Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans Qiongmei Ji a , Cheng-Han Huang a,⁎, Jianbin Peng a , Sarwar Hashmi b , Tianzhang Ye a , Ying Chen a a Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021, USA b Developmental Biology, Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021, USA

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

We report here the identification and characterization of STIP, a multi-domain nuclear

Received 27 November 2006

protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan

Revised version received

repeats highly conserved in metazoan species. To analyze their functional role in vivo, we

28 December 2006

cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis

Accepted 3 January 2007

elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was

Available online 10 January 2007

produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types

Keywords:

including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms

STIP

large polymers with a rod-like shape when expressed in mammalian cells. Using deletion

Nuclear localization

mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer

Coiled-coil

assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference

G-patch

arrested development and resulted in morphologic abnormalities around the 16-cell stage

Gene evolution

followed by 100% lethality, suggesting its essential role in worm embryogenesis.

Protein structure

Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila

Embryonic development

and human genes via transgenic expression. Our data provide the first direct evidence that

Cross-species complementation

STIP have a conserved essential nuclear function across metazoans from worms to humans. © 2007 Elsevier Inc. All rights reserved.

Introduction STIP (septin and tuftelin interacting proteins) are G-patch domain proteins that bear an array of six conserved glycines [1–3]. The Drosophila and mouse homologs were found via yeast two-hybrid (Y2H) screens as a partner of septin protein

Peanut [2] and tooth matrix protein tuftelin [3], respectively, but their function and direct physical association remain uncharacterized. Human STIP is a nuclear phosphoprotein in HeLa cells as shown by large-scale proteomic studies [4]. Mouse STIP was initially located in the apical secretory pool of amleoblasts [2], but recently localized to a novel subnuclear

⁎ Corresponding author. Fax: +1 212 570 3251. E-mail address: [email protected] (C.-H. Huang). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.01.003

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structure in close proximity to the SC35 speckles [5]. The origin and species distribution of STIP are obscure, since few homologs have been identified. Genome-wide Y2H data showed that Drosophila and Caenorhabditis elegans STIP interacted with entirely different proteins [6,7], whereas proteomic studies indicated that human STIP were associated with the spliceosome [8–10]. Moreover, little is known about whether STIP are essential for animal survival and are involved in fundamental organismal processes, e.g., embryogenesis and development. We report here the structure, function, and evolution of STIP homologs mainly from metazoan species including four nematodes. We show that metazoan STIP arose during unicellular eukaryotic evolution and have gained novel structural features in addition to the G-patch domain. We then explore C. elegans as model to address whether STIP have a conserved essential function for animal development and viability. We characterize C. elegans STIP (STIP-1) on a biochemical and molecular level, define its spatiotemporal expression, and analyze its functional role in embryonic development. We find that stip-1 is expressed at all developmental stages and in many tissue/cell types, is a nuclear protein capable of forming large rod-shaped polymers, and has intrinsic nuclear localization signals (NLS) for this compartmentalization. We demonstrate that suppression of stip-1 expression by RNA interference (RNAi) resulted in 100% embryonic lethality, suggesting its absolute requirement for embryogenesis in C. elegans. Importantly, we show via cross-species gene complementation that the lethal embryonic phenotype of the worm could be rescued by transgenic expression of either the Drosophila or human stip homolog. These data provide the first direct evidence that STIP have a conserved essential function across metazoans from worms and flies to humans.

Materials and methods Culture of nematodes, Drosophila S2 cells, and mammalian cells Nematode strains were from Caenorhabditis Genetics Center (University of Minnesota) and cultured as described [11]. Drosophila S2 cells were cultured at room temperature in Schneider's Drosophila medium (Invitrogen) supplied with 15% fetal calf serum (FCS). Human embryonic kidney 293T (HEK293T) cells and green monkey kidney COS1 cells (ATCC) were cultured in DMEM supplied with 10% FCS and maintained at 37 °C in a humidified 5% CO2 incubator, as previously described [12].

Cloning and sequencing of metazoan STIP genes Metazoan total RNA samples [13], prepared with TRIzol reagent (Invitrogen), were used for cloning stip genes by RTPCR. The open reading frame (ORF) of C. elegans stip-1 was PCRamplified with two gene-specific primers (GSP): stip1s (sense 5-ATGGAGGACGATGATGGACG-3) and stip1a (antisense 5TCCTTGTGCACCTTGAGCCATTTG-3′). 35 cycles of PCR were run at 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 3 min, using a

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high fidelity DNA polymerase. The product was cloned into vector pCRII™ TOPO and sequenced. The 5′ or 3′ untranslated region (UTR) of stip-1 mRNA was analyzed by rapid amplification of cDNA ends (RACE) from total RNAs. Oligo(dT)17 (5′acgtcatgactcgagtcgacatcgattttttttttttttttt-3′) was used for cDNA synthesis. In 3′ RACE, stip2s (5′-GGACTCCAAGCCACTCCGATT-3′) and adaptor (5′-atgactcgagtcgacatcgatt-3′) were coupled. In 5′ RACE, splice leader 1 (SL1, 5-ggtttaattacccaagtttgag-3) and stip2a (5-CTCCTCTCATCCAGCTACCGAATT-3) were paired. PCR products were cloned into vector pCRII™ TOPO for sequencing.

