2932  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
MBoC  |  ARTICLE
Identification and characterizat...
Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2933 
cytoplasm of larval Malpighian tubules and
reduced lev...
2934  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
sometimes in nuclear Cajal bodies
(Narayanan et a...
Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2935 
functions as the cytoplasmic recycling factor
for SPN1...
2936  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
In mammalian cells, Cajal bodies are the
first de...
Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2937 
Moleskin-null mutants are larval lethals
(Lorenzen et ...
2938  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
in mammals. Previous results from our lab
show th...
Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2939 
protein in vertebrates, definitive identifica-
tion of...
2940  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
Recombinant protein expression and S2 cell transf...
Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2941 
RNAi
dSNUP dsRNAs were transcribed in vitro from PCR p...
2942  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell
complex with snurportin1 and importin beta. Hum M...
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Natalizio_MBOC_2013

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  • 1. 2932  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell MBoC  |  ARTICLE Identification and characterization of Drosophila Snurportin reveals a role for the import receptor Moleskin/importin-7 in snRNP biogenesis Amanda Hicks Natalizioa and A. Gregory Materaa,b,c,d,e a Curriculum in Genetics and Molecular Biology, Departments of b Biology and c Genetics, d Program in Molecular Biology and Biotechnology, and e Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 ABSTRACT  Nuclear import is an essential step in small nuclear ribonucleoprotein (snRNP) biogenesis. Snurportin1 (SPN1), the import adaptor, binds to trimethylguanosine (TMG) caps on spliceosomal small nuclear RNAs. Previous studies indicated that vertebrate snRNP import requires importin-β, the transport receptor that binds directly to SPN1. We identify CG42303/ snup as the Drosophila orthologue of human snurportin1 (SNUPN). Of interest, the importin- β binding (IBB) domain of SPN1, which is essential for TMG cap–mediated snRNP import in humans, is not well conserved in flies. Consistent with its lack of an IBB domain, we find that Drosophila SNUP (dSNUP) does not interact with Ketel/importin-β. Fruit fly snRNPs also fail to bind Ketel; however, the importin-7 orthologue Moleskin (Msk) physically associates with both dSNUP and spliceosomal snRNPs and localizes to nuclear Cajal bodies. Strikingly, we find that msk-null mutants are depleted of the snRNP assembly factor, survival motor neuron, and the Cajal body marker, coilin. Consistent with a loss of snRNP import function, long-lived msk larvae show an accumulation of TMG cap signal in the cytoplasm. These data indicate that Ketel/importin-β does not play a significant role in Drosophila snRNP import and demon- strate a crucial function for Msk in snRNP biogenesis. INTRODUCTION Biogenesis of uridine-rich small nuclear ribonucleoproteins (U snRNPs) is biphasic, taking place in two distinct cellular subcom- partments (reviewed in Matera et  al., 2007). Small nuclear RNAs (snRNAs) of the Sm class are transcribed by a specialized form of RNA polymerase II (Hernandez and Weiner, 1986) and then ex- ported to the cytoplasm for assembly into pre-snRNPs by the export adaptor, PHAX (Ohno et al., 2000). Once in the cytoplasm, the sur- vival motor neuron (SMN) complex mediates the assembly of the Sm core RNP by loading seven Sm proteins onto the snRNA (Meister et al., 2002; Pellizzoni et al., 2002). After Sm core assembly, the 5′-end methylguanosine cap struc- ture of the snRNA is hypermethylated to form a trimethylguanosine (TMG) cap by the RNA methyltransferase (Tgs1; Mouaikel et  al., 2002), and this modification is believed to be a signal for nuclear import (Mattaj and De Robertis, 1985; Hamm et al., 1990; Fischer et al., 1993; Palacios et al., 1997). The partially assembled snRNPs are then transported back into the nucleus via the import adaptor, snurportin1 (SPN1) and the import receptor, importin-β (Impβ; Palacios et al., 1997; Huber et al., 1998). SPN1 contains two copla- nar β-sheets linked by two crossing β-strands (Strasser et al., 2005) that selectively bind the TMG cap. Once in the nucleus, snRNPs undergo additional maturation steps within the nucleoplasm and/or in Cajal bodies (Jády et al., 2003). RNP import is a crucial step in the biogenesis of snRNPs, as these factors cannot participate in active splicing without proper import into the nucleus. Monitoring Editor Karsten Weis University of California, Berkeley Received: Mar 1, 2013 Revised: Jul 15, 2013 Accepted: Jul 15, 2013 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E13-03-0118) on July 24, 2013. Address correspondence to: A. Gregory Matera (matera@unc.edu). © 2013 Natalizio and Matera. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is avail- able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB® ,” “The American Society for Cell Biology® ,” and “Molecular Biology of the Cell® ” are registered trademarks of The American Society of Cell Biology. Abbreviations used: DAPI, 4’,6-diamidino-2-phenylindole; dSmB, Drosophila Smith B antigen; dSMN, Drosophila survival motor neuron; dSNUP, Drosophila snurportin; dsRNA, double-stranded RNA; GFP, green fluorescent protein; IBB, Importin-β binding domain; Imp7, Importin-7; Impβ, Importin-β; IP, immunopre- cipitation; Msk, Moleskin; PHAX, phosphorylated adaptor for snRNA export; RNAi, RNA interference; RNP, ribonucleoprotein; RT-PCR, reverse transcription- PCR; SMN, survival motor neuron; snRNP, small nuclear RNP; SNUPN, human Snurportin; SPN1, Snurportin1; TMG, 2,2,7-trimethylguanosine; U snRNA, uridine- rich small nuclear RNA; VFP, Venus fluorescent protein; WT, wild type; Xpo1/ Crm1, Exportin1.
