Small Bristles, the Drosophila Ortholog of NXF-1, Is Essential for Mrna Export Throughout Development

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Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development

GAVIN S. WILKIE,1,4 VITALY ZIMYANIN,1,5 RUTH KIRBY,1 CHRISTOPHER KOREY,2 HELEN FRANCIS-LANG,3,6 DAVID VAN VACTOR,2 and ILAN DAVIS1 1 Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, King’s Buildings, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom 2 Harvard Medical School, Harvard University, Boston, Massachusetts 02115, USA 3 Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California 95064, USA

ABSTRACT We identified a temperature-sensitive allele of small bristles (sbr ), the Drosophila ortholog of human TAP/NXF-1 and yeast Mex67, in a screen for mutants defective in mRNA export. We show that sbr is essential for the nuclear export of all mRNAs tested in a wide range of tissues and times in development. High resolution and sensitive in situ hybridization detect the rapid accumulation of individual mRNA species in sbr mutant nuclei in particles that are distinct from nascent transcript foci and resemble wild-type export intermediates. The particles become more numerous and intense with increasing time at the restrictive temperature and are exported very rapidly after shifting back to the permissive temperature. The mRNA export block is not due indirectly to a defect in splicing, nuclear protein import, or aberrant nuclear ultrastructure, suggesting that in sbr mutants, mRNA is competent for export but fails to dock or translocate through NPCs. We conclude that NXF-1 is an essential ubiquitous export factor for all mRNAs throughout development in higher eukaryotes. Keywords: Drosophila; intracellular mRNA localization; Mex67; mRNA nuclear export; NXF-1; small bristles (sbr); TAP

INTRODUCTION a role in mRNA export from the nucleus (reviewed in Conti & Izaurralde, 2001)+ For example, microinjection mRNA export from the nucleus is essential for gene of anti-GLE1 antibodies into cultured cells causes nu- expression and most of the factors required for this clear accumulation of poly(A)ϩ RNA (Watkins et al+, process have been characterized in yeast+ Although 1998)+ Transcript export efficiency is reduced when mu- many orthologs of the yeast proteins have also been tant forms of the DBP5 protein are injected into Xeno- identified in higher eukaryotes, it has proven difficult to pus oocytes (Schmitt et al+, 1999)+ RAE1, the ortholog study their role in mRNA export due to the lack of an of yeast GLE2, shuttles between the nucleus and cyto- in vitro nuclear mRNA export assay and absence of plasm in Xenopus oocytes and binds the nucleoporin mutations in the genes+ Nevertheless, in mammalian NUP98 (Pritchard et al+, 1999)+ Overexpression of the cultured cells, considerable circumstantial evidence sug- domain of NUP98 that binds to RAE1 causes nuclear gests that GLE1, DBP5, RAE1, and TAP/NXF1 all have accumulation of poly(A)ϩ RNA, a phenotype that is abrogated by excess RAE1 (Pritchard et al+, 1999)+ It is Reprint requests to: Ilan Davis, Wellcome Trust Centre for Cell , , , therefore likely that all these factors contribute to mRNA Biology Institute of Cell and Molecular Biology King’s Buildings Uni- + versity of Edinburgh, Edinburgh EH9 3JR, Scotland, United King- export dom; e-mail: ilan+davis@ed+ac+uk+ The best characterized mRNA export factor in higher 4 Present address: MRC Human Genetics Unit, Western General eukaryotes is TAP/NXF1, the cellular cofactor required Hospital, Crewe Road, Edinburgh, EH4 2XU, Scotland, United Kingdom+ for the nuclear export of unspliced retroviral RNA con- 5 Present address: Wellcome Trust/CRC Centre for Developmen- taining a constitutive transport element (CTE; Gruter tal Biology, Tennis Court Road, University of Cambridge, Cambridge +, + , + et al 1998) TAP is a member of a family of five human CB2 1QR United Kingdom ; +, , 6 Present address: Exelixis, Inc+, 170 Harbor Avenue, P+O+ Box nuclear export factors (NXFs Herold et al 2000 re- 511 S+, San Francisco, California 94083, USA+ viewed in Conti & Izaurralde, 2001)+ In Saccharomyces 1781 Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