Database, phylogenetic, and bioinformatics analyses Cloned STIP were used as queries to search for public databases with BLAST [14]. Both cloned and retrieved metazoan STIP were aligned using MUSCLE [15]; their phylogenetic tree was reconstructed using the neighbor-joining method [16] and rooted with homologs from unicellular organisms Chlamydomonas and Dictyostelium. Prediction of coiled-coil regions in STIP was performed as previously described [17,18].

RNA preparation and stage-specific profile of the stip-1 transcript using RT-PCR Total RNA was isolated from cultured Drosophila S2 cells using TRIzol Reagent and used for cloning of the full-length cDNA of Dm-stip (see below). C. elegans stage-specific total RNAs were prepared from a synchronized population of all developmental stages [12,19]. The synchronous population of arrested first-stage larvae (L1) was prepared by treatment of gravid hermaphrodites with sodium hypochlorite and subsequent hatching from the embryos overnight in water. The arrested L1 were then transferred onto nematode growth medium (NGM) agarose plates seeded with E. coli OP50, which allowed the worm to develop through L2, L3, L4, and adult stages during a 40 h period. These synchronous worms were then collected at their specific developmental stages [19]. Singlestrand cDNA was reverse-transcribed from 1 μg of total RNA using ThermoScript RT-PCR kit and random hexamers (Invitrogen). The specific cDNA fragment of stip-1 was then PCR-amplified using two GSP as follows: 5′-GCGGATGCACAGAAAAGAAT-3′ (forward) and 5′-AACAGTTTCAAGAGAATACA-3′ (reverse). Cel-ama-1, which encodes the large subunit of RNA polymerase II and is expressed constitutively, was used as a control for RT-PCR as previously described [19].

Polyclonal antibody production and Western blot analysis To produce an antibody for Cel-STIP-1, the sequence encoding the amino acid (aa) 680–830 was amplified by PCR with stip3s (5′-atcgaagcttGGTGTCGATTACAATGAAGT-3′, HindIII) and stip3a (5′-agctctcgagTCCTTGTGCACCTTGAGCCAT-3′, XhoI). The DNA was digested by HindIII/XhoI and inserted 5′ to 6xHis-tag in pET28b vector (Norvagen). The stip-1::6xHis plasmid was transformed into E. coli BL21 cells and induced with 0.3 mM IPTG (30 °C, 4 h). After sonication and cell lysis, the STIP-1::6xHis fusion protein was passed through a Ni-NTA column (Qiagen). A total of ∼ 300 μg of the purified protein was emulsified with adjuvant and injected into rabbits using the

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standard protocol [20]. Western blot analysis was performed using extracts of HEK293T cells transiently expressing the STIP-1 protein. The blot was probed with the above antibody (1:3000) and stained with HRP-linked donkey anti-rabbit IgG (1:5000) (Amersham). Protein bands were stained with a chemiluminescent kit (Pierce).

Generation and expression of stip-1 reporters The reporters stip-1::lacZ and stip-1::gfp were made as follows. The 2510 bp genomic fragment retaining the 1900 bp promoter to the 5′ portion of exon 3 of stip-1 was PCR-amplified with

stip4′s (5′-acgcgtcgacTGAAGGTGGAAGGTACAA-3′, SalI) and stip4a (5′-cgggatccGTCTCCTCTCATCCAGCTACC-3′, BamHI). Similarly, the 2085 bp fragment retaining the 1475 bp promoter to the 5′ portion of exon 3 was amplified with stip4s (5′acgcgtcgacATGTTGGAAATGTTTCGTGATGGG-3′, SalI) and stip4a above. The products were digested with SalI/BamHI and separately cloned into the GFP reporter pPD95.77. For lacZ fusion, either stip4′s or stip4s, shown above, was coupled with stip5a (5′-cgggatccCCGTCTCCTCTCATCCAGCTAC-3′, BamHI) for PCR, and the products were digested with SalI/BamHI and cloned into the lacZ reporter pPD90.23. Transformation of worms was done by microinjecting the stip-1::gfp or stip-1::lacZ

Fig. 1 – Metazoan STIP identify a novel subset of G-patch domain-containing proteins. (a) Phylogenetic tree of STIP members from nematode and arthropod to vertebrate. The tree was reconstructed using a neighbor-joining method and rooted with low homologues from green alga C. reinhardtii and slime mold D. discoideum. Numerals at nodes are bootstrap proportions. The scale bar denotes 0.1 substitutions per amino acid site. (b) The sequence profile of G-patch domains found in 42 metazoan STIP and two STIP-like proteins from unicellular eukaryotes. The starting and ending amino acid positions of each G-patch are given. Conserved glycine residues are highlighted. The residues conserved at the level of 70, 90, and 100% of STIP and STIP-like homologs are shown on the top. Dashes denote gaps and dots insertions. Note that the whole data set of metazoan STIP and their sequence alignment are presented in detail in the Supplementary data.