  • 2. Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2933  cytoplasm of larval Malpighian tubules and reduced levels of snRNP biogenesis mark- ers coilin and dSMN. These results demon- strate a novel and conserved interaction between Snurportin and Msk/Imp7. We discuss implications for studies of verte- brate nuclear import. RESULTS Identification and characterization of Drosophila Snurportin Bioinformatic analysis of the Drosophila ge- nome predicts that the fruit fly orthologue of human Snurportin1 maps to the computed gene locus CG42303/CG42304, near band position 62E on chromosome 3L. The current FlyBase gene model predicts the existence of a dicistronic transcript with two nonoverlapping open reading frames (ORFs) present within CG42303. Reverse transcription (RT)-PCR and 5′-RACE data from the modEncode project (Sue Celniker lab; www.modencode.org/celniker/) support the existence of two transcription start sites (Figure 1A), one for each ORF. The protein predicted by CG42303 is encoded by a single exon and is 35% iden- tical to SPN1. The CG42304 protein product is also highly similar (37% identity) to the human terminal deoxynucleotidyltransferase interacting factor 1 (TdIF1 or DNTTIP1), which binds and negatively regulatestheactivityofterminaldeoxynucleotidyltransferase(Kubota et al., 2007). This same genetic architecture exists in all other se- quenced Drosophilid genomes but is not conserved in Anopheles gambiae, Apis mellifera, or Caenorhabditis elegans, as SPN1 and DNTTIP1 homologues are unlinked in these organisms. These data suggest that the two genes became linked sometime after the Drosophila radiation from other Diptera such as Anopheles or Apis. Alignment of CG42303 with human SPN1 reveals extensive simi- larity throughout the length of the two sequences, especially within the TMG cap–binding domain (Huber et al., 1998; Ospina et al., 2005; Strasser et al., 2005; and data not shown). Perhaps the most striking feature is that critical residues known to interact with Impβ in the N-terminal region of human SPN1 are missing from the putative fruit fly protein (Figure 1B; Ospina et al., 2005). Human SPN1 en- codes a 360–amino acid (aa) protein of 43-kDa molecular weight; the SPN1-like ORF in CG42303 is predicted to generate a protein of 351 aa and 42 kDa. Using bacterially expressed protein targeting the upstream ORF in CG42303, we generated two polyclonal antibodies (one in rabbit, one in guinea pig) and tested them by Western blotting. As shown in Figure 2A (lane 1), the rabbit antise- rum recognizes a prominent 42-kDa band, along with three other minor polypeptides. The guinea pig antiserum does not recognize endogenous snurportin in Western blots but does detect recombi- nant and exogenously expressed fly snurportin; it also works in other assays (see later discussion). Previous gene models suggested different architectural scenar- ios for CG42303 and CG42304. One model posited the existence of two completely separate transcripts (CG1247, CG1248), whereas another (CG32297) suggested that there is a single mRNA that generates a fused ORF encoding a predicted protein of ∼100 kDa. To examine the specificity of our antibody and test the various gene models, we designed double-stranded RNAs (dsRNAs) tar- geted against putative exons 1 and 3 of CG42303 (Figure 1A). RNA U snRNPs do not contain a classical nuclear localization signal. Instead, U snRNP import depends on two noncanonical signals: the TMG cap and the Sm core (Fischer et  al., 1993; Marshallsay and Lührmann, 1994). SPN1 is the import adaptor for the TMG cap pathway (Huber et al., 1998), whereas the SMN complex (or some component thereof) is believed to function as the import adaptor for the Sm core pathway (Narayanan et al., 2004). Thus bipartite import signals are believed to ensure that only functional RNPs are im- ported into the nucleus. U snRNP import is complicated by the fact that individual U snRNPs have distinct import requirements. Although the TMG cap is required for U1 and U2 snRNP import in frog oocytes, it is not required in somatic cells or for U4 and U5 snRNPs in oocytes (Fischer et al., 1991, 1993; Wersig et al., 1992). The observed TMG cap dependence of snRNP import is cell-type specific rather than species specific (Fischer et al., 1994). In digitonin-permeabilized human cells, recombinant SPN and Impβ are necessary and suffi- cient for U1 snRNP import (Huber et al., 2002). Moreover, an SPN mutant that is incapable of binding to Impβ does not interfere with U1 import via the Sm core–dependent pathway (Ospina et  al., 2005). These observations show that the two import pathways are redundant in vitro, but they fail to elucidate the need for two inde- pendent snRNP import pathways. An in vivo model system of snRNP import is therefore needed to fully dissect the complex na- ture of this pathway. As a first step toward developing an in vivo model system of snRNP nuclear import, we identified and characterized Drosophila Snurportin (dSNUP). We found that, like its human counterpart, dSNUP binds to snRNAs and to dSmB and dSMN in an RNA- dependent manner. Surprisingly, dSNUP lacks an obvious IBB do- main and fails to bind to Impβ in vivo and in vitro. Furthermore, fruit fly Impβ does not interact with snRNAs. We also identified Moleskin (Msk), the Drosophila orthologue of the vertebrate trans- port factor importin-7 (Imp7), as the putative snRNP import recep- tor. Msk localizes to Cajal bodies and physically interacts with snRNPs. In addition, we discovered that Msk-null mutant larvae display a significant accumulation of TMG-capped RNAs in the FIGURE 1:  CG42303 is the Drosophila Snurportin orthologue. (A) Cartoon of bicistronic transcripts predicted from dSNUP/DNTTIP1 locus. Translated regions are shown in black and untranslated regions in gray. Black bars indicate regions targeted by dsRNAs or dSPN antibodies. (B) Alignment of N-termini SPN orthologues. The IBB of hSPN is defined as amino acid residues 26–65, based on similarity with the IBB of importin-α (Huber et al., 1998). Homo sapiens, Xenopus laevis, C. elegans, and D. melanogaster SPN proteins are aligned, with identities in dark gray and similarities in light gray. Asterisk indicates human residue R27, which abolishes importin-β binding when mutated (Ospina et al., 2005).
