fig. 4A-E). For both, four stages of localization occur: (1) early transport of RNA into the presumptive oocyte, filling the oocyte (stages 1-7); (2) restriction of RNA to the oocyte cortex

(stage 8); (3) further restriction of RNA to the anterior oocyte cortex (stages 9-10a); and (4) maintenance of RNA in the anterior cortex of late stage oocytes (stages 10b-14). We conclude that the N4 3′UTR is necessary and sufficient for all aspects of hts transcript transport into, and localization within, the oocyte. A core 3′′UTR element is essential for transport and localization in stages 1-8 ovarian follicles To map sequences within the N4 3′UTR that are necessary for transcript transport and localization, we assayed a series of transgenic reporter transcripts containing 5′, 3′ or internal deletions within the N4 3′UTR (Fig. 5). These reporter lines were first used to map 3′UTR sequences necessary for transport from the nurse cells into the oocyte at stages 1-7 and for cortical localization within the oocyte at stage 8. Deletion of the first 150 nt of the N4 3′UTR had no effect on the transport of the reporter transcript into the oocyte at stages 1-7 or on cortical localization at stage 8 (Fig. 6A). However, deletion of another 50 nt (or more) abolished transport and localization (Fig. 6B,H). Successive deletions inward from the 3′ end of the 3′UTR showed that deletion of the 3′-most 95 nt of the 3′UTR, leaving nt 1-250 intact, had no

3392 K. L. Whittaker and others Fig. 6. Localization of reporter transcripts from hts N4 3′UTR deletion transgenes during stages 1-8. In situ hybridizations using an antisense β-galactosidase RNA probe on ovaries from hts N4 3′UTR transgenic females. (A) 5′∆3 (containing nt 150-345 of the hts N4 3′UTR). Early transport and localization of reporter RNA to the oocyte are normal (arrowheads). (B) 5′∆4 (nt 200-345). Deletion of more than 150 nt from the 5′ end of the 3′UTR abolishes early transport and localization of reporter transcripts to the oocyte. (C) 3′∆2 (nt 1-250). Deletion of the last 95 nt of the 3′UTR has no effect on the ability of reporter transcripts to be transported to, and localized within, the oocyte at stages 1-7 (arrowheads). (D) 3′∆3 (nt 1-200). Removal of more than 95 nt from the 3′ end abolishes early transport to, and localization of reporter transcripts within, the oocyte. (E) ∆50-1 (deletion of nt 250-300). Deletions outside the hts N4 TLE (nt 150-250) do not affect early transport or localization of reporter transcripts to stage 1-7 oocytes (arrowheads). (F) ∆50-2 (deletion of nt 200-250). Transport and localization are abolished, as is the case with all deletions overlapping the TLE. (G) ∆50-5 (deletion of nt 50-100). Cortical localization at stage 8 is normal for all deletions outside the TLE. (H) 5′∆4 (nt 200-345). Deletions that overlap the TLE abolish nurse cell-oocyte transport of reporter transcripts in stage 8 oocytes.

effect on reporter transcript localization during stages 1-8 of oogenesis (Fig. 6C). Removal of an additional 50 nt (or more) from the 3′ end abolished reporter RNA localization (Fig. 6D). To confirm that 3′UTR sequences from 150-250 are essential for transport and localization of RNA in the oocyte, we assayed the effects of internal deletions. Deletions that leave 3′UTR nt 150-250 intact (∆50-1, ∆50-4, ∆50-5; see Fig. 6E) had no effect, while all internal deletions that overlap nt 150-250 abolished transport and localization (for ∆50-2 see Fig. 6F; for ∆50-3, ∆100-1, ∆100-2, ∆100-3 see Fig. 5). RT-PCR was used to verify, for selected lines, that full-length reporter RNA was present even when no localization was detectable in the oocyte (data not shown; see Materials and Methods). We conclude that the 100 nt region from 150-250, which we will refer to as hts N4 transport and localization element (TLE), is essential for RNA transport from the nurse cells into the oocyte during stages 1-8 of oogenesis and for cortical localization within the oocyte at stage 8. Additional 3′′UTR elements are necessary together with the TLE for anterior localization of transcripts in stage 9-10a oocytes At stages 9-10a, endogenous hts transcripts, as well as reporter transcripts carrying the full 345 nt N4 3′UTR, are tightly confined to the anterior of the oocyte (Figs 3F, 4C,D). Deletion of nt 250-345 of the N4 3′UTR does not affect transcript localization (Fig. 7A-C). All deletions that remove part of the TLE (nt 150-250) result in failure of transcript transport from the nurse cells into the oocyte at these stages (data not shown), indicating that the TLE is essential. Deletion analyses also indicate that two regions act in concert with the TLE to enhance normal anterior localization of hts N4 transcripts at stages 9-10a. We will refer to these as hts N4 localization elements A (LE-A; nt 1-100) and B (LE-B; nt 100-150). Removal of LE-A (nt 1-100, 5′∆2) results in decreased levels of anterior localized RNA (Fig. 7D). Deletion of either the 5′ half (nt 1-50, 5′∆1; Fig. 7E) or the 3′ half (nt 50-100, ∆50-5; Fig. 7F) of LE-A has no effect on transcript localization. Removal of LE-B (nt 100-150, ∆50-4) results in severely diminished levels of anterior localized reporter transcript (Fig. 7G). Deletion of both LE-A and LE-B (nt 1-150, 5′∆3) completely abolishes anterior localization at stages 9-10a (Fig. 7H).