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reporter into the distal arm of the gonad of young hermaphrodites [21,22]. Each construct (final concentration 15– 25 μg/ml) was co-injected with the pRF4 plasmid (final concentration 80 μg/ml) having a dominant mutant allele of rol-6 gene [23]. Transgenic worms were identified by their roller phenotype. Lines in which F2 progeny and later generation showed the roller phenotype were maintained and picked for GFP imaging or β-galactosidase staining. GFP images were captured through a fluorescence microscope by mounting live worms on slide in 0.01% sodium azide that inhibits worm movement. β-galactosidase staining was carried out as described [24], and stained worms were observed using nomarski optics. At least three independent transgenic lines were examined for each reporter construct.

Construction of stip-1 vectors and their expression in mammalian cell lines Stip-1 cDNA was cloned in pEGFP-C3 and pEGFP-N3 vectors (Clontech) to produce C- and N-fused GFP plasmids: STIP1–830 (full-length), STIP1–213, STIP1–423, STIP214–422, STIP214–830 and STIP423–830. GSP were added with HindIII (aagctt) or KpnI (ggtacc) site in pair, as follows. STIP1–830: stip1F (forward, 5′acgcaagcttATGGAGGACGATGATGGACG-3′) and stip830R (reverse, 5′-acgtggtaccTCCTTGTGCACCTTGAGCCA-3′); STIP1–213: stip1F and stip213R (5′-acgtggtaccCGCTGCAGATTCTCCAAACT-3′); STIP1–422: stip1F and stip422R (5′-acgtggtaccAACAGTTTCAAGAGAATACA-3′); STIP214–422: stip422R and stip214F (5′-acgcaagcttGATGCACAGAAAAGAAT-3′); STIP214–830: stip214F and stip830R; STIP423–830: stip423F (5′-acgcaagcttGCTATACCAACTGTTCTT-3′) and stip830R. All PCR-amplified regions cloned in expression vectors were verified by DNA sequencing. For transient expression of stip-1 in mammalian cells, each plasmid was transfected using Lipofectamine2000 (Invitrogen) into HEK293T or COS1 cells grown on four-well chamber slides (Lab-Tek). After 24 h cell culture, GFP images were captured on a Nikon PCM 2000 confocal microscope and processed using the image browser software.

Preparation of double-strand RNA and RNAi RNAi was carried out as described [25,26]. The exons 2 and 3 (47–213aa) of stip-1 were amplified with stip47F (5′-taatacgactcactatagggGATGACGATGAACAAGGAA-3′, T7 promoter) and stip213R (5′-atttaggtgacactatagCGCTGCAGATTCTCCAAACT-3′, SP6 promoter). This region was chosen because it shares little homology with the equivalent part of fly and human stip genes. The product was purified and served as a template for in vitro transcription of single-strand RNA (ssRNA) using the T7 or SP6 RNA polymerase supplied in the MEGAscript kit (Ambicon). The ssRNA molecules were purified, and equimolar amounts of them were mixed, heated (68 °C, 10 min), and annealed (37 °C, 30 min) to allow double-strand RNA (dsRNA) formation. RNAi using the soaking protocol was performed on L4 worms, as described [26]. 12–16 worms were picked and incubated in 15 μl of soaking buffer (0.2 M sucrose/0.1× PBS) containing 3 mg/ml dsRNA. Larvae soaked in dsRNA-free buffer (0.2 M sucrose/ 0.1× PBS) were used as a control. After 24 h, soaked larvae were transferred to NGM agarose plates seeded with E. coli

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OP50. The F1 progeny were quantified and examined for embryonic development.