1782 G.S. Wilkie et al. cerevisiae, the only NXF gene, MEX67, is required for NXF1, whereas NXF3 does not (Herold et al+, 2000)+ In mRNA export together with MTR2 (Santos-Rosa et al+, C. elegans, RNAi against NXF1 causes lethality in both 1998)+ Both genes are essential and their mutations adult and embryonic stages and leads to nuclear ac- lead to mRNA export defects (Segref et al+, 1997)+ In cumulation of poly(A)ϩ RNA (Tan et al+, 2000)+ How- contrast, mex67 ϩ, the only NXF gene in Schizosac- ever, this defect occurs a relatively long time after RNAi charomyces pombe, is not essential for growth or mRNA treatment, and it is unclear whether the block in mRNA nuclear export, but mex67 mutations are synthetic lethal export is due to indirect affects, such as aberrant splic- with rae1 (Yoon et al+, 2000)+ S. pombe rae1 ϩ is required ing, or defective nuclear ultrastructure+ Furthermore, for mRNA export (Brown et al+, 1995), as is GLE2, the the effects on individual mRNAs have not been inves- S. cerevisiae ortholog (Murphy et al+, 1996)+ These re- tigated in nematodes+ Moreover, in yeast, the precise sults indicate that Mex67 protein has a contributory intranuclear location of specific yeast mRNAs is diffi- rather than key role in mRNA export in S. pombe and cult to determine under the light microscope because may be an accessory factor for Rae1-mediated export of the small size of the nucleus+ Although some studies (Yoon et al+, 2000)+ suggest that mRNAs accumulate all over the nucleo- There is considerable circumstantial evidence sug- plasm when mRNA export is blocked (Hurt et al+, 2000), gesting that TAP/NXF1 is required for cellular mRNA other experiments show accumulation only at the site exported in higher eukaryotes+ Excess CTE RNA in- of transcription (Jensen et al+, 2001)+ hibits mRNA export in Xenopus oocytes, a phenotype Here, we address the role of TAP/NXF1 in Drosoph- abrogated by excess TAP (Gruter et al+, 1998)+ TAP ila in the export of specific transcripts by studying heterodimerizes with p15, the analog of yeast Mtr2p, mutations in the gene using high resolution in situ hy- and also interacts with RAE1 and the nucleoporins bridization+ We identified a temperature-sensitive (ts) NUP98, NUP214, NUP88, and NUP93 (Katahira et al+, allele of NXF1 in a screen for mutations that disrupts 1999; Bachi et al+, 2000; Suyama et al+, 2000)+ Further- mRNA export and show that the gene is encoded by more, TAP can be crosslinked to Poly(A)ϩ RNA and small bristles (sbr ), a previously uncloned locus with shuttles between the nucleus and cytoplasm by trans- several existing mutations (Lindsley & Zimm, 1992)+ portin-mediated nuclear import and export as a com- We show that Sbr is required for the nuclear export of plex with mRNA (Bear et al+, 1999; Kang & Cullen, all mRNA tested in many different kinds of tissues 1999; Bachi et al+, 2000)+ TAP also interacts with REF throughout development+ Two different ts alleles of sbr proteins, which are recruited to pre-mRNA processing cause a rapid nuclear accumulation of mRNA from all complexes and target mature mRNA for export (Le Hir genes tested in distinct particles, which are distributed et al+, 2000; Stutz et al+, 2000; Zhou et al+, 2000; Rod- throughout the nucleoplasm+ The accumulated intra- rigues et al+, 2001)+ Human TAP and p15 together can nuclear particles do not contain introns, and the phe- functionally complement mex67mtr2 double knockout notype is rapidly reversible by shifting back to the in yeast, and are therefore likely to perform conserved permissive temperature, suggesting that the mRNA is functions in nuclear mRNA export (Katahira et al+, 1999)+ competent for export+ We also show that the pheno- Furthermore, overexpression of TAP/p15 heterodimers type of sbr alleles is not due to an aberrant nuclear stimulates export of RNAs that are otherwise ineffi- ultrastructure or defect in nuclear protein import+ To- ciently exported in cultured cells and in Xenopus oo- gether, our results suggest that sbr mutations prevent cytes (Braun et al+, 2001; Guzik et al+, 2001)+ the docking or translocation of mRNA export intermedi- However, TAP/NXF1 has not been shown directly to ates that are otherwise competent export cargo+ be required for mRNA export in vertebrates+ Further- more, TAP/NXF1 is a member of a family of two in Caenorhabditis elegans, four in Drosophila, and at least RESULTS five in humans (Herold et al+, 2000)+ There are also two p15 orthologs (p15-1/NXT1 p15-2/NXT2) in humans, Identification of a ts mutation in all of which bind multiple NXF proteins (Herold et al+, small bristles that disrupts mRNA 2000)+ It is therefore unclear what combinations of NXF export from blastoderm nuclei and p15 are universally required for mRNA export and whether different members of the family have special- To isolate mutations that disrupt mRNA export in Dro- ized roles in the export of subclasses of mRNAs or at sophila, we screened a collection of X-linked ts lethal different times or tissues in development+ Although mutations (MacDougall et al+, 2001) generated by Helen Mex67p and Mtr2p are likely to form an export receptor Francis-Lang and William Sullivan by isolating EMS- for all mRNAs in yeast (Segref et al+, 1997; Hurtetal+, induced mutations that are male viable at 21 8C but 2000), it is not clear whether TAP/NXF1 and p15-1/ lethal at 29 8C+ We examined 100 ts alleles from the NXT1 have an equivalent universal function in higher collection that were sufficiently viable and fertile at 21 8C eukaryotes+ Indeed, the human protein NXF2 displays to allow the collection of eggs from cages of