We conclude that the TLE (nt 150-250), LE-A (nt 1-100) and LE-B (nt 100-150) function additively to direct anterior localization of transcripts at stage 9-10a. Deletion of part or all of the TLE eliminates nurse cell-oocyte transport and thus there are no transcripts within the oocyte. In contrast, deletion

Fig. 7. Localization of reporter transcripts from hts N4 3′UTR deletion transgenes during stages 9-10a. In situ hybridizations using an antisense β-galactosidase RNA probe on ovaries from transgenic females. (A) 3′∆1 (nt 1-300). Reporter transcripts are localized normally to the anterior. (B) 3′∆2 (nt 1-250). Reporter transcripts are localized normally to the anterior. (C) ∆50-1 (deletion of nt 250-300). Reporter transcripts are localized normally to the anterior. (D) 5′∆2 (nt 100-345). Diminished levels of anterior localized reporter transcripts are present. (E) 5′∆1 (nt 50-345). Reporter transcripts show normal anterior localization within the oocyte at stages 9-10a. (F) ∆50-5 (deletion of nt 50-100). Deletion of nt 50-100 has no effect on reporter transcript localization at this stage. (G) ∆50-4 (deletion of nt 100-150). When nt 100-150 are removed, severely diminished levels of anterior localized reporter transcripts are seen. (H) 5′∆3 (nt 150-345). Deletion of nt 1-150 abolishes anterior localization.


Fig. 8. Localization of reporter transcripts from transgenes containing N4 3′UTR nt 100-250 (LE-B + TLE) in stage 1-12 oocytes. In situ hybridizations using an antisense βgalactosidase RNA probe on ovaries from transgenic females. (A) A transgene containing nt 100-250 is sufficient to drive normal transport to the oocyte and localization within the oocyte at stages 1-7 (arrowheads). (B) Cortical localization of reporter RNA occurs in stage 8 oocytes. (C) Stage 9-10a oocytes show anterior localization of reporter transcripts but at a slightly diminished level, consistent with the fact that LE-A (nt 1-100) was not included in the transgene. (D) Reporter transcripts are clearly enriched at the anterior but are also partially delocalized in stage 10b11 oocytes. (E) A slight diffusion from the anterior is also seen at stage 12.

of LE-A and/or LE-B does not disrupt nurse cell-oocyte transport. Loss of both LE-A and LE-B completely eliminates anterior transcript localization. Individually, they affect the amount of anterior localized RNA but not the localization pattern (with LE-B playing a more important role than LE-A). A 150 nt fragment containing the TLE and LE-B is sufficient for nurse cell-oocyte transport and intraoocyte transcript localization at stages 1-10a Transport of specific transcripts, such as hts N4, from the nurse cells into the oocyte is an active process during stages 1-10a of oogenesis. In order to define a minimal element sufficient for most aspects of transport and localization at these stages we asked whether a 150 nt fragment containing both the TLE (nt 150-250) and LE-B (nt 100-150) could direct transcript transport and localization at stages 1-10a. This 150 nt fragment is sufficient for transport from the nurse cells into the oocyte from stages 1-10a and for most aspects of localization within the oocyte at stages 8-10a (Fig. 8A-C). The level of [TLE + LE-B] reporter RNA at the anterior of stage 9-10a oocytes is slightly diminished relative to transcripts that carry the fulllength hts N4 3′UTR (Fig. 8C; cf. Fig. 4C) consistent with the fact that LE-A was not included. In contrast, a construct containing [LE-A + LE-B + TLE] (3′∆2, nt 1-250) localizes completely normally from stage 1-10a (Figs 6C, 7B). Maintenance of transcripts at the anterior of stage 10b-14 oocytes requires TLE, LE-A and LE-B, together with additional 3′′UTR elements At stage 10b-11, the nurse cells begin bulk transfer of their cytoplasmic contents to the oocyte, including RNAs not