Rescue of stip-1 RNAi phenotypes with fly and human genes The rescue constructs, pDmStipR and pHsStipR, were generated as follows. The 1.9 kb stip promoter was amplified from DNA with primers 5′-TGAAGGTGGAAGGTACAA-3′ (forward) and 5′CTGAAAATTACGAAATTA-3′ (reverse), and cloned in pGEM®TEasy vector to generate the pT-Prom plasmid. The stip-1 3′-UTR (429 bp) was also amplified from DNA but using two pairs of primers to facilitate subsequent cloning steps: Pair 1, 5′atcgtctagaATCTTGTTTAAACTTACGAT-3′ (sense strand with XbaI site), and 5′-atcgcatatgCCGTCAGAAAGAGTAGCTGT-3′ (antisense strand with NdeI site); and Pair 2, 5′-atcgctcgagTCATTTTTAACATATCATATGA-3′ (sense strand with XhoI site), and 5′-atcggagctcCCGTCAGAAAGAGTAGCTGT-3′ (antisense strand with SacI site). Fly STIP full-length cDNA was amplified by RT-PCR from Drosophila S2 total RNA using DmStip1s (SalI, 5′acgtgtcgacATGTCGGACAACGATTACGA-3′) and DmStip1a (XbaI, 5′-atcgtctagaTTAAAGAATTCCCGTTTGA-3′). Human STIP was amplified from cDNA clone BC011599 (ATCC) with HsStip1s (5′atcgactagtATGTCATTGTCCCACTTAT-3′, SpeI) and HsStip1a (5′atcgctcgagTCACTTGGCCATGTCGATCA-3′, XhoI). DmSTIP and Cel-stip-1 3′-UTR were cloned in SalI/NdeI-digested pT-Prom vector to generate the rescue plasmid pDmStipR. HsSTIP and Cel-stip-1 3′UTR were cloned in SpeI/SacI-digested pT-Prom to produce the rescue plasmid pHsStipR. All PCR reactions were done with a high-fidelity DNA polymerase. The orientation and sequence of the cloned DNA fragments in the two rescue constructs were verified by sequencing. For cross-species gene complementation, the pDmStipR or pHsStipR plasmid (15–20 μg/ml) was injected into the distal arm of the gonad of "wild type N2 hermaphrodites as above. Each rescue construct was co-injected with a pRF4 plasmid (80 μg/ml) for roller selection. The pRF4 plasmid was also injected alone as a control to monitor non-specific effect that might result from the rol-6 mutation. Transformants were identified by the roller phenotype and by single worm PCR with GSP. Transgenic lines whose F2 and later generations showed this phenotype were maintained. Using the soaking method, RNAi was then performed on L4 hermaphrodites of the wild type N2 strain as well as transgenic strains [27] expressing pRF4, pDmStipR/pRF4, and pHsStipR/pRF4 plasmids, respectively. F1 progeny were scored for each type of worms to assess the effects of knockdown and complementation.

Results Metazoan STIP are single-copy genes that encode a novel subset of G-patch proteins and arise during unicellular eukaryotic evolution To determine their species distribution and evolutionary pathway, 42 STIP were identified in metazoans through cDNA cloning (28 genes) and database mining (14 genes) (Fig. 1a, and Supplementary Fig. 1). We found that all stip genes are of single-copy in the sequenced metazoan

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genomes, indicating them to be functional orthologs. This conclusion is substantiated by the statistics and topology of the phylogenetic tree for STIP, whose branching conforms to

the species order from nematodes and arthropods to vertebrates (Fig. 1a). Metazoan STIP are conserved proteins and may thus possess an important biological function.

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Fig. 3 – Genomic organization, RNA processing and stage-specific expression of stip-1. (a) Stip-1 is organized in seven exons (upper numerals denote start and ending nucleotides and lower ones the size of introns). Its mRNA contains the 5′ SL1, ORF, 3′-UTR and poly(A) + tail. The bar shows the position and span of two primers for stip-1 expression analysis. (b) Developmental stage-specific expression of stip-1 transcripts. The specific stages of the wild type N2 strain are labeled (Emb, embryonic stage; L1 to L4, larval stages 1 to 4; adult, adult stage; and dauer, starvation stage). Total RNA was converted to cDNA using random primers and amplified with stip-1 GSP. The size of stip-1 and control Cel-ama-1 products is denoted. Note the presence of stip-1 transcripts in all RNA samples from different stages. (c) Western blots of Flag-tagged STIP-1 expressed in HEK293T cells. The blots were probed with anti-STIP-1 polyclonal antibodies (lane 1) and anti-Flag monoclonal (lane 2). Molecular weight markers and the 95-kDa STIP-1 are indicated at margins.

Genuine stip-like genes are present in unicellular organisms (i.e., green alga and slime mold) (Fig. 1a), but not in “deepbranching” eukaryotes (e.g., Giardia and others), indicating that stip genes arose lately or lost secondarily during unicellular eukaryotic evolution. To define the sequence conservation and structural features of metazoan STIP, we analyzed their G-patch domains together with those unicellular homologs. Consistent with the typical G-patch [1], all STIP retain the regular spacing of six glycines (Fig. 1b). Notably, most metazoan STIP contain six extra conserved glycines flanking the G-patch core, three on the left side and three on the right side (Fig. 1b). In addition, metazoan STIP G-patch domains retain a shorter gap between the 5th and 6th glycines (11aa in STIP vs. 14–18aa in others [1]) and have a distinct amino acid composition around the conserved glycines. All these features are partially conserved in unicellular STIP homologs (Fig. 1b) Thus, metazoan STIP form a subset of G-patch proteins, raising the

possibility that this module might mediate novel interactions with both nucleic acids and proteins.