temporary similar RNA and nucleoporin binding properties to TAP/ homozygous stocks+ Embryos, 1+5–2+5 h old, were col- Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Drosophila small bristles/NXF1 and mRNA export 1783 lected at the permissive temperature (18 8C), shifted for combination against multiply marked X chromosomes+ 1h,and fixed at the restrictive temperature (33 8C)+ In These showed that l(1)ts148 maps to 9F5-10 (data not situ hybridization using fushi tarazu ( ftz) antisense shown)+ Complementation analysis with available al- probes and nonfluorescent histochemical detection were leles in the region showed that five lethal alleles of performed to identify any changes in the intracellular small bristles (sbr 5, sbr 6, sbr 10, sbr 12, and sbr mgln; distribution of the mRNA+ Pair-rule transcripts are nor- Arking, 1975; Zhimulev & Ilyina, 1980; Zhimulev et al+, mally detected by this method as intranuclear foci of 1981; Eeken et al+, 1985; Korey et al+, 2001) fail to fluorescence and localized apical cytoplasmic signal complement the lethality of l(1)ts148 at 29 8C+ Further- (Davis & Ish-Horowicz, 1991)+ Given that ftz mRNA has more, we found that another ts allele, sbr 10 (Arking, a half-life of only 6 min (Edgar et al+, 1986), changes in 1975), the trans-heterozygous combination l(1)ts148/ its distribution could be detected easily after 60 min at sbr 10 and l(1)ts148 over various deficiencies that un- 33 8C+ cover sbr (see Materials and Methods for details) all One mutant, l(1)ts148, showed a dramatic excess of cause accumulation of mRNA in the nucleus at 33 8C, nuclear ftz signal at 33 8C (data not shown)+ We ana- in a similar manner to l(1)ts148 (data not shown)+ We lyzed this phenotype in greater detail using high reso- conclude that the ts alleles are likely to be loss of func- lution fluorescent in situ hybridization (Wilkie & Davis, tion alleles and that Sbr is required for the export of 1998; Wilkie et al+, 1999) with probes against a variety mRNA from the nucleus+ This function of Sbr also ex- of different transcripts (see below) including, runt (run; plains the previous observations that sbr alleles cause Fig+ 1)+ In all cases, we observed that mRNA accumu- a posttranscriptional defect in heat shock protein syn- lates in many discrete particles throughout the nucleus thesis (Evgen’ev & Denisenko, 1990; see Discussion)+ at 33 8C and the amount of cytoplasmic mRNA is re- To identify and clone the wild-type gene mutated in duced+ In all cases throughout this study, l(1)ts148 em- l(1)ts148, we used a candidate gene approach by map- bryos at the 18 8C (Fig+ 1A), yw 67 embryos (the ping 10 ESTs encoding Drosophila orthologs of genes isogenized starting stock for making the ts collection) likely to be required for mRNA export in yeast+ The after 30 min or 60 min at 33 8C showed a normal dis- Drosophila ortholog of human NXF1/TAP and Mex67 tribution of mRNA (data not shown) indistinguishable mapped to region 9F5 and identified full-length cDNA from wild type (Wilkie et al+, 1999; Wilkie & Davis, 2001)+ clones that corresponded well with the length of the To determine the gene mutated in l(1)ts148, we used major sbr transcript seen in Northern blots (Fig+ 2A)+ complementation analysis against deficiencies and re- We found only one change in the entire DmNXF1 coding region in l(1)ts148, corresponding to a nonconser- vative change from valine154 to glutamic acid (V154E; Fig+ 2B,C)+ The crystal structure of human TAP predicts that the mutation V154E in l(1)ts148 would disrupt the folding of the RNA binding domain, since it falls at position 6 within the octameric RNP1 motif (Fig+ 2C)+ The residue at this position points to the interior of the RNP domain and is important for structural stability as it lies against helices in the hydrophobic core of the domain (Liker et al+, 2000)+ We also found a similar change further upstream in the RNA-binding domain of sbr 10 (Fig+ 2B,C)+ Furthermore, a mutation in sbr mgln intro- duces a premature stop codon, truncating the protein by 256 amino acids, thus deleting the entire UBA domain and part of the NTF2 domain (Korey et al+, 2001)+ Sequence alignments of the full-length sbr coding region against Mex67 and NXF1-5 show that sbr is an FIGURE 1. l(1)ts148 mutant embryos accumulate unexported run pair-rule and other mRNAs in embryonic nuclei at 33 8C+ run mRNA ortholog of yeast Mex67 and is the closest ortholog of (red) is visualized by high resolution fluorescent in situ hybridization TAP/NXF1 in Drosophila (data not shown; Herold et al+, and the nuclear envelope with AlexaFluor488 coupled wheat germ 2000)+ We conclude that l(1)ts148 is a ts loss of func- agglutinin (green) (A) 1(1)ts148 male syncytial midinterphase 14 ts148+ blastoderm embryo raised at 18 8C showing the normal distribution tion allele of sbr and have renamed it sbr of run pair-rule mRNA with one bright nascent transcript (arrow) foci in the interior of expressing nuclei (run is on the X chromosome), and cytoplasmic mRNA almost exclusively in the apical cytoplasm (out of the plane of focus)+ Fainter export intermediates are occasionally Sbr is required directly for mRNA export detected (arrowheads)+ B: 1(1)ts148 mutant embryo shifted to 33 8C in the blastoderm embryo for 30 min and fixed at midinterphase 14+ run mRNA that is not exported accumulates at many intranuclear sites (arrowhead) but some localized apical mRNA is also detected (out of the plane of The nuclear accumulation of mRNA in sbr mutants could focus)+ be due to the direct involvement of Sbr in mRNA ex- Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