previously transported. Cis-acting localization signals functioning during this time period are therefore more likely to be necessary for the tethering of hts N4 RNA at the anterior of the oocyte rather than for specific transport. Subsequently, at stages 12-14 when bulk transfer is complete, cis-acting signals are likely to act to maintain anterior transcript localization. At stages 10b-14, the 150 nt fragment containing [TLE + LE-B] is sufficient to direct anterior localization; however, as at stages 9-10a, this region is not sufficient to confer fully wildtype transcript localization. Although these reporter transcripts are enriched at the anterior, they are partially delocalized (Fig. 8D,E) or on occasion may show ectopic localization (data not shown). However, in contrast to stage 9-10a, addition of both LE-A and LE-B to TLE (3′∆2, nt 1-250) is not sufficient to restore normal anterior localization. Rather, additional elements 3′ to the TLE contribute to maintenance of transcripts at the anterior of stage 10b-14 oocytes, such that the entire N4 3′UTR is required for normal localization. All deletions that overlap the TLE (nt 150-250) completely abolish anterior anchoring of reporter RNA. Successive 50-nt deletions from the 5′ end of the N4 3′UTR result in changes in both the pattern and amount of localized reporter RNA. Removal of the first 50 nt of the 3′UTR (i.e. the 5′ half of LEA) still allows anterior localization but transcripts are not as tightly localized as normal at stage 10-11 (Fig. 9A). At stage 12-14, transcripts lacking the first 50 nt of the 3′UTR show both anterior localization and diffusion throughout the oocyte (Fig. 9B). While nt 1-50 are essential for normal anchoring of transcripts at the anterior of stage 10b-14 oocytes, they are not sufficient for any aspect of transcript anchoring (3′∆6, data not shown). Complete removal of LE-A (5′∆2) results in partial delocalization of the transcripts from the anterior (similar to removal of only the 5′-half of LE-A in 5′∆1) and a lower overall level of localized mRNA (Fig. 9C). Separate deletions of the 3′ half of LE-A (nt 50-100, ∆50-5) or of LE-B (nt 100150, ∆50-4) each impair localization, resulting in a drop in the amount of localized RNA (Fig. 9D,E). Simultaneous deletion of both LE-A and LE-B abolishes localization (5′∆3, Fig. 9F). A region that includes both LE-A and LE-B (but without the TLE) is incapable of directing anterior transcript localization (3′∆4, data not shown). Deletions 3′ to the TLE (i.e. in the 250-345 region) result in abnormal reporter RNA distribution at stages 10b-14. We refer to the region from nt 250-300 as LE-C and from nt 301-345 as LE-D. Loss of LE-C alone (∆50-1) has no effect at stages 10b11 (Fig. 9G) but impairs anterior anchoring at stage 12-14, appearing as diffusion of RNA from the anterior (Fig. 9H) or, occasionally, as delocalization from all but the anterior dorsal ‘corner’ of the stage 12 oocyte cortex (data not shown). Removal of LE-D (3′∆1) causes reporter transcript diffusion from the anterior in some oocytes (Fig. 9I,J) and ectopic localization in others (Fig. 9K). Removal of LE-C + LE-D (3′∆2) results in a significant reduction in the amount of anterior anchored RNA (Fig. 9L), and can also produce ectopic localization similar to loss of LE-D alone (data not shown). Thus LE-C and LE-D are essential for normal anchoring of transcripts at the anterior of stage 10b-14 oocytes. However, LE-C + LE-D alone are not sufficient for anterior localization since transgenic transcripts containing only LE-C + LE-D are not localized (5′∆5, data not shown).