Worm STIP are homologous to human STIP with further shared structural features besides the G-patch domain To develop an animal model for studying the function of STIP, we explored C. elegans due to its decided advantages and available powerful research tools. As worm STIP homologs were little studied, we characterized them in four species. Fig. 2 shows the structure of worm proteins and their identity/ similarity to selected homologs. We found that the worm proteins like other animal homologs are mostly acidic and their pIs fall between 5.33 (C. elegans) and 6.05 (B. malayi) (Supplementary Table 1). Besides a G-patch, worm proteins carry short clusters of positive charged residues as potential NLS and a coiled-coil, located in the N and C-terminus of the G-patch, respectively (Fig. 2a). We further found that worm

Fig. 2 – Worm STIP share conserved structural and organizational features representative of all metazoans. (a) The domain division (upper) and amino acid sequence of STIP (lower) in four nematodes. Ce, C. elegans ; Cb, C. brigssae ; Cr, C. remanei ; Bm, B. malayi . Cel-STIP-1 sequence is in bold. Identical residues are shaded in yellow. Dashes denote gaps. The two clusters of negative charged residues, two putative NLS, G-patch, coiled-coil, and six W(Xaa13–15)W(F) repeats are diagrammed. (b) Percent identity (red) and similarity (blue) of STIP between worm and other species. Hs, human; Gg, chicken G. gallus ; Xt, frog X. tropicalis ; Dr, zebrafish D. rerio ; Ag, mosquito A. gambiae ; Dm, fruit fly D. menalogaster ; Cr, unicellular green alga C. reinhardtii ; and Dd, unicellular slime mold D. discoideum . The designation numbers are the same as that compiled in Supplementary Table 1 where all accession numbers are given.

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Fig. 4 – Spatiotemporal expression patterns of STIP-1 in C. elegans determined by using transcriptional reporter constructs. (a) Schematic representation of the reporter constructs, stip-1::lacZ and stip-1::gfp . The 1475 bp and 1900 bp promoters yielded the same LacZ staining patterns and the same GFP imaging patterns (images for the 1900 bp promoter are not shown for brevity). (b) The histochemical staining for β-galactosidase produces insoluble deposits in nuclei transcribing stip-1 . C. elegans were photographed using nomarski optics. The STIP-1::LacZ signal is observed in worm embryo (I) and adult (II–IV): II, head and body region, where the fusion protein exists in head neurons, pharyngeal muscle cells, and pharyngeal gland cells. Pharyngeal and body muscle cells are arrow-pointed as examples; III, body region, where fusion protein is seen in body muscle cells, vulval muscle cells (arrow-pointed) and hypodermal cells; and IV, tail region, where fusion protein is expressed in tail neurons and hypodermal cells. (c) Expression of STIP-1::GFP fusion protein was observed with a Zeiss fluorescent microscope using the isothiocyanate filter set and photographed at ×200 magnifications. Note that STIP-1::GFP shows the same pattern as STIP-1::LacZ and is seen in all developmental stages. Embryonic stages: I, multi-cell stage embryo; II, lima bean stage embryo; III, comma stage embryo; IV, pretzel stage embryo. Larval stages: V, L1 larva (for example). Adult worms: VI, whole adult; VII, head region; VIII, body region; and IX, tail region. The two arrows indicate head and tail neuron cells, respectively. (d) STIP-1::GFP fusion protein is also expressed in the oocyte of C. elegans and is apparently located in the nuclear compartment: I, GFP image; and II, DIC image. Arrows point to the oocytes.

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proteins share a hitherto unrecognized, conserved motif, i.e., six short W(Xaa13–15)W(F/Y) repeats C-terminal to the coiledcoil region (Fig. 2a). Systematic analyses revealed that the arrangement of these repeats is the identifying feature for all metazoans (Supplementary Fig. 1), suggesting that they represent a new sequence module potentially mediating novel protein-protein interactions. The alignment of worm STIP shows that they are highly conserved with identical sequences run through their polypeptides (Fig. 2a). The overall identify/similarity of worm proteins falls in the range of 74–83/84–91% among C. elegans, C. brigssae, and C. remanei but 38–39/60–62% with respect to B. malayi (Fig. 2b). Nevertheless, the degrees of their sequence conservation to human or other metazoan proteins are all on a similar level, with B. malayi STIP bearing a slightly higher identity likely due to its higher species order. Given the comparable conservation level and the known single-copy status in C. elegans and human genomes, Cel-STIP-1 and human STIP are probably functional orthologs.

STIP-1 is expressed at all developmental stages, produced via trans-splicing, and translated as a 95-kDa protein We next focused on the characterization of stip-1, whose 2551 bp cDNA is spread in seven exons and contains no 5′-UTR but a splice leader, an 830aa ORF, and a 58 bp 3′-UTR followed by a poly(A)+tail (Fig. 3). Thus, like 70% genes of C. elegans [28], Cel-stip-1 mRNA is produced via SL1 trans-splicing. To determine its stage-specific expression, stip-1 was amplified from total RNA isolated from embryos, L1 through L4 stage, dauer, and adult worms. As shown, stip-1 transcripts were readily detected, revealing its expression at all developmental stages (Fig. 3). We also examined STIP-1 expression in human cells. Western blots of whole extracts from HEK293T cells transiently expressing the worm homologue showed a protein product of expected size when probed with the anti-Flag or anti-STIP-1 antibody (Fig. 3). The results demonstrate a faithful translation of STIP-1 in a heterologous expression system.