  • 3. 2934  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell sometimes in nuclear Cajal bodies (Narayanan et al., 2002; Ospina et al., 2005). Using the UAS-Gal4 system (Brand and Per- rimon, 1993), we expressed Venus fluores- cent protein (VFP)–tagged dSNUP in trans- genic flies and analyzed its localization by fluorescence microscopy. Using a variety of Gal4 drivers, we find that VFP-dSNUP local- izes to the nucleus and the cytoplasm, with a pronounced accumulation at the nuclear periphery (Figure 3A). Of note, VFP-dSNUP localizes to snRNP-rich structures that costain with anti-dSmB in the oocyte germi- nal vesicle (Figure 3B) and in the nurse cell cytoplasm, where it accumulates in U bodies (Liu and Gall, 2007) identified by anti-dSMN (Figure 3C). In addition, dSNUP enrichment in U bodies was also confirmed in the follicle cell cytoplasm of egg chambers, visualized by anti-dSmB (Figure 3D). In larval Mal- pighian tubules, VFP-dSNUP frequently lo- calizes to Cajal bodies (Figure 3A). This lo- calization pattern is similar to that of human SPN mutants that contain deletions or sub- stitutions in the IBB domain (Narayanan et al., 2002; Ospina et al., 2005). We there- fore decided to examine the interaction be- tween dSNUP and Ketel/Impβ. Drosophila snRNP import is importin- β independent Studies in vertebrates show that SPN1 inter- acts with Impβ and that this interaction is mediated via the IBB domain (Huber et al., 1998, 2002; Bhardwaj and Cingolani, 2010). The bipartite IBB of SPN1 is contained within residues 1–65 (Mitrousis et al., 2008), and crystal structures reveal that residues 1–16 also contain a nuclear export signal recognized by the export receptor, Xpo1/Crm1 (Dong et al., 2009; Monecke et al., 2009). Sequence analysis indicates that dSNUP lacks important residues in the IBB (Figure 1B; Huber et al., 2002; Ospina et al., 2005; Mitrousis et al., 2008), suggesting that it might not bind to Impβ. Specifically, a highly conserved arginine residue, mutation of which disrupts the interaction of SPN1 with Impβ (Ospina et al., 2005), is not conserved in dSNUP (Figure 1B, asterisk). In the absence of an IBB, dSNUP could potentially interact with Ketel/Impβ indirectly through the Sm core (Fischer et al., 1993). Human SPN1 also forms a preimport snRNP complex with SMN (Narayanan et  al., 2002). To enrich for import competent assem- blies, we used cytoplasmic extracts to carry out immunoprecipita- tion and pull-down assays. As a positive control for coimmunopre- cipitation, we show that, like its human counterpart, dSNUP forms a complex with dSMN (Figure 4A). Consistent with its lack of an ap- parent IBB domain, however, dSNUP fails to coimmunoprecipitate Ketel/Impβ (Figure 4A). We also found that Ketel is capable of interacting with an IBB domain by transfecting S2 cells with various Flag-tagged constructs and coexpressing them with green fluorescent protein (GFP)–Ketel. As shown in Figure 4B, Flag-tagged human SPN1 (Flag-hSPN) or the human SPN1 IBB domain fused to the TMG cap–binding domain of dSNUP (Flag-hIBB-dSNUP) coimmunoprecipitates GFP-Ketel, whereas the empty Flag vector (negative control) and Flag-dSNUP do not. Finally, we tested whether Ketel interacts with snRNAs. interference (RNAi) analysis in Drosophila Schneider 2 (S2) cells shows that the 42-kDa band is specifically depleted by dsRNAs targeting either exon 1 or exon 3 (Figure 2A). This result supports the prediction that the CG42303 transcript contains a relatively long 3′ flanking region and shows that the 90-kDa band on the Western blot is not a fused SPN1/DNTTIP1-like chimeric protein. We conclude that the exonic organization in the CG42303 gene model is correct. The mRNA encoding the downstream CG42304 protein product is thus likely to originate from an alternative tran- scription start site (Figure 1A). The CG42303 protein product is expressed during all stages of development, most prominently in embryos (Figure 2B). We found that although it does not work well for detection of endogenous dSNUP by Western blotting, guinea pig anti-dSNUP was functional in immunoprecipitation assays, as shown in Figure 2D. Using gluta- thione S-transferase (GST) pull downs and coimmunoprecipitation assays from S2 cell cytoplasmic lysates, we show that CG42303 in- teracts with both RNA and protein components of snRNPs, as well as with the snRNP biogenesis factor, dSMN (Figure 2, C and D). Furthermore, RNase treatment of the S2 lysate abolishes these pro- tein interactions, demonstrating that they are RNA dependent (Figure 2E). These results provide strong evidence that CG42303 is the Drosophila orthologue of human SPN1. To avoid confusion with the abbreviations for the Spinophilin gene (Spn) and the spindle gene family (spn-A, spn-B, etc.) in Drosophila, we decided to desig- nate the CG42303 gene as Snurportin (Snup). Previously, we showed that human SPN1 primarily localizes to the cytoplasm, concentrating around the nuclear periphery and FIGURE 2:  Drosophila Snurportin interacts with snRNPs. (A) dSNUP RNAi. Predicted 42-kDa band recognized by dSNUP rabbit antibody is specifically knocked down by dsRNAs targeting dSNUP exon 1 or 3 in S2 cell culture. (B) Developmental Western blot. dSNUP is expressed at all Drosophila developmental stages. (C) GST IP Northern blot. Bacterially purified GST-dSNUP interacts with U1, U2, and U4 snRNAs from S2 cell cytoplasmic lysate. (D) dSNUP Guinea pig IP. Guinea pig dSNUP antibody coimmunoprecipitates dSMN and dSmB in S2 cell cytoplasmic lysate. (E) RNase dSNUP Guinea pig IP. RNase treatment of cytoplasmic S2 lysate abolishes interaction of dSNUP with dSmB and dSMN.