3394 K. L. Whittaker and others Fig. 9. Localization of reporter transcripts from hts N4 3′UTR deletion transgenes during stages 10b-14. In situ hybridizations using an antisense β-galactosidase RNA probe on ovaries from transgenic females. (A) 5′∆1 (nt 50-345 present). Reporter transcripts are localized to the anterior but are not as tightly localized as normal in stage 10b-11 oocytes. (B) 5′∆1 (nt 50345 present). At stage 13, 5′∆1 reporter transcripts are enriched at the anterior but show diffusion from the anterior and a low background level throughout the oocyte. (C) 5′∆2 (nt 100-345 present). Reporter transcripts are localized to the anterior but show a significant decrease in level. (D) ∆50-5 (deletion of nt 50-100). Reporter transcripts are found at the anterior (arrowheads) but the level is reduced. (E) ∆50-4 (deletion of nt 100-150). Reporter transcripts are localized to the anterior but in diminished amounts. (F) 5′∆3 (nt 150-345 present). Localization of reporter transcripts within the oocyte is abolished. (G) ∆50-1 (deletion of nt 250-300). Anterior anchoring of reporter transcripts is slightly impaired at stage 10-11, with reporter transcripts diffusing from the anterior of the oocyte. (H) ∆50-1 (deletion of nt 250-300). At stage 12-14, the diffusion of reporter mRNA from the anterior is more noticeable than at stage 10-11. (I) 3′∆1 (nt 1-300 present). Reporter transcripts are localized to the anterior but transcripts are not as tightly localized as normal in stage 10b-11 oocytes. (J) 3′∆1 (nt 1-300 present). At stages 12-14, reporter transcripts are strongly enriched at the anterior of the oocyte but diffusion from the anterior is also seen. (K) 3′∆1 (nt 1-300 present). Occasionally, ectopic localization of 3′∆1 reporter mRNA is seen in stage 10-11 oocytes. This oocyte has been understained to show localization within the oocyte; expression is also present in nurse cells although it is not visible in this photograph. (L) 3′∆2 (nt 1-250 present). Deletion of the last 95 nt of the N4 3′UTR results in a significant drop in the amount of reporter mRNA localized to the anterior of stage 10b-14 oocytes.

We conclude that LE-C and LE-D act additively, together with the TLE, LE-A and LE-B, and function to enhance transcript maintenance at the anterior of stage 10b-14 oocytes.

swallow acts through the hts N4 3′′UTR swallow (sww) is required for hts N4 RNA localization during oogenesis (Ding et al., 1993). In light of the present results, the SWALLOW protein would be expected to influence hts localization by direct or indirect function through the N4 3′UTR. To test this possibility, flies transgenic for pAdd3′UTR were crossed into sww mutant backgrounds and transgenic reporter transcripts were examined by whole-mount in situ hybridization (Fig. 10). In sww mutant ovaries pAdd3′UTR reporter transcript localization was normal from stages 1 through 8: reporter RNA was correctly transported into the oocyte at these stages and was restricted to the oocyte cortex at stage 8 (Fig. 10A,B). However, by stage 9-10, while reporter RNA was still enriched at the anterior, it was more diffuse and less tightly localized than normal (Fig. 10C,D). At stage 10-11, reporter RNA was localized primarily at the dorsal anterior of the oocyte, rather than throughout the anterior (Fig. 10D,E). The remaining anterior transgenic transcripts continued to delocalize during stage 11-12 and were completely delocalized by the end of oogenesis (Fig. 10F,G). Transcript localization in sww1/sww1 ovaries is slightly more defective than that in sww1/sww6 ovaries as would be expected since sww1 is one of the strongest sww alleles while sww6 is a weaker allele (Hegdé and Stephenson, 1993). Reporter transcripts were completely delocalized by stage 12 in sww1/sww1 oocytes (data not shown) while some transcripts were still detectable at the anterior of stage 12 sww1/sww6 oocytes (Fig. 10F). Thus in sww mutant