STIP-1 is expressed in many tissues and cell types including oocytes in C. elegans Next we used LacZ and GFP fusion reporters under the direction of either the 1900 bp or 1475 bp promoter (Fig. 4a) to characterize the spatiotemporal expression pattern of STIP-1 in C. elegans. The use of the two promoters yielded the same results. Consistent with RNA analyses (Fig. 3b), STIP-1::LacZ protein was well expressed at all developmental stages from embryos to adults and distributed in many tissue/cell types. High level expression of STIP-1 was observed in many pharyngeal cells including muscles, neuron and gland cells (Fig. 4b). In addition, STIP-1 expression was observed in tail and head neurons, some body muscle cells, and occasionally hypodermal cells (Fig. 4b and c). The expression of STIP-1::GFP protein revealed the same distribution pattern (Fig. 4c); but significantly it was observed also in those microscopic images that highlight the oocyte of C. elegans (Fig. 4d). Careful inspection of the images indicated that STIP-1 is present in the nucleus of

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oocytes (Fig. 4d), a finding that conforms to its nuclear location when expressed in heterologous cells (see below). Given its broad tissue/cell distribution and its residence in the oocyte nucleus, we hypothesize that stip has an important nuclear function.

STIP-1 is a nuclear protein, retains NLS, and assembles large polymers with a rod-like morphology Although human and mouse STIP are nuclear proteins [4,5], the sequence responsible for their nuclear location is unknown. Given its apparent location in oocyte nuclei, we asked whether STIP-1 could be targeted to the nucleus and, if so, what regions serve as NLS to specify this subcellular compartmentalization. A series of constructs in either GFP:: STIP-1 or STIP-1::GFP configuration were generated (Fig. 5a), transfected into mammalian cells (COS1 and HEK293T), and analyzed with confocal microscopic imaging. The images of full-length STIP1–830::GFP and GFP::STIP1–830 showed that both fusion proteins are mostly located in the nucleus (Fig. 5b, panels I and II), while the GFP control is evenly dispersed in the cytoplasm and nucleus. Like mouse STIP [5], C. elegans STIP1–830 is capable of forming large polymers in the nucleus, and these presumed supramolecular complexes are essentially excluded from the nucleolus (Fig. 5b). However, the expression of truncated forms of STIP-1 showed strikingly different patterns in both location and polymerization. As shown, STIP1–213::GFP was targeted exclusively to the nucleus but was distributed evenly with no polymer formation (Fig. 5c, panel III), but STIP1–422::GFP was localized to the nucleus and displayed a speckle-like shape, similar to the full-length STIP1–830::GFP (Fig. 5c, panel IV). In contrast, all other truncated fusions, STIP214–422::GFP, STIP214– 830::GFP and STIP423–830::GFP, were similar to GFP controls that distributed in the cytoplasm and nucleus with no polymer formation (Fig. 5c, panels V, VI, and VII). The reconstruction of 3D images revealed that STIP1–830::GFP forms a rod-like structure, while STIP1–213::GFP is dispersed in the nucleoplasm (Fig. 5d, panel III). The results were the same, when the Cterminal fused GFP::STIP-1 constructs were used (data not shown). Thus, the NLS must reside in the 1–213aa region, consistent with their position in the aa22–121 region Nterminal to the G-patch (Fig. 2a). The N-terminal 1–422aa likely retains all the sequence elements necessary for STIP-1 to polymerize in the nucleus. Since STIP1–213 retains the NLS and G-patch and STIP214–422 retains the coiled-coil, these domains are crucial for the coupling of nuclear localization and polymerization of the protein. Moreover, these results indicate that the coiled-coil domain is necessary but not sufficient for polymer formation of STIP-1 protein.

STIP-1 is essential for embryogenesis in C. elegans Because loss-of-function alleles of stip-1 are currently not available, we performed RNAi to down-regulate its expression to assess its function. We made in vitro a 500 bp dsRNA corresponding to the coding region of stip-1 exons 2 and 3 (Fig. 6a) and soaked L4-stage hermaphrodites of the wild type N2 strain directly with this dsRNA. Twenty-four hours later, these worms were transferred to individual plates, and their F1

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progenies examined and quantified for indexing embryonic development. The nomarski DIC images of the wild type N2 embryos showed that they were normally divided into 2 to 4 to 16 cells, reached the comma stage (Fig. 6b, upper), and then

developed further to larvae and adult worms. Controls soaked in PBS only showed the same pattern as the wild type (not shown). The RNAi embryos showed normal cell division from 2 to 16 cells; however, after the 16-cell stage, they began to

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stop progression and displayed remarkable morphological abnormalities (Fig. 6b, lower). All the embryos with stip-1 suppression were arrested in the multi-cell stage and failed to undergo any further morphogenesis, indicating that knockdown of stip-1 by RNAi caused 100% embryonic lethality. This shows that stip-1 is an essential gene absolutely required for embryonic development (and likely animal viability) in the worm C. elegans. Our data also verified the essential nature of stip-1 gene (CO7E3.1) indexed by genome-wide RNAi screens in C. elegans [29], although those screens provided no details with each gene.