  • 4. Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2935  functions as the cytoplasmic recycling factor for SPN1 once it deposits its cargo in the nucleus (Paraskeva et al., 1999). The interac- tion between SPN1 and Imp7, however, was believed to be indirect due to the fact that Imp7 (formerly RanBP7) was shown to het- erodimerize with Impβ (Görlich et al., 1997). Imp7 also binds directly to the nuclear pore complex (Görlich et al., 1997), however, and can transport cargoes independently (Jäkel and Görlich, 1998), thus satisfying an impor- tant criterion noted earlier. The Drosophila Imp7 homologue (Mole- skin [Msk]) is 53% identical to the human protein and was identified in a dominant suppressor screen for wing blisters caused by the misexpression of αPS integrin (Baker et al., 2002). All of the alleles that were dis- covered in this suppression screen (msk2 , msk4 , msk5 ) are late embryonic or larval le- thal. It is interesting to note that although msk and ketel are both essential genes, there are cell types in which Ketel expres- sion is very low (FlyBase; Lippai et al., 2000). On the other hand, Msk is ubiquitously ex- pressed (FlyBase), satisfying the other afore- mentioned criterion. Thus it is possible that Msk/Imp7 plays a more direct role in snRNP import than previously imagined. To investigate whether Imp7/Msk forms complexes with snRNP biogenesis markers, we carried out coimmunoprecipitation anal- yses. As shown in Figure 5A, anti-dSNUP coprecipitates Msk; dSMN and Ketel are shown as positive and negative controls, re- spectively. S2 cells transfected with various GFP-tagged constructs also coprecipitated Msk. Figure 5B shows that Msk interacts with GFP-dSNUP, -dSMN, and -dSmB. GFP-Msk also coprecipitated with U1, U2, and U4 snRNAs, as shown in Figure 5C. In addition, RanQ69L (a Ran mutant that is unable to hy- drolyze bound GTP; Bischoff et  al., 1994) disrupts the interaction of Flag-dSNUP with Msk (Figure 5D). This interaction also depends on RNA, as RNase treatment of cytoplasmic lysate abolishes binding of endogenous Msk to either GFP-dSNUP or GFP-dSMN (Figure 5E). These results clearly demonstrate that Msk can physically interact with snRNPs and that Msk interacts with dSNUP in a Ran- and RNA-dependent manner. Moleskin/importin-7 localizes to snRNP-rich structures in the nucleus As a nucleocytoplasmic transport factor, Msk shuttles from the cy- toplasm to the nucleus. As such, previous investigations showed that the subcellular localization of Msk (also known as DIM-7) is dynamic; in certain cells the protein was primarily found in the cy- toplasm, whereas in others it was predominantly nuclear (Lorenzen et al., 2001; James et al., 2007). Given that Msk forms complexes with snRNP components and biogenesis factors, we carried out immunofluorescence analyses in Drosophila larval and adult tis- sues. Msk is primarily cytoplasmic within the egg chambers of the Immunoprecipitation analysis, followed by Northern blotting, showed that, whereas GFP-dSNUP coimmunoprecipitated U1, U2, and U4 snRNAs (Figure 4C), GFP-Ketel failed to do so. Thus neither the RNA nor the protein components of snRNPs interact with Ketel in Drosophila. Taken together with the fact that we were unable to detect Ketel in a complex with dSNUP, these experiments strongly support the interpretation that Ketel does not serve as the snRNP import receptor in Drosophila cells. Moleskin/importin-7 interacts with snRNPs and Snurportin The failure of Ketel/Impβ to associate with dSNUP or snRNAs sug- gests the involvement of another import factor. Because splicing is a cell essential function, we reasoned that potential snRNP import re- ceptors not only must be ubiquitously expressed, but also should be able to function independently of Impβ. Of interest, Paraskeva et al. (1999) originally showed that epitope-tagged human SPN1 copuri- fies with three major proteins: Impβ, the export receptor CRM1, and the transport factor Imp7. The authors went on to show that CRM1 FIGURE 3:  Localization of dSNUP. (A) Immunofluorescence with dSmB (Y12) antibody in Malpighian tubules expressing VFP-dSNUP driven by tubulin-Gal4. dSNUP localizes primarily to the nucleus, with a relatively pronounced staining of the nuclear periphery, and can be found in nuclear foci that are often Cajal bodies (marked by arrows). (B) Immunofluorescence with dSmB (Y12) in egg chambers expressing VFP-dSNUP driven by nanos-Gal4. VFP-dSNUP is enriched in the germinal vesicle (arrow). (C) Immunofluorescence with dSMN antibody in egg chambers expressing VFP-dSNUP driven by nanos Gal4. VFP-dSNUP is enriched in U bodies visualized with dSMN antibody (arrow). (D) Immunofluorescence with dSmB (Y12) and dSNUP Guinea pig antibodies in egg chambers. dSNUP is enriched in U bodies of follicle cells. Image in D was kindly provided by Zhipeng Lu (University of North Carolina, Chapel Hill). Scale bars, 10 μm.