ovaries, reporter transcripts bearing only the hts N4 3′UTR show the same defects in localization as endogenous transcripts (Ding et al., 1993). SWW thus plays a role in maintenance of transcripts at the anterior of the oocyte by acting, directly or indirectly, through the hts N4 3′UTR. Deletion of the site(s) in the 3′UTR through which SWW acts would be expected to result in decreased and diffuse anterior localization at stage 9-10, anterior-dorsal localization at stage 10-11 and subsequent transcript delocalization. None of the deletion transgenes studied here matched this phenotype exactly, as none showed dorsal anterior localization of reporter transcripts at stage 10-11. At stage 1214, removal of LE-C (nt 250-300; ∆50-1) impairs anterior localization and results in occasional anterior dorsal localization (data not shown; see Fig. 5). Thus it is possible that SWW acts in part through LE-C at stages 12-14 (see Discussion). DISCUSSION Structure and expression of the Drosophila hts locus We have recovered four classes of hts cDNAs (R1, N4, R2, N32) from ovarian and early embryonic cDNA libraries, representing alternatively processed hts transcripts. There appear to be two hts promoters, one at or near the 5′ end of exon 1, and one at or near the 5′ end of exon 4A. Since all classes of cDNAs include 3′ poly(dA) tracts, there appear to be four transcription termination and polyadenylation sites, at the 3′ ends of exons 6A, 6B, 11 and 12. Previous analyses (Yue and Spradling, 1992) were able to define the 3′ part of exon 2

hts mRNA expression and localization 3395 Fig. 10. Localization of reporter transcripts containing the full-length hts N4 3′UTR in swallow mutant ovaries. In situ hybridizations of antisense β-galactosidase RNA probe to ovaries from sww1//sww6 females homozygous for the pAdd3′UTR transgene. (A) Reporter transcripts are localized normally to the oocyte in a sww1/sww6 mutant background during stages 1-7, and correctly restricted to the oocyte’s cortex at stage 8 (B). (C) Defects in reporter transcript localization are first seen at stage 9, when reporter RNA is found at the anterior of the oocyte but localization is impaired (arrowheads). (D) The effects of the transheterozygous mutant background on reporter RNA localization are very noticeable at stage 10, when reporter RNA is distributed over the anteriormost 20% of the oocyte rather than being confined tightly to the anterior cortex. Arrowheads here mark the boundary of posterior spreading of reporter RNA at stage 10. (E) By stage 10b-11, reporter transcripts are found only at the dorsal anterior ‘corner’ of the oocyte (arrowhead) and spread posteriorly along the dorsal side of the oocyte. (F) By stage 11-12 there is very little reporter RNA remaining at the antero-dorsal ‘corner’ (arrowhead). (G) Finally, reporter RNA is completely delocalized during the last stages of oogenesis (stage 12-14) in sww1/sww6 mutant ovaries.

as well as exons 3-11. Novel exons defined here are exon 1, 4A, 6A, 6B and 12, as well as the 5′ part of exon 2. Since we have defined several novel exons, we have revised and updated the numbering system for hts exons used previously (Yue and Spradling, 1992). The possibility that a promoter exists just 5′ to exon 4A is of interest in light of extant mutants (Yue and Spradling, 1992). All of these are transposable element insertions into intron 2 (hts1, hts2) or intron 3 (htsRK) and thus might not be expected to affect the presumptive exon 4A promoter, which is well downstream of these insertions. In other words, all transcripts that derive from the 4A promoter would still be produced in hts mutants; hence these hts alleles are not expected to be null. This might explain why the existing hts alleles are female-

A LE-A

nt 1

TLE

LE-B

50

100

150

200

LE-C

250

LE-D

300

345

Stage of oogenesis at which required

B

1 -8

9 -1 0 a 10b-14 T LE L E- A

hts N4 3'UTR localization element

L E- B L E- C LE- D

Fig. 11. RNA localization elements in the hts N4 3′UTR and their temporal functions during oogenesis. (A) Spatial organization of localization elements in the hts N4 3′UTR. LE-A (nt 1-100) is shown in green, LE-B (nt 100-150) in red, TLE (nt 150-250) in blue, LE-C (nt 250-300) in purple and LE-D (nt 300-345) in orange. (B) Stage of oogenesis at which each element is required. The TLE is required for RNA localization at all 14 stages of oogenesis. LE-A and LE-B are required from stages 9-14. LE-C and LE-D are required from stages 10b-14.