The worm lethal phenotype could be rescued with fly and human genes Having shown stip-1 to serve an essential function for C. elegans embryogenesis, we asked whether this function is species-specific or conserved, given its moderate identity to fly to human proteins (∼ 28%, Fig. 2b). A cross-species complementation approach was taken to address this question by putting back the fly or human gene via transgenic expression. The rescue constructs, in which the coding sequence of fly or human stip was under the control of the native stip-1 promoter and 3′-UTR (Fig. 6a), were transformed into worms to establish transgenic lines. Comparison of the knockdown effects of stip-1 by RNAi among the wild type N2 strain, the pRF4 (rol-6) strain, and the fly (pDmStipR) or human (pHsStipR) transgenic lines is summarized in Fig. 6c. The results showed again that after 24 h of dsRNA soaking, the wild type N2 C. elegans exhibited 100% embryonic lethality. The embryonic lethality of pRF4 transgenic worms also was 100%, proving that the rol-6 allele did not cause the lethal phenotype. In contrast, the rate of embryonic lethality of the transgenic lines carrying a fly gene was only 18.0%, and that carrying a human gene was merely 33.4% (Fig. 6c). Although there was a difference between fly and human genes in complementation, likely due to their evolutionary distance from C. elegans, these results demonstrated that they rescued most of the knockdown effects on stip-1. In conclusion, our studies provided the first, direct evidence that metazoan stip homologues are orthologues sharing a conserved and essential function.

Discussion In this study metazoan STIP have been characterized as a conserved group of multi-domain proteins that contain the NLS, G-patch, coiled-coil, and short WW repeats. With the

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insight obtained from their protein structure, gene evolution, species distribution, spatial temporal expression pattern, and subcellular location, we addressed two key questions about this group of proteins. (1) Do STIP have an essential biological function? (2) If so, is this function conserved in metazoans and involved in fundamental organismal processes such as embryogenesis and development? The results reported here demonstrate that stip genes (and proteins) are evolutionarily conserved orthologs that possess an essential in vivo function required for metazoan viability. The lethal phenotype of stip-1 knockdown in C. elegans embryos and its rescue through transgenic expression of both fly and human stip genes provide direct evidence in support of this conclusion. Our approach to investigating the function of STIP illustrates the power of systematic analyses combined with the use of appropriate model organisms. We showed that all STIP retain the G-patch core, a string of six conserved glycines, consistent with the identification of fly and mouse homologs as lone G-patch proteins [1–3]. More significantly, our systemic analyses have revealed additional conserved features that pertain to metazoan homologous proteins as a whole. In metazoan, the region flanking the G-patch core contains six extra conserved glycines that are also partially conserved in lower homologs but apparently absent from unrelated G-patch domain proteins. Moreover, besides this G-patch, metazoan STIP contain unique sequence features, implying a functional coordination via specific domain combination, like other Gpatch proteins [30–33]. Significantly, despite a moderate amino acid sequence identity between the worm and human proteins (i.e., 28–30%), the G-patch, NLS, coiled-coil, and short WW repeats are conserved and arranged similarly in all metazoans. The NLS and coiled-coil region are invariant modules despite their variation in exact position and sequence composition. As a newly evolved sequence novelty, the WW repeat motifs serve as the identifying feature for metazoan proteins and distinguish STIP from the known WW domain-containing proteins by a length variation between the two tryptophan residues [34]. Their apparent absence in stip-harboring unicellular homologs suggests that these short WW repeats may play a novel functional role in metazoan animals specifically. Nematode stip genes were little studied metazoan homologs until now. Our results derived from four species have defined the genuine features representative of all metazoans. We demonstrate that C. elegans stip-1 gene is constitutively expressed at all stages during development, and its protein is broadly distributed in a variety of cell/tissue types. This wide spectrum of gene expression and protein distribution is

Fig. 5 – Subcellular localization of STIP-1 using GFP fusion constructs. (a) Schematics of stip-1::gfp constructs in full-length (I and II) and truncation forms (III through VII). Stip-1 was fused either 5′ or 3′ to gfp. Because the two fusions gave the same results, only the 5′-stip-1::gfp-3′ set of constructs is shown for brevity. Each image presented below is labeled with the construct (I to VII) used. (b) Subcellular localization of STIP-1::GFP expressed in HEK293T cells was determined by confocal microscopy. Arrows point to the location of the nucleolus. Left I and II are GFP images, and right I and II are GFP plus DIC images. GFP images at the bottom are produced with p EGFP-C3 and pEGFP-N3 controls. (c) Subcellular localization of full-length STIP1–830:: GFP and truncated STIP-1::GFP fusion proteins expressed in HEK293T cells. Note the difference between the full-length and truncated versions in subcellular location and polymer formation. (d) Reconstruction of the 3D images of the full-length STIP1–830::GFP and truncated STIP1–213::GFP proteins (right panel). Note the rod-like shape of the full-length form.

