Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (2001), 7:1781–1792+ Cambridge University Press+ Printed in the USA+ Copyright © 2001 RNA Society+ DOI: 10+1017+S1355838201014121 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 nu- merous 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)1 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)1 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)1 RNA (Tan et al+, 2000)+ How- contrast, mex67 1, 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 1 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)1 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