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FIGURE 2. (Continued from facing page.) l(1)ts148 is an allele of sbr and encodes the Drosophila ortholog of human TAP/NXF1 and yeast Mex67+ A: A northern blot of wild-type embryonic mRNA probed with sbr antisense probe and showing a major 3+2 Kb transcript+ B: A map of the intron/exon structure of the sbr locus and the position of two sbr mutations that map to the RNA-binding domain+ The different domains of the protein are shown in different colors, based on Herold et al+ (2000)+ C: An alignment of the RNA-binding domains of human TAP/NXF1, C. elegans NXF1, and Drosophila Sbr/NXF1, based on Liker et al+ (2000) showing the known structural elements of human NXF1 and the amino acid changes in the RNP domain (also called RRM or RBD domain) present in sbr ts148 and sbr 10+ The positions of the four beta sheets and two alpha helices of the RNP domain are also shown as are the positions of RNP1 and RNP2 consensus motifs of the b sheets+ sbr ts148 and sbr 10 are likely to destabilize the structure of the protein+ port+ Alternatively, the sbr phenotype could be due in- transport+ Such indirect effects have been observed in directly to splicing defects, aberrant nuclear or NPC many cases in yeast and other organisms (reviewed in morphology or to a defect in protein nucleocytoplasmic Doye & Hurt, 1997)+ Indeed, although Mex67p is thought Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Drosophila small bristles/NXF1 and mRNA export 1785 to play a direct role in mRNA export in yeast, various indirect roles have not been ruled out in the case of NXF orthologs in higher eukaryotes+ To distinguish between these possibilities, we first determined how rapidly the mRNA export defect appears and disappears in sbr mutants+ We found that an excess of nuclear run mRNA was first detectable in the nucleus after a 5-min shift to 33 8C (Fig+ 3)+ In a time course of shifts to 33 8C, greater numbers of mRNA particles were visualized in the nucleus after succes- sively longer temperature shifts (Fig+ 3)+ Many of the particles of mRNA that accumulate within the nucleus after 5 or 15 min at 33 8C are similar in size and fluorescence intensity to nuclear mRNA export intermediates and cytoplasmic mRNA localization intermediates detected at 18 8C (Fig+ 3A) and in wild-type embryos (Wilkie & Davis, 2001)+ Previously we showed that such nuclear sites of hybridization are genuine export intermediates, distinct from background fluorescence (Wilkie & Davis, 2001)+ We found that transcription is not disrupted in sbr mutants, as the intensity of nascent transcript foci within the nuclei are not diminished (Fig+ 3B,C)+ However, after a 30-min shift to 33 8C, the additional nuclear mRNA often forms particles that are as bright as nascent transcripts (Fig+ 3D)+ We conclude that in sbr mutants, mRNA export intermediates are unable to exit the nucleus and accumulate throughout the nucleoplasm because of a direct requirement for Sbr+ To test whether the nuclear mRNA particles that accumulate in sbr mutants are likely to be due to a defect in the mRNA export machinery rather than the compe- tence of the mRNA cargo to be exported, we tested how rapidly the phenotype is reversed upon a shift back to 18 8C+ sbr embryos were incubated at 33 8C for 60 min, then shifted back to 18 8C and fixed after a range of different times (Fig+ 4)+ The results show that a shift down of 10 min at 18 8C, which is equivalent to 5 min of developmental time at 25 8C, renders embryos phenotypically indistinguishable from wild-type con- trols (Fig+ 4E), with similar numbers of run mRNA export intermediates (Wilkie & Davis, 2001)+ After a 5-min shift down to 18 8C, about half the embryos show normal numbers of export intermediates (Fig+ 4D)+ The speed of the reversal of the phenotype is too great to allow transcription of new mRNA or synthesis of any new proteins required for mRNA export+ Therefore, it is most likely that the defect in the sbr ts148 mutation is FIGURE 3. Sbr mutant embryos show an mRNA export block very rapidly after a shift to 33 8C+ sbr ts148 embryonic nuclei showing the due to a temperature-dependent disrupt in Sbr protein + : , + nuclear envelope in green and run mRNA in red A At 18 8C nascent function which is reversed upon shifting to 18 8C This transcript foci and some rare export intermediates (arrowhead) are allows the export of presynthesized and processed detected in the nuclei+ B: After a 5-min shift to 33 8C additional nuclear export intermediates and cytoplasmic localization interme- mRNAs that are competent for export but have accu- + : + diates are detected (arrowheads) C After a 15-min shift to 33 8C mulated in the nucleus many more export intermediates and cytoplasmic localization inter- In mammalian cells, TAP/NXF1 binds directly to the mediates are visible (arrowheads) mRNA accumulate in the nuclei+ Simian Type D retrovirus via the CTE sequence, thus D: After a 30-min shift to 33 8C (arrowheads) numerous mRNA export intermediates accumulate in the nuclei, but very few cytoplasmic allowing the export of unspliced viral genomic RNA localization intermediates (arrowhead)+ through the normal mRNA export pathway (Gruter et al+, 1998; Braun et al+, 1999), but it has not been tested Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