  • 5. 2936  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell In mammalian cells, Cajal bodies are the first detectable sites of nuclear accumula- tion of newly imported snRNPs (Sleeman and Lamond, 1999). Given the interaction data described earlier, we suspected that these Msk-positive nuclear foci were Cajal bodies. Indeed, costaining for Sm proteins and other Cajal body markers demonstrates that the foci correspond to Cajal bodies (Figure 6 and data not shown). Note that the oocyte nucleoplasm typically does not stain uniformly with 4′,6-diamidino-2-phenylin- dole (DAPI); only the karyosome is well stained (Liu et al., 2006a). Within the germi- nal vesicle, Cajal bodies can often be found proximal to the karyosome (Figure 6B), although they can also be distally located (Figure 6A). Owing to their relative promi- nence within larval Malpighian tubule nuclei, Cajal bodies are perhaps best visualized in this tissue (Liu et al., 2006b). In Malpighian tubules (Figure 6C), we found that Msk is primarily nucleoplasmic and accumulates in bright nuclear foci. The bright Msk foci colo- calize with the snRNP core component, dSmB (Figure 6C) or coilin (data not shown). In S2 cell cultures, only a fraction of the cells display Cajal bodies. Whenever we observed the bright nuclear foci that stained with anti-Msk, however, they invariably also stained positive for coilin, the Cajal body marker protein (Figure 6D). These results provide strong support for the notion that Msk is involved in import of Sm-class snRNPs. Moleskin depletion disrupts snRNP biogenesis and import RNA interference (RNAi) analysis in S2 cells using dsRNAs targeting Msk revealed that Cajal bodies were disrupted by Msk deple- tion (data not shown). This finding is con- sistent with previous results showing that Cajal body homeostasis requires ongoing ovary (Figure 6A), but both nurse and follicle cell nuclei remain largely unstained. Msk also shows prominent localization to the nurse cell nuclear periphery and to bright foci within the oocyte germinal vesicle (Figure 6A). FIGURE 4:  Ketel/Impβ does not interact with Drosophila snRNPs. (A) Anti-dSNUP guinea pig IP Western blot. dSNUP guinea pig antibody does not coimmunoprecipitate Ketel from cytoplasmic S2 cell lysate. (B) Flag-conjugated beads IP Western blot. Transfected Flag-tagged proteins hSPN and hIBB-dSNUP, but not dSNUP, coimmunoprecipitate GFP-Ketel in S2 cell cytoplasmic lysate. The amounts of Flag-tagged proteins immunoprecipitated are shown with anti-Flag (bottom). (C) GFP IP Northern blot. Transfected GFP-dSNUP coimmunoprecipitates snRNAs U2, U1, and U4, but GFP-Ketel does not, from S2 cell cytoplasmic lysate. The amounts of GFP-tagged proteins immunoprecipitated are shown with anti-GFP (bottom). FIGURE 5:  Moleskin interacts with Drosophila snRNPs. (A) dSNUP Guinea pig IP Western blot. dSNUP Guinea pig antibody coimmunoprecipitates Msk but not Ketel from S2 cell cytoplasmic lysate. (B) GFP IP Western blot. Msk coimmunoprecipitates with transfected GFP-dSMN, GFP-dSmB, and GFP-dSNUP from S2 cell cytoplasmic lysate. (C) Anti-GFP IP Northern blot. Major U snRNAs U1, U1, and U4 coimmunoprecipitate with GFP-Msk and GFP-dSNUP but not GFP-Ketel. (D) Anti-Flag IP Western blot. Transfected Flag-dSNUP coimmuno­ precipitates Msk in the absence of RanQL. This interaction is disrupted by the addition of RanQL. Nonconjugated protein A beads serves as negative control IP (–). (E) RNase anti-GFP IP Western blot. RNase treatment of cytoplasmic S2 lysate abolishes interaction of transfected GFP-dSNUP and GFP-dSMN with endogenous Msk.
  • 6. Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2937  Moleskin-null mutants are larval lethals (Lorenzen et  al., 2001); a small fraction of mutant larvae survive >10 d, but they do not develop past the second instar. The ex- tended survival of msk mutants suggests that, like Ketel protein (Villányi et al., 2008), Msk protein also has a long half-life. To determine whether there are snRNP- specific phenotypes associated with loss of Msk, we carried out immunofluorescence with anti-TMG cap antibodies. Wild-type, Ketelnull/− , and UAS-msk transgenic rescue animals were used as controls. Moleskin mutants displayed a slight but reproducible cytoplasmic TMG accumulation in the Mal- pighian tubules (Figure 8), suggesting a dis- ruption in snRNP import and/or biogenesis. This accumulation was not simply due to the developmental arrest, as Ketelnull/− mutants do not display this phenotype, and expres- sion of UAS-msk rescues it (Figure 8). In an effort to bypass the Msk dependence of this observed snRNP import defect, we gener- ated transgenic flies expressing VFP-dSNUP or VFP-hIBB-dSNUP from UAS promoters. Because we previously showed that hIBB- dSNUP forms a complex with Ketel (Figure 4B), we hypothesized that its expression might rescue snRNP import in Malpighian tubules. We therefore expressed these transgenes in both wild-type and msk−/− backgrounds. Using either a ubiquitous tu- bulin-Gal4 driver or a gut-specific Mal- pighian tubule driver, we found that expression of VFP-hIBB-dSNUP was domi- nantly lethal in both backgrounds. It is un- likely that the dominant-negative pheno- type of the hIBB-dSNUP construct is due to VFP tagging because expression of the con- trol VFP-dSNUP construct had no such dom- inant effects and was able to rescue dSNUP RNAi (data not shown). The dominant lethal- ity of the hIBB-dSNUP fusion precluded us from testing whether targeting dSNUP to an alternative nuclear import receptor pathway (in this case Ketel) might alleviate the appar- ent block to snRNP import. We therefore conducted immunofluores- cence with anti-dSMN and anti-coilin anti- bodies in control and msk mutant larvae. Confirming the results noted for S2 cells, we found that in the Malpighian tubules of msk mutants, dSMN and coilin staining was dramatically reduced and Cajal bodies were disrupted (Figure 9). Staining for both dSMN and Cajal bodies (anti-coilin) was restored upon expression of (untagged) Msk using a UAS-msk transgene (Figure 9). As shown in Figure 7B, the loss of dSMN is fairly extensive, as it can be detected by West- ern blotting using total larval lysates. Of importance, the expression of UAS-msk transgene partially rescues both Msk and dSMN expres- sion (Figure 7, B and C), as well as development of the organism beyond larval stages (Lorenzen et al., 2001; this work). In addition, coilin and dSMN reduction is detectable by day 1 post egg laying snRNP biogenesis (Shpargel and Matera, 2005; Lemm et  al., 2006). Because U snRNPs are extremely stable complexes, how- ever, with half-lives on the order of 3–5 d (Sauterer et al., 1988), this analysis was not very informative with regard to snRNP pheno- types. We therefore obtained a presumptive msk-null mutant from the Bloomington Drosophila Stock Center (msk−/− ), which contains a piggyback transposon insertion in exon 1. We con- firmed by Western blotting that this allele is indeed a null, demon- strating the absence of Msk protein in homozygous mutant larvae (Figure 7A). FIGURE 6:  Moleskin is enriched in Cajal bodies. (A) Immunofluorescence in egg chambers with Msk antibody. Msk is enriched in the germinal vesicle (arrow). (B) Immunofluorescence in egg chambers with dSmB (Y12) and Msk antibodies. Msk is enriched in the germinal vesicle (arrow). (C) Immunofluorescence in Malpighian tubules with dSmB (Y12) and Msk antibodies. Msk is enriched in Cajal bodies of Malpighian tubules. (D) Immunofluorescence in S2 cells with coilin and Msk antibodies. Msk is enriched in Cajal bodies of S2 cells. Scale bars, 10 μm.