sterile rather than lethal (Yue and Spradling, 1992), despite evidence that Drosophila has a single gene (hts) encoding adducin-related products (Ding et al., 1993; Yue and Spradling, 1992) and that adducin-related proteins are ubiquitously expressed during development (Zaccai and Lipshitz, 1996a,b). Whole-mount in situ hybridizations using probes specific to the hts R1, N4 and R2/N32 3′UTRs demonstrated that transcripts bearing these 3′UTRs are expressed in and targeted to separate locations within the developing egg chamber (the follicle cells, the oocyte and the nurse cells, respectively). It should be noted that these in situ hybridization experiments using 3′UTR-specific probes do not prove that the specific transcripts represented by our cDNAs are in fact expressed in these regions, rather they suggest that the majority of transcripts bearing these particular 3′UTRs are expressed there. The Drosophila HTS family of proteins The four classes of hts cDNAs identified here encode three classes of adducin-related protein isoforms. All three predicted HTS protein isoforms have the same N-terminal region but are different lengths and have different C termini. Since the probes we used for library screening included the adducinhomologous sequences, we cannot exclude the possibility that additional hts splice variant mRNAs exist that encode proteins without the amino-terminal adducin homology. Of the presumptive Drosophila adducin-related proteins identified here, HTSR1 is most closely related to the human adducins. HTSR1 is the only Drosophila adducin-related protein predicted to contain a MARCKS motif at the Cterminal end. The core sequence of the MARCKS motif is identical in Drosophila HTSR1 and human adducins, including the serine residue that has been shown to be the target for PKA

3396 K. L. Whittaker and others and PKC phosphorylation in human adducins. The MARCKSrelated sequence also functions in human adducins to bind calmodulin (Matsuoka et al., 1996). Both calmodulin binding and PKA phosphorylation of human adducin inhibit binding of adducin to spectrin-actin complexes and recruitment of additional spectrin molecules to the complexes by adducin (Gardner and Bennett, 1987; Matsuoka et al., 1996). Calmodulin binding downregulates the ability of adducin to act as a barbed-end capping protein for actin (Kuhlman et al., 1996) and PKC phosphorylation of human adducin redistributes adducin away from cell-cell contact sites in cultured epithelial cells (Kaiser et al., 1989). The specific functions of HTSR1 protein in the follicle cell epithelium during Drosophila oogenesis remain unknown. Given the high sequence conservation between the Drosophila HTSR1 and human adducins, we speculate that HTSR1 protein activity in the follicle cells is likely to be regulated by signal transduction pathways similar to those affecting human adducins. Since exon 12 encodes the MARCKS-homologous C-terminal region of the protein, one strategy for defining these functions will involve identification of exon 12-specific mutations. We previously reported two monoclonal antibodies, 1B1 and 2C1, that recognize adducin-related proteins in the follicle cells (Zaccai and Lipshitz, 1996a,b). We have shown that both of these antibodies recognize HTSN4 and HTSR1 but not HTSR2/N32, suggesting that they recognize the adducinrelated regions encoded by exons 7 to 10 (D. Lin and H. D. L., unpublished data). Interestingly, the proteins recognized by these antibodies are membrane-associated in the follicle cells and are most highly expressed in the follicle cells that abut the oocyte (Zaccai and Lipshitz, 1996a). Human adducins are capable of promoting local cytoskeletal rearrangements and dynamic changes in actin-spectrin cytoskeletal stability at cellcell contact junctions (Kaiser et al., 1989; Seidel et al., 1995). We speculate that the HTSR1 protein might function in cytoskeletal remodeling of follicle cells during their extensive shape changes, migrations and interactions with the oocyte during Drosophila oogenesis. Transcripts encoding the other two classes of adducinrelated proteins described here are expressed, not in the follicle cells, but in the germ line. These proteins share N-terminal homology to mammalian adducin, but are either truncated (HTSR2/N32) or encode an extensive C-terminal region that is not homologous to mammalian adducin (HTSN4). In the case of the HTSN4 protein, it is known that the C-terminal polypeptide becomes associated with, and functions at, the ring canals during oogenesis, possibly after proteolytic cleavage from the full-length HTSN4 protein (Robinson et al., 1994). It is also known that adducin-related proteins are important components of the fusome during early germ line development (Lin and Spradling, 1995; Lin et al., 1994; Yue and Spradling, 1992; Zaccai and Lipshitz, 1996a,b). It is not yet known which specific HTS-family proteins are present in the fusome. However, since antibodies 1B1 and 2C1 bind to the fusome and recognize adducin-related epitopes (see above), the HTS components of the fusome must include adducin-related regions. Cytoplasmic localization of hts transcripts and the organization of 3′′UTR transcript localization signals We have shown that alternative processing of hts 3′ exons is