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nascent transcript foci (Davis & Ish-Horowicz, 1991; Shermoen & O’Farrell, 1991; Kopczynski & Muska- vitch, 1992) where transcription and processing occur (LeMaire & Thummel, 1990; Bauren & Wieslander, 1994; Custodio et al+, 1999)+ We covisualized intron RNA and mRNA from the h gene by in situ hybridization (Fig+ 5) with two color fluorescent detection (Wilkie & Davis, 1998)+ As expected, in wild-type embryos and control sbr embryos, h intron RNA is only detected at nascent transcript foci (data not shown)+ In sbr mutant embryos at 33 8C, h intron RNA is confined to nascent transcript foci and the h mRNA particles that accumulate throughout the nucleus lack detectable intron sequences (Fig+ 5A–C)+ We conclude that h mRNA is not retained in the nucleus due to inefficient removal of introns at 33 8C and that the sbr mRNA export defect is unlikely to be caused indirectly by a splicing defect (see Discussion)+ To test whether the mRNA particles whose export is blocked in sbr mutants accumulate in any specific nuclear sites, we covisualized the transcripts of two different genes in sbr embryos at 33 8C+ Some pair-rule genes such as run and ftz, or eve and h, are expressed in overlapping stripes at the syncytial blastoderm stage, so are often expressed in the same nuclei+ After a 30- min shift to 33 8C, sbr embryos showed h and eve mRNA accumulation in discrete nuclear particles that do not overlap significantly (Fig+ 5D)+ We conclude that different mRNAs accumulate at independent sites in sbr mutant nuclei supporting the idea that the unexported mRNAs are competent export cargo diffusing within the nucleoplasm+ Many mRNA export mutants isolated in yeast have also been found to disrupt nuclear ultrastructure (see Introduction and Discussion)+ For example, Mex67 shows some abnormal nuclear structures after2hat the restrictive temperature (Segref et al+, 1997)+ We determined whether sbr mutants show such morpho- FIGURE 4. The sbr mutant phenotype is very rapidly reversed upon logical abnormalities that could lead indirectly to the shift back to 18 8C+ A–E: Nuclear envelope (green) run mRNA (red) mRNA export defect using transmission electron micros- in sbr mutant embryos shifted to 33 8C for 60 min and then shifted + back down to 18 8C for (A) 0 min, (B) 1 min, (C) 2 min, (D) 5 min, or copy on wild-type and sbr embryos at 18 8C and 33 8C (E)10min+The phenotype is almost completely reversed after 10 min We found no detectable changes in ultrastructure of sbr at 18 8C (equivalent to 5 min at 25 8C)+ embryos shifted to 33 8C for 30 min+ NPCs were visible around the entire nucleus, were not found in clusters or herniated, and their gross structure was normal (Fig+ 6)+ directly whether the role of TAP/NXF1 in host mRNA As in wild type, sbr nuclei contain a single unfrag- export is in someway also related to splicing+ Further- mented nucleolus, visualized as an electron-dense re- more, the presence of splicing signals and introns is gion of the nucleoplasm on the apical side of the nucleus known to prevent the nuclear export of pre-mRNA (Le- where rDNA genes are positioned (Fig+ 6)+ We con- grain & Rosbash, 1989) and mutations in splicing ma- clude that Sbr is required directly for the nuclear export chinery such as prp22 in yeast inhibit pre-mRNA splicing, of mRNA, rather than being involved in maintaining the thus causing an mRNA export defect indirectly (Lee & structural integrity of NPCs or any kind of other nuclear Silver, 1997)+ We therefore tested whether splicing was structure that could be required for mRNA export+ disrupted in sbr mutants and found that the nuclear To test whether sbr also functions in other nuclear mRNA particles that accumulate in sbr mutations lack transport processes, we investigated nuclear protein introns+ In wild-type embryos, in situ hybridization using import in sbr mutants+ We used a rapid and sensitive intron probes gives a signal that is restricted only to assay for nuclear protein import that we developed pre- Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Drosophila small bristles/NXF1 and mRNA export 1787