  • 7. 2938  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell in mammals. Previous results from our lab show that SMN can bind directly to Impβ in vitro and that purified SMN complexes are required for SPN1 independent snRNP im- port (Narayanan et  al., 2004). The precise identity of the import adaptor for the Sm- core mediated import pathway, however, is not known. Whether the Impβ-binding site of SMN is masked while the protein is in the SMN import complex is also unknown. Sev- eral possibilities thus exist in vivo: Impβ may bind directly to SMN or indirectly through an unidentified adaptor protein (e.g., Imp7/ Msk), or some combination of both scenar- ios might hold, as they are not mutually exclusive. We envision two models by which Imp7 could function in the nuclear import of snRNPs in vertebrates. In one scenario, Imp7 and Impβ could have partially redundant functions, in which they could each inde- pendently function as import receptors in single snRNP import events (Figure 11A). Alternatively, Imp7 could serve as an import adaptor for Impβ, functioning with it in the same import cycle (Figure 11B). Curiously, we find that an unidentified band of the ap- propriate size copurifies with the SMN com- plex in numerous publications (Baccon et al., 2002; Pellizzoni et al., 2002; Yong et al., 2004; Carissimi et al., 2005, 2006a,b). Thus it is possible that Imp7 is the unidentified Sm core import adaptor (Figure 7D). Thus Msk is required for the stability of dSMN and coilin. SMN plays a crucial role in snRNP biogenesis, and its depletion disrupts Cajal bodies in HeLa cells (Shpargel and Matera, 2005). Therefore the significant reduction of dSMN in Msk mutant larvae could be responsible for the Cajal body and TMG cap phenotypes. To investigate this possibility, we overexpressed Flag-tagged dSMN in the Msk-mutant background (Figure 7E). Overexpression of Flag- dSMN failed to rescue organismal viability, Cajal body disruption, or cytoplasmic TMG cap localization (Figure 10). Therefore Msk func- tion in vivo is not limited to SMN stability. Taken together with the subcellular localization and biochemical interaction analyses de- scribed earlier, these genetic results provide strong evidence linking Msk to a role in snRNP biogenesis. DISCUSSION Vertebrate Imp7 and Impβ form an abundant heterodimeric com- plex (Görlich et  al., 1997). Because Impβ is entirely sufficient for snRNP import in vitro and in Xenopus oocytes (Huber et al., 1998; Palacios et al., 1997), it was assumed that the copurification of Imp7 with SPN1 in HeLa cells was simply an indirect consequence of its interaction with Impβ (Paraskeva et al., 1999). In this study, we show that dSNUP is the Drosophila orthologue of human SPN1 and pro- vide convincing evidence that it fails to bind Ketel/Impβ in vitro and in vivo. Our results strongly favor the interpretation that Drosophila snRNP import uses the import receptor Msk/Imp7 in place of Ketel/ Impβ. Thus the physical interaction between Imp7/Msk and SPN1/ dSNUP is conserved in humans and Drosophila, raising the question of whether Imp7 might play a previously unrecognized role in verte- brate snRNP import. In mammalian cells, Imp7 functions as an import receptor for various protein cargoes, independent of its role as an adaptor for Impβ (Jäkel et al., 1999; Freedman and Yamamoto, 2004). Thus it is possible that Imp7 plays a similar role in the snRNP import pathway FIGURE 7:  Moleskin mutant characterization. (A–C) Western blot of second-instar larvae. (A) msk−/− larvae have significantly reduced Msk protein levels. (B) Ketelnull/− larvae have WT levels of Msk. UAS-msk driven by armadillo-Gal4 in the msk−/− background shows recovery of Msk protein. (C) msk−/− larvae have significantly reduced levels of dSMN, which can be recovered by UAS-msk driven by armadillo-Gal4. (D) Western blot of first-instar larvae. msk−/− larvae have significantly reduced levels of dSMN and coilin by day 1 post egg laying (DPE). (E) Western blot of first-instar larvae. UAS-flag-dSMN driven by armadillo-Gal4 in the msk−/− background shows Flag-dSMN expression. Long exposure (top) and short exposure (middle) with dSMN antibody. FIGURE 8:  Moleskin-null mutant Malpighian tubules display TMG cap cytoplasmic accumulation. Immunofluorescence in second-instar larvae. Long-lived msk−/− larvae show cytoplasmic accumulation of TMG in Malpighian tubules, whereas similar long-lived Ketelnull/– larvae do not. UAS-msk driven by armadillo-Gal4 in the msk−/− background partially rescues cytoplasmic TMG phenotype. Scale bars, 10 μm.