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consistent with the data of Northern blot analysis of stip genes in mouse and human [3], suggesting their function at a cellular and subcellular level. According to our results, the expression of STIP-1 starts from early embryos throughout adult worms, and this expression is persistent in multiple tissues, including head, pharynx, hypodermis, vulva, body, and tail. Likewise, STIP-1 has been localized to multiple cell types in those

tissues, such as neuronal cells, muscle cells, gland cells, hypodermal cells, and most significantly also in oocytes. The detailed studies enabled us to develop the first metazoan model in which to investigate the conserved in vivo function of stip genes. Using various tag–fusion constructs, we determined STIP1 to be a nuclear protein capable of forming rod-like polymers

Fig. 6 – Stip-1 is required for C. elegans development and its knockdown results in 100% embryonic lethality. (a) Schematics of dsRNA for RNAi and configuration of the rescue constructs for cross-species gene complementation. The region for dsRNA production was chosen, because it shares little homology to the fly or human stip gene. (b) DIC images of the wild type embryos and stip-1 RNAi embryos. The wild type N2 embryos undergo normal cell division from 2 to 4 to 16-cell stage and comma stage (upper panels) followed by further development to larvae and adult worms. RNAi embryos in which stip-1 is knocked down undergo a similar developmental progression as wild type embryos at early stages from one to four cells. But these embryos all begin to show cellular disruption and morphological abnormalities beginning at the 16-cell stage, and are entirely arrested at multi-cell stages with no further morphogenesis or tissue differentiation (lower panels). Stages are: I, 2-cell; II, 4-cell; III, 16-cell (note the round shape of cells and loss of intracellular granules in the stip-1 RNAi embryo); IV, multi-cell. (c) Summary of cross-species gene complementation using the fly and human stip homologous genes to rescue the stip-1 knockdown phenotypes.

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in the nuclear compartment. This finding conforms to human STIP being a nuclear phosphoprotein [4] and mouse STIP forming a subnuclear structure close to SC35 speckles [5]. Thus, our studies and others reinforce the concept that STIP share an important and conserved nuclear function. Importantly also, we discovered that the nuclear localization and intranuclear polymerization are intimately coupled processes, and the coupling is likely linked to the nuclear function of STIP. We showed that the coupling is dependent on 1–422aa of STIP-1, which retains the NLS, G-patch, and coiled-coil, but independent of the C-terminal half, which is dominated by WW repeats and spacers. Using deletion mutants, we showed that the N-terminal 1–213aa of STIP-1 is responsible for nuclear import and the central coiled-coil domain has a key role in polymer formation, while the Cterminal 422–830aa is evidently not involved in the two nuclear processes. We note that the coiled-coil domain is necessary but not sufficient for polymer formation, because STIP-1 could not assemble a rod-shaped structure when it failed to anchor in the nucleus upon NLS removal from the Nterminus and retained in the cytoplasm. These findings entice a hypothesis that the N-terminal sequence determinants of STIP concertedly promote its nuclear import and polymerization in assembling a rod-like structure through interactions with additional nuclear components. We here dub the STIP-centered supramolecular ensemble “stiposome” and envisage its potential engagement in crucial subnuclear functions. Further studies are needed to fully appreciate the functional roles of individual domains and tease out candidate nuclear factors that are dedicated to the stiposome formation and organization. To assess its functional roles, we used the RNAi approach to down-regulate stip-1 and then analyzed the knockdown effects on developmental processes in C. elegans. We have shown that stip-1 is an essential gene, whose biological function is absolutely required for embryogenesis in C. elegans and conceivably contributes to animal viability. Its essential nature is reflected by the loss-of-function inducing 100% embryonic lethality and by the remarkable phenotypes of development arrest and morphologic abnormalities beginning at the 16-cell stage. We find that the stip-1 RNAi embryos showed normal cell division at early stages, but were all arrested at a later multi-cell stage, beyond which the embryos collapsed and failed to undergo any morphogenesis. Such pleiotropic effects echo the wide expression of stip-1 in worm embryos and adult tissues, implicating for the ablation of a fundamentally important function in this organism. To define if this function is broadly conserved in metazoans, we used cross-species gene complementation to rescue the worm embryonic lethal phenotype. Both fly and human genes could rescue most of the phenotypic effects caused by stip-1 down-regulation. Given the single-copy status and observed complementation, metazoan stip genes must be orthologs that have reserved the same function throughout evolution, despite a moderate sequence identity of their proteins. Although the precise function of STIP remains to be deciphered, our studies are the first to show that STIP is essential and required for vital organismal processes. Since STIP is a possible spliceosome-associated factor [8,9] but does not directly mediate RNA splicing [5], it will be key to determine whether it is engaged in postspliceosomal or even other nuclear

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processes. The availability of a C. elegans model should pave the way for future studies aimed at tackling the unanswered questions about this conserved essential function in metazoans from worms to humans.

Acknowledgments We thank the Central Facility of the New York Blood Center for DNA sequencing and confocal analyses. This work was supported in part by the National Institutes of Health Grant HD62704 to C.-H. H. The nucleotide sequences reported herein have been deposited in GenBank Database with accession numbers provided in Supplementary Table 1.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2007.01.003.

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