FIGURE 6. sbr ts148 does not affect nuclear ultrastructure+ Thin section transmission electron micrographs+ A: Wild-type embryo showing a dense apical single nucleolus and even distribution of NPCs on all sides of the nucleus+ B: sbr mutant after 30 min at 33 8C showing a similar ultrastructure to wild type+ C: High magnification glancing section through an sbr mutant embryo showing that NPC gross structure is similar to wild type (data not shown)+

viously (Kumar et al+, 2001)+ The assay relies on the use of a small fusion between GFP and a nuclear localization signal (nls) leading to the accumulation of GFP in the nucleus (Davis et al+, 1995)+ However, the fusion protein diffuses continuously out of the nucleus, so that disruption of protein import by Ran mutants or competition with an excess of nlsBSA import substrate leads to a rapid breakdown of its nuclear accumulation (Kumar et al+, 2001)+ We crossed nlsGFP chromosome FIGURE 5. sbr ts148 does not disrupt splicing, and unexported mRNA into the sbr ts148 homozygous mutant background and from different genes accumulates at independent intranuclear sites+ In A and B, the nuclear envelope is shown in green and mRNA is in imaged GFP fluorescence in living embryos using a red+ In C and D, the nuclear envelope is shown in blue and RNA in temperature-controlled chamber+ We found that nlsGFP green and red+ A: sbr mutant embryo showing h mRNA accumulation nuclear import was not disrupted in interphase 14 blasto- in the nucleus at 33 8C+ B: sbr mutant embryo showing h intron localizing only to one intranuclear site in a male embryo at 33 8C+ derm embryos even after a 30-min shift to 33 8C (data C: A cross section through sbr mutant embryos in which h intron not shown)+ We conclude that sbr is not involved in (green) and h mRNA (red) sequences are covisualized and show nls-mediated nuclear protein import and its phenotype that h intron localizes to two nascent transcript foci in each nucleus+ D: sbr mutant embryo showing eve mRNA in green and ftz mRNA in is not caused indirectly by a general block in nuclear red+ The two mRNAs do not colocalize within the nucleus+ trafficking+ Taking all these results together, we conclude that Sbr is a factor required specifically and directly for mRNA export in the blastoderm embryo+ Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

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Sbr is required for nuclear export of many different mRNAs and tissues, throughout development