  • 8. Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2939  protein in vertebrates, definitive identifica- tion of which remains a subject of consider- able interest. Traditionally, import receptors have been believed to be bound immediately by RanGTP in the nucleus; subsequently the re- ceptors are recycled back into the cytoplasm. There is evidence, however, that Imp7 may be a bit different from traditional nuclear im- port receptors. Unlike Impβ, Imp7 does not require RanGTP for histone H1 nuclear im- port (Jäkel et al., 1999). The lower affinity for RanGTP is hypothesized to be a potential advantage. Jäkel et  al. (1999) suggested that by delaying the dissociation of Imp7 from H1, Imp7 could accompany the histone to the chromosome for assembly into chro- matin. The same idea could be applied to our surprising finding that Msk/Imp7 local- izes to Cajal bodies in both Drosophila and human cells (Figure 6 and Supplemental Figure S1, respectively). Hence Msk/Imp7 might act in a chaperonin-like manner inside the nucleus, ferrying snRNPs to Cajal bodies for potential interaction with coilin and/or SMN (Liu et al., 2000; Narayanan et al., 2004; Ospina et  al., 2005; Shpargel and Martel, 2005; Tanackovic and Kramer, 2005). Navigating the complex nature of snRNP import mechanisms will require precise molecular dissection of the interactions be- tween snRNPs, their transport receptors, and their downstream effectors. Our finding that loss of msk function leads (directly or indi- rectly) to codepletion of dSMN is particularly significant in this regard. Collectively, our studies provide strong evidence that Ketel/ Impβ is not the TMG cap import receptor in Drosophila and that Msk/Imp7 is required for ongoing snRNP biogenesis. Furthermore, we provide important food for thought re- garding a potential role for Imp7/Msk in mammalian snRNP import. Imp7/Msk may have different binding capacities than Impβ/ Ketel in particular tissues or for individual species of U snRNPs. Additional experiments are needed to clarify these and other impor- tant questions. Understanding the role of FIGURE 9:  Coilin and dSMN are reduced in Moleskin-mutant Malpighian tubules. Immunofluorescence in second-instar larvae. Long lived msk−/− larvae have reduced dSMN and Cajal bodies (anti-coilin) in Malpighian tubules, whereas similar long-lived Ketelnull/– larvae do not. UAS-msk driven by armadillo-Gal4 in the msk−/− background shows recovery of both coilin and dSMN (UAS-msk; msk−/− ). Scale bars, 10 μm. FIGURE 10:  Overexpression of Flag-dSMN does not rescue coilin and dSMN reduction in Moleskin-mutant Malpighian tubules. Immunofluorescence in second-instar larvae. Long-lived msk−/− larvae have reduced dSMN and Cajal bodies (anti-coilin) in Malpighian tubules compared with wild type. UAS-msk driven by armadillo-Gal4 in the msk−/− background shows recovery of both coilin and dSMN (UAS-msk; msk−/− ). UAS-flag-dSMN driven by armadillo-Gal4 in the msk−/− background fails to rescue coilin or dSMN (UAS-flag-dSMN; msk−/− ). Scale bars, 20 μm.
  • 9. 2940  |  A. H. Natalizio and A. G. Matera Molecular Biology of the Cell Recombinant protein expression and S2 cell transfections GST-dSNUP was expressed in BL21-star bacteria (Invitrogen) by 1 mM isopropyl-β-d-thiogalactoside induction for 3 h. Lysate was extracted by sonication and passed over glutathione beads. S2 cells were transfected using Cellfectin as directed (Invitrogen), and cells were harvested 4 d after transfection. Antibodies A rabbit polyclonal anti-dSNUP antibody (dSNUP) was generated (Pacific Immunology, Ramona, CA) using GST-tagged dSNUP. A guinea pig polyclonal anti-dSNUP antibody was generated (Pocono Rabbit Farm and Laboratory, Canadensis, PA) using dSNUP. GST (Santa Cruz Biotechnology, Santa Cruz, CA; anti-mouse; 1:1000), GFP (Roche, Indianapolis, IN; anti-mouse; 1:1000, and Ab- cam, Cambridge, MA; anti-rabbit, 1:1000), dSmB (Y12) anti-sDMA (a gift from J. Steitz, Yale University, New Haven, CT; anti-mouse; 1:3000), dSMN (Praveen et  al., 2012; affinity-purified anti-rabbit; 1:2000), dSNUP (affinity-purified anti-rabbit; 1:3000), Msk (a gift of L. Perkins, Harvard Medical School Boston, MA; anti-rabbit; 1:2000), Ketel (a gift from J. Szabad, Faculty of Medicine, University of Szeged, Szeged, Hungary; anti-rabbit; 1:5000), bellwether (Abcam, Cambridge, MA; anti-mouse, 1:5000), Flag (Sigma-Aldrich, St. Louis, MO; horseradish peroxidase–conjugated anti-Flag; 1:8000), and tubulin (Sigma-Aldrich; anti-rabbit; 1:10,000) antibodies were used for Western blotting. Secondary antibodies used were goat anti- mouse–, anti–guinea pig–, and anti-rabbit–conjugated horseradish peroxidase at 1:5000 (Pierce, Rockford, IL). Msk (a gift of L. Perkins; rabbit; 1:1000), coilin (a gift of J. Gall, Carnegie Institution for Science, Baltimore, MD; guinea pig; 1:1000), dSMN (Praveen et  al., 2012; affinity-purified rabbit; 1:200), dSNUP (guinea pig; 1:200), dSmB (Y12) anti-sDMA (a gift from J. Steitz, Yale University, New Haven, CT; mouse monoclo- nal; 1:200), Imp7 (Sigma-Aldrich; rabbit; 1:250), and hSMN (mouse monoclonal; clone 8, BD Biosciences, San Diego, CA; 1:250) were used for immunofluorescence. GFP (Abcam, Cambridge, MA; rabbit; 1.5 