used to control transcript localization within the germ line, representing a novel method for differential intracellular targeting of transcripts encoded by a single gene. hts transcripts that carry the R2/N32 classes of 3′UTR are synthesized in the nurse cells but are not transported into the oocyte or localized therein. In contrast, transcripts that carry the N4 class of 3′UTR are transported into the oocyte and localized first to its cortex and, later, to its anterior pole. This transcript localization pattern is reflected in the pattern of localization of the 87 kDa adducin-related protein recognized by the 1B1 antibody (Zaccai and Lipshitz, 1996a,b). We have exploited reporter transgenes to map the transport and localization signals in the hts N4 mRNA. Specifically, we have shown that the 345 nt 3′UTR of the N4 transcript is necessary and sufficient for all phases of localization (Fig. 11). Detailed analysis of the 3′UTR has enabled us to define a 150 nt core localization region (comprising hts N4 TLE + LE-B) that is sufficient to direct transport from the nurse cells into the oocyte, cortical localization in stage 8 oocytes and anterior transcript localization in stage 9-14 oocytes. This region is predicted by the GCG MFOLD program to form the largest and most stable stem-loop structure in the hts N4 3′UTR (data not shown). We do not yet know whether this structure is conserved in related Drosophila species, neither have we carried out mutagenesis of this region to define whether the stem or loops are functionally important for localization. While this core region is sufficient to direct most aspects of transcript localization throughout oogenesis, additional elements in the 3′UTR (LE-A, LE-C and LE-D) are required to enhance transcript concentration and anchoring at the anterior of later stage oocytes. These elements reside both 5′ and 3′ of the core region and are not by themselves sufficient for any aspects of localization. Rather, they act in concert with the core region, enhancing its ability to anchor transcripts at the anterior. Thus the ‘design’ of the N4 3′UTR is modular: it has a core localization element of 150 nt while the adjacent 5′ 100 nt and 3′ 95 nt contain additional elements that enhance anchoring of transcripts at the anterior.

Trans-acting factors in hts N4 RNA localization The swallow gene has previously been shown to be required for hts N4 (Ding et al., 1993) and bicoid mRNA (Berleth et al., 1988; St Johnston et al., 1989) localization. Additional factors known to function in anterior localization or anchoring of other transcripts do not function in hts N4 transcript localization (e.g. EXUPERANTIA and STAUFEN; Ding et al., 1993). swallow mutations disrupt hts transcript localization and affect the distribution of an 87kD adducin-related protein recognized by the 1B1 antibody (Ding et al., 1993; Zaccai and Lipshitz, 1996b). swallow mutations also delocalize bicoid transcripts from the anterior pole and polar granules from the posterior (Berleth et al., 1988; Hegdé and Stephenson, 1993; St Johnston et al., 1989; Zaccai and Lipshitz, 1996a). We have speculated that SWALLOW may function directly or indirectly to anchor RNAs and RNPs in the cortex (Zaccai and Lipshitz, 1996b). Although putative RNA-recognition motifs (RRMs) have been identified in the SWALLOW protein, these motifs are very divergent from consensus RRMs (Chao et al., 1991) and it is not known whether SWALLOW is capable of binding RNA directly. We have shown here that all localization signals in the hts

hts mRNA expression and localization 3397 N4 transcript are contained within its 3′UTR and that SWALLOW acts, directly or indirectly, through the N4 3′UTR. Our data suggest a role for LE-C in mediating SWALLOW function at stages 12-14. However, since deletion of LE-C does not result in swallow-like transcript localization defects at stages 9-11 it is possible that, at these stages, SWALLOW acts through the TLE or there are multiple redundant regions in the N4 3′UTR through which SWALLOW acts. Conclusions We have defined a novel mechanism that is used to target different mRNA products of a single gene to distinct intracellular destinations. This is accomplished through alternative splicing and/or polyadenylation of 3′UTRs that either do or do not contain transport and localization elements. In the case of the hts gene studied here, the transcripts encode different protein isoforms, suggesting that the targeting mechanism may be used to direct local synthesis of these proteins in distinct cytoplasmic regions. Further genetic and molecular studies will be required to define the mechanisms of hts transcript localization and the functions of the encoded protein isoforms. We thank Angelo Karaiskakis and Weili Fu for expert technical assistance. Plasmid p701, which encodes the α-tubulin 3′UTR, was a gift from Paul Macdonald. We acknowledge the Berkeley Drosophila Genome Project for genomic sequence of the hts locus and the Bloomington Drosophila Stock Center for sww mutants. We thank Henry Krause, Arash Bashirullah, Ramona Cooperstock, Hua Luo and Wael Tadros for critical reading of the manuscript. This research was funded from a grant to H.D.L. from the Medical Research Council of Canada (MT-14409).

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