To test whether sbr is essential in all tissues and stages of development, we studied the effects of the sbr ts148 mutation on different stages of development at 33 8C+ The hatch rates of sbr and yw 67 flies were measured at different temperatures+ We found that sbr embryos do not hatch at all at 23 8C and higher temperatures, but show a normal hatch rate of approximately 90% at lower temperatures+ sbr embryos shifted to 33 8C were fixed and imaged, and were found to die at various stages of development consistent with arrest shortly after the shift to 33 8C+ No specific defects in the pattern of the cuticle were observed in sbr embryos+ To test the effect of sbr at other stages of the Dro- sophila life cycle, larvae, pupae, and adult flies were shifted to 29 8C+ We found that sbr larvae fail to pupate, die, and became necrotic at 29 8C+ Furthermore, no adult flies eclose from sbr pupae shifted to 29 8C+ Male sbr flies die after approximately 3 days at 29 8C, whereas female flies survive for a week at 29 8C but lay fewer and fewer small eggs that do not develop or hatch (data not shown)+ Consistent with this result, many egg chambers from sbr females shifted to 29 8C for 3 days show a severe defect in oogenesis after stage 7, when + + the egg chambers appear to enter apoptosis (Fig 7A) FIGURE 7. Sbr is essential throughout development and required Furthermore, previous work on sbr mutations has shown for the nuclear export of all tested mRNAs in many embryonic and that sbr 10 (also called ts403) is cell lethal and that most larval tissues+ A: sbr mutant egg chambers from mothers shifted to , ; , 33 8C for 3 days stained with DAPI to highlight the nuclei and phalloidin- sbr alleles are lethal (Arking 1975 Lindsley & Zimm rhodamine to visualize actin+ Early oogenesis proceeds relatively 1992)+ We conclude that sbr is an essential gene at all normally, but the egg chambers degenerate after stage 7+ B: sbr stages of development+ mutant embryos showing nuclear accumulation of wg mRNA (red) in epithelial nuclei+ The nuclear envelope is shown in green+ To determine whether the lethality we observe in sbr mutants is due to an mRNA export defect, we studied the distribution of many different transcripts in sbr mutants+ We first performed in situ hybridization with probes is the ubiquitous mRNA export factor required in all against several classes of mRNA in the blastoderm tissues and stages of development and is likely to act embryo+ In all the cases tested, mRNA accumulated in as a receptor for mRNA export+ the nuclei of sbr mutant embryos at 33 8C+ Transcripts tested include the gap genes hunchback and Kruppel, DISCUSSION the pair-rule genes ftz, hairy, run, even-skipped, and paired, the segment polarity genes wingless and en- In this article, we show that sbr, the Drosophila ortholog grailed, the homeotic gene Ultrabithorax, the cell cycle of NXF1, is required directly and specifically for the regulator string, and the neurogenic gene atonal (Fig+ 7; export of many different kinds of mRNAs in a variety data not shown)+ We also studied the effects of tem- of tissues and times in development+ Strong loss of perature shifts at various different times and tissues function ts alleles of sbr that map to the RNA-binding during development+ In all cases, we found that sbr domain cause the accumulation of mRNA in the nu- mutations cause the abnormal accumulation of mRNA cleoplasm, mostly away from the nuclear periphery, in in nuclei in a manner similar to that observed in em- particles of RNA that increase in number and intensity bryos+ For example, we studied wg in the embryonic with time after the shift to 33 8C+ We show that sbr is epidermis and mesoderm, atonal in embryonic and likely to play a direct role in mRNA export, rather than imaginal discs peripheral nervous system cells (Fig+ 7C), its mutant phenotype being an indirect consequence of and hairy in tracheal pits (data not shown)+ a splicing defect, altered nuclear ultrastructure, or a Considering all our results in the context of what is block in nuclear protein import+ We conclude that Sbr/ known about Mex67p and NXF1/TAP, we conclude that NXF1 is the major general NXF export factor for all sbr is the NXF family member in higher eukaryotes that mRNAs throughout development+ Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Drosophila small bristles/NXF1 and mRNA export 1789

NXF1 is a general mRNA export factor ificity still remain unanswered in C. elegans and in other in all eukaryotes higher eukaryotes+ Moreover, in Drosophila and humans, there are many more NXF family members (four Despite the Sbr gene not being previously cloned, a and five, respectively) and it is not clear which is the number of different sbr mutations were described and closest to CeNXF1, as the multiplication of NXF genes known to be mainly lethal or semiviable alleles+ The appears to have occurred independently in different name of the mutations originates from the fact that a evolutionary lineages (Herold et al+, 2000)+ few viable and semiviable alleles have missing or small Although the work on human NXF1/TAP, the closest scutellar bristles (Zhimulev & Ilyina, 1980; Lindsley & ortholog to sbr in humans, suggests strongly that the Zimm, 1992)+ We interpret this phenotype as indicat- gene is involved in mRNA export, it is circumstantial in ing that bristle development is particularly sensitive the absence of biochemical depletion experiments in to a reduction in the efficiency of mRNA export (Korey combination with in vitro mRNA export assays or the et al+, 2001)+ This interpretation is consistent with the ability to follow the fate of individual transcripts in mu- fact that bristle defects caused by nonspecific effects tants+ Our experiments in Drosophila are complemen- are extremely common in flies (Lindsley & Zimm, 1992)+ tary to the existing data on NXF1/TAP and test the role sbr 10 was previously reported to have a posttranscrip- of sbr directly in the export of individual transcripts+ We tional defect in heat shock protein synthesis at the re- have also addressed directly the question of tissue and strictive temperature, and to cause prolonged synthesis developmental specificity in vivo and show a require- of Hsp proteins after heat shock, compared to wild type ment in all tissues and developmental times tested+ (Evgen’ev & Denisenko, 1990; Mamon & Kutskova, Moreover, the excellent cytology of the Drosophila em- 1993)+ This is likely to result from nuclear accumulation bryo has allowed us to study the detailed distribution of transcripts encoding heat shock proteins at the re- and kinetics of the mRNA export block induced by sbr strictive temperature, which are later exported to the mutations in a way that has not been possible in yeast cytoplasm and translated after shift down to the per- due to the small size of the cell+ missive temperature+ A similar effect has been shown to occur in a ts allele of the yeast MEX67 gene (Hurt Are NXFs true receptors for mRNA export? et al+, 2000), and is consistent with the finding that nuclear mRNA accumulation in sbr ts148 is rapidly re- For a factor to act as a receptor, it needs to fulfill a versible by shifting back to 18 8C+ Furthermore, hsp70 number of criteria (see below), but first and foremost, it and hsp83 transcripts have been found to accumulate must be shown to play a direct, rather than indirect role in the nucleus of Drosophila SL2 cells after depletion of in the transport process+ Although many yeast mutants NXF1/Sbr levels by dsRNAi (Herold et al+, 2001)+ have been identified in screens for mRNA export de- Our results are consistent with previous studies in S. fects, a number of these have turned out to show pleo- cerevisiae showing that Mex67p, the only member of tropic effects+ Indeed, in several cases, the mRNA export the NXF family of export factors in yeast, is essential block is likely to be a consequence of defects in splic- for poly(A)ϩ mRNA export (Segref et al+, 1997) and the ing or aberrant nuclear pore morphology+ For example, export of specific polymerase II transcripts such as Ash1 a conditional allele of the nucleoporins rat7/nup159 and (Hurt et al+, 2000)+ Like Sbr, Mex67p is not required for rat2/nup120 causes both a rapid block in mRNA export nuclear protein import+ Surprisingly, the only NXF fam- and reversible clustering of NPCs (Gorsch et al+, 1995; ily member in S. pombe, mex67 ϩ, is not essential for Heath et al+, 1995)+ Unspliced mRNAs are unable to be growth or viability, but is synthetic lethal with rae1 be- exported and mRNAs that have not been through the cause of an mRNA export defect (see Introduction)+ splicing pathway are inefficiently exported+ Further- These results indicate that S. pombe Mex67 protein more, it is likely that some mRNA export factors be- has a contributory, but nevertheless important role in come associated with the mRNA during splicing+ We mRNA export and may indicate that mRNA export re- show that in sbr mutants, h mRNA that is not exported quires many factors and that their relative contributions lacks a h intron+ Although we cannot exclude the pos- vary between different organisms+ sibility that a more subtle defect in splicing is present, Our data are also in agreement with a considerable it is most likely that h and other mRNAs that are trapped volume of evidence in other higher eukaryotes sug- in sbr mutant nuclei are competent for export, and the gesting that NXF1 probably functions in the export of phenotype is due to a defect in the export machinery cellular mRNAs+ Prior to our study, the most direct evi- itself+ This interpretation is strengthened by the fact dence was in C. elegans, which has two NXF orthologs: that sbr mutants do not show aberrant NPC morphol- CeNXF1 and CeNXF2+ RNAi experiments have shown ogy or nuclear ultrastructure and show normal nuclear that CeNXF1 is required for poly(A)ϩ mRNA export protein import+ whereas CeNXF2 is not (Tan et al+, 2000)+ However, It is interesting to ask whether TAP together with p15 the distribution of specific mRNAs was not tested and act like a true mRNA export receptor+ The strict defini- the questions of tissue specificity or developmental spec- tion of a receptor is a molecule that is required for Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