μl), GFP (mouse; Roche; 1.5 μl), and dSNUP (guinea pig; 10 μl) antibodies and Flag-conjugated aga- rose beads (Sigma-Aldrich; 15 μl per immunoprecipitation [IP]) were used for IP in buffer A (10 mM 4-(2-hydroxyethyl)-1-pipera- zineethanesulfonic acid, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol). Immunoprecipitation S2 cell cytoplasmic lysate was prepared by resuspending cells in 5× pellet volume of buffer A. Resuspended cells were incubated on ice for 30 min to allow swelling, mixed 10× with a p200 pipette, and incubated for an additional 10 min on ice before passing through a 27.5-gauge needle 40×. Cells were spun for 1 min at 13,000 rpm in a microfuge, and the cytoplasmic supernatant was treated with pro- tease inhibitor cocktail (Pierce). For RNase experiments, S2 cell cy- toplasmic lysate was divided into equal fractions, which were un- treated or treated with 1 μg of RNase/5 μg lysate for 1 h at 37°C. For RanQ69L experiments, bacterially expressed GST-RanQ69L was added to cytoplasmic lysate. Cytoplasmic fractions were incubated with antibody for 1 h (no antibody added for negative control IP) at 4°C before incubation overnight at 4°C with 15 μl of protein A beads (Pierce). Bound proteins were washed 5× with 1 ml of buffer A. For IP Northern experiments, bound RNA was directly phenol/ chloroform extracted off beads, denatured in formamide loading buffer, run on a 10% polyacrylamide-urea gel (Invitrogen), transferred to a nylon membrane, and probed with 32 P-labeled PCR products corresponding to the D. melanogaster U1, U2, and U4 snRNAs. Imp7/Msk in snRNP biogenesis in both vertebrate and invertebrate systems should elucidate how the two different nuclear import path- ways complement one another. MATERIALS AND METHODS DNA constructs dSNUP, hIBB-dSNUP, dSMN, dSmB, Msk, and Ketel full-length cD- NAs were PCR amplified with appropriate primers flanked by Gate- way recombination sequences (Invitrogen, Carlsbad, CA). These products were recombined initially into pDONR221 (Invitrogen) be- fore entry into GFP-tagged pAGW, Flag-tagged pAFW (Drosophila Genome Research Center, Indiana University, Bloomington, IN), or pBI-UASC-mVenus (a gift from Brian McCabe, Columbia University, New York, NY). FIGURE 11:  Models of Imp7 role in snRNP import. (A) Imp7 and Impβ could function redundantly as autonomous snRNP import receptors. (B) Alternatively, Imp7 could function as an Sm core snRNP import adaptor for Impβ.
  • 10. Volume 24  September 15, 2013 Importin-7 in snRNP import  |  2941  RNAi dSNUP dsRNAs were transcribed in vitro from PCR products flanked with T7 promoters. Drosophila S2 cells were placed in SF-900 media and treated with fresh 14 μg/ml dsRNA each day for 4 d before harvesting. Cytoplasmic extracts were generated 4 d after transfec- tion. A 50-μg portion of cytoplasmic extract was loaded on a poly- acrylamide gel for Western blotting analysis to confirm knockdown. Fly stocks Oregon-R was used as the wild-type strain. A Msk-null line contain- ing a piggyback insertion in intron 1 of Msk (msk−/− ), MskB185 , w1118 ; PBac{5HPw+ }mskB185 /TM3, Sb1 Ser1 , and a line containing msk with a UAS promoter (UAS-msk), w*; P{UAS-msk.L}47M1/CyO, previously characterized (Lorenzen et al., 2001), were obtained from the Bloom- ington Drosophila Stock Center (Bloomington, IN). Armadillo-Gal4 was recombined with msk−/− and crossed to UAS-msk for rescue of previously characterized UAS-flag-dSMN (Chang et al., 2008). Previ- ously characterized Ketelnull/− (Villányi et al., 2008) was a gift from J. Szabad. (The – sign stands for a small deficiency [ketelrx32 ] that removes Ketel and a few of the adjacent loci, whereas the Ketel null [ketelrx13 ] is a complete loss-of-function mutant allele; Erdélyi et al., 1997.) The dSNUP and hIBB-dSNUP transgenic constructs were cloned into pBI-UASC-mVenus (Wang et al., 2012) and sent to BestGene (Chino Hills, CA) for embryo injection using the phiC31 system. Transgenes were integrated at site 86fB (Bischof et al., 2007). Flies bearing a UAS:VFP-Snup transgene were crossed to a variety of Gal4 drivers, including tubulin-Gal4 and nanos-Gal4. The msk−/− flies were recombined with either VFP-dSNUP or VFP-hIBB-dSNUP trans- genic lines and with Gal4 drivers. Timed matings were allowed to proceed for 6 h, and larvae were collected for phenotypic analyses on subsequent days. Immunofluorescence Drosophila tissues and HeLa and S2 cells were fixed at room tem- perature for 10 min in 3.7% paraformaldehyde in phosphate-buff- ered saline (PBS; 135 mM NaCl, 2.5 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.2). Tissues/cells were then permeabilized with 1% Triton 100×, blocked in PBST (PBS with 0.1% Triton 100×) containing 5% NGS (blocking solution) and then washed with PBST. The primary antibody, diluted in PBST, was incubated with the sam- ples overnight at 4°C. After being washed with PBST, the secondary antibody, diluted in blocking solution, was incubated with the sam- ples for 2 h at room temperature. The samples were stained with DAPI, washed with PBST, and mounted in antifade solution (0.233 g of DABCO, 800 μl of water, 200 μl of 1 M Tris-HCl, pH 8.0, 9 ml of glycerol). 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