1790 G.S. Wilkie et al. transport, binds the cargo (directly or indirectly), shut- previously described (Wilkie & Davis, 1998, 2001; Wilkie et al+, tles in and out of the nucleus, and associates with NPCs+ 1999), except all solutions including fix were preheated to Yeast Mex67p fulfills all these tests when in the pres- 33 8C+ For the initial screen, histochemical in situ hybridiza- ence of Mtr2p, thus fitting the narrow definition of a tion was performed with alkaline phosphatase histochemical , + receptor for mRNA export (Conti & Izaurralde, 2001)+ detection (Tautz & Pfeifle 1989) For high resolution detec- , Although human TAP/NXF1 and p15 also fulfill most of tion of mRNA in situ hybridization with fluorescent TSA detection and covisualization of the nuclear envelope was the definitions of mRNA export receptors, they have performed as described previously (Wilkie & Davis, 1998, not been shown directly to be essential for cellular 2001; Wilkie et al+, 1999)+ Multichannel three-dimensional im- + / mRNA export Our data shows that Sbr NXF1 is es- age stacks were acquired and deconvolved as previously sential for mRNA export in Drosophila and, by implica- described (Davis, 2000)+ Ovaries were prepared and stained tion, in humans+ Therefore, it seems very likely that with rhodamine phalloidin (Molecular Probes) to detect f-actin+ human and Drosophila NXF1 act as true receptors for mRNA export+ If NXF1 is indeed a receptor for mRNA export, this Thin section transmission does not exclude additional roles for other factors, some electron microscopy + of which could be receptors in their own right It is likely Dechorionated embryos were fixed in a two-phase mixture of that there are many essential factors that are required freshly prepared 2% paraformaldehyde and 0+5% gluteralde- in vivo to function synergistically to export mRNA+ These hyde in 0+1 M phosphate buffer, pH 7+5, under heptane for could include additional NXFs, but it is equally possible 1h+The vitelline membrane was removed by hand using a that Drosophila NXF2-4 have specialized roles in spe- needle, and embryos were then postfixed for 3 h+ Embryos cific tissues or times in development or function in other were then fixed in Osmium Tetroxide, dehydrated, and em- aspects of mRNA metabolism and transport+ Although bedded in Spurrs’ resin+ Ninety-nanometer sections were cut in humans, NXF2-5 share some properties with NXF1 on a Reichart Jung Ultracut microtome, collected on speci- , + (Herold et al+, 2000), recent data suggests that Dro- men grids and stained in uranyl acetate and lead citrate sophila NXF2 does not play a role in mRNA export in Specimens were viewed with a Philips CM120 Biotwin transmission electron microscope at an accelerating voltage of Drosophila tissue culture cells (Herold et al+, 2001)+ 100 kV+ Future in vivo experiments studying the role of other NXF family members in mRNA export in Drosophila embryos are likely to reveal the answers to these Sequence analysis of sbr questions+ The full-length wild-type sbr cDNA sequence was identified by screening a Drosophila embryonic poly(A) selected cDNA + MATERIALS AND METHODS library (BDGP) The size of the cDNA corresponds well to the size of the major sbr transcript detected by northern analysis+ To identify mutations within the DmNXF1 ORF in sbr mutant Drosophila strains strains, total RNA was prepared from batches of 30 wild-type or homozygous mutant sbr ts148 or sbr 10 adult flies at 18 8C, All fly stocks were raised on standard cornmeal-agar medium using an RNeasy mini kit (Qiagen)+ sbr cDNA was synthe- , , , + at 18 8C 21 8C 25 8C or 29 8C depending on the experiment sized from total RNA by RT-PCR using a Titan RT-PCR kit 67g + OregonR and y,Df(1)w were used as the wild-type strains (Roche) with appropriate primers to amplify full-length sbr 5 6 +, sbr (l(1)K4 ) and sbr (l(1)K5 ) (Zhimulev et al 1987) were cDNA+ To eliminate the chances of mistakes arising from + + 10 , kindly sent by I Zhimulev sbr (l(1)ts403) (Arking 1975) PCR errors, PCR products were always sequenced on both 12 +, and sbr (24/45A) (Eeken et al 1985) were kindly sent by strands and from two independent PCR reactions+ Further- + + , , , L Mamon Df(1)v64f Df(1)v-L4 and Df(1)v-L11 (Lefevre more, mutations detected in sbr cDNA were confirmed by ts148 1969) fail to complement sbr at 33 8C and were received sequencing PCR products produced from genomic DNA+ The + , + , , from J Roote L Mamon and the Bloomington Stock center full length cDNA sequence of sbr has been submitted to the + respectively Transgenic Drosophila lines yw; nlsGFPM; European Molecular Biology Laboratory data base (acces- nlsGFPN that express nuclear-localized GFP have been de- sion number AJ251947)+ Identical full-length coding sequences +, + scribed previously (Davis et al 1995) The collection of were later obtained independently by A+ Herold and E+ Izau- , , X-linked ts male lethals which we screened were originally rralde (Herold et al+, 2001)+ generated by Helen Francis Lang and William Sullivan and first described in MacDougall et al+ (2001)+ Northern blotting Fixation of Drosophila tissues and Thirty micrograms of fly total RNA were electrophoresed on mRNA in situ hybridization denaturing agarose gels and transferred to a Hybond N membrane by capillary transfer and crosslinked to the membrane Temperature-sensitive mutant embryos were shifted to 33 8C with UV light+ The membrane was hybridized with a DIG by immersing parafilm-wrapped collection plates in a water labeled RNA probe synthesized from the dmNXF1 EST bath at 33 8C+ Embryos were then dechorionated and fixed as LD11064 (BDGP), detected with an alkaline phosphatase con- Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Drosophila small bristles/NXF1 and mRNA export 1791 jugated anti-DIG antibody (Roche) and visualized by chemi- a delay in the appearance of zygotic fluorescence+ Dev Biol luminescent detection+ 170:726–729+ Davis I, Ish-Horowicz D+ 1991+ Apical localization of pair-rule transcripts requires 39 sequences and limits protein diffusion in the Drosophila blastoderm embryo+ Cell 67:927–940+ ACKNOWLEDGMENTS Doye V, Hurt E+ 1997+ From nucleoporins to nuclear pore complexes+ Curr Op Cell Biol 9:401–411+ , , , + + We thank Andrea Herold and Elisa Izaurralde for sharing Edgar BA Weir MP Schubiger G Kornberg T 1986 Repression and , , turnover pattern of fushi tarazu RNA in the early Drosophila em- unpublished information discussions and comments on the bryo+ Cell 47:747–754+ manuscript+ We are grateful to Hildegard Tekotte, Joe Lewis, Eeken JC, Sobels FH, Hyland V, Schalet AP+ 1985+ Distribution of and David Tollervey for discussions+ We also thank Ludmilla MR-induced sex-linked recessive lethal mutations in Drosophila Mamon, Igor Zhimulev, and John Roote for various sbr mu- melanogaster+ Mutat Res 150:261–275+ , + + tants and deficiencies, and Elena Kiseleva for fly stock safe- Evgen’ev MB Denisenko ON 1990 The effect of ts-mutation on + expression of genes induced by heat shock in Drosophila mela- keeping while in transit We are indebted to John Findley and nogaster+ Genetika, Moscow 26:266–271+ Chris Jeffree for help and advice with electron microscopy+ Gorsch LC, Dockendorff TC, Cole CN+ 1995+ A conditional allele This work was supported by a Wellcome Trust career devel- of the novel repeat-containing yeast nucleoporin Rat7/Nup159 opment fellowship and a Lister Institute senior fellowship to causes both rapid cessation of messenger-RNA export and re- + : + +, + + + versible clustering of nuclear pore complexes J Cell Biol 129 I D an MRC studentship to G S W as well as a National 939–955+ Institutes of Mental Health Predoctoral National Research Gruter P, Tabernero C, vonKobbe C, Schmitt C, Saavedra C, Bachi A, Service Award to C+A+K+ and National Institutes of Health Wilm M, Felber BK, Izaurralde E+ 1998+ TAP, the human homolog (NIH) grants NS35909 and NS40043 to D+V+V+ C+A+K was a of Mex67p, mediates CTE-dependent RNA export from the nu- + + cleus+ Mol Cell 1:649–659+ National Science Foundation predoctoral fellow and D V Vis , , , , , , + Guzik BW Levesque L Prasad S Bor YC Black BE Paschal BM a Leukemia and Lymphoma Society scholar Generation of Rekosh D, Hammarskjold ML+ 2001+ NXT1 (p15) is a crucial cel- the ts collection of alleles was supported by an NIH grant lular cofactor in TAP-dependent export of intron-containing RNA (GM46409) to William Sullivan and a Human Frontiers grant in mammalian cells+ Mol Cell Biol 21:2545–2554+ to H+F+-L+ Heath CV, Copeland CS, Amberg DC, Delpriore V, Snyder M, Cole CN+ 1995+ Nuclear pore complex clustering and nuclear accumulation of poly(A)ϩ RNA associated with mutation of the Saccha- Received August 17, 2001; 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Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development.

G S Wilkie, V Zimyanin, R Kirby, et al.

RNA 2001 7: 1781-1792

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