- Email: [email protected]
Identification and characterization of a long isoform of human IFT80, IFT80-L
Biochemical and Biophysical Research Communications 373 (2008) 653–658
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification and characterization of a long isoform of human IFT80, IFT80-L Weihua Huang, Justin K. Kane, Ming D. Li * Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, VA 22911, USA
a r t i c l e
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Article history: Received 12 June 2008 Available online 2 July 2008
Keywords: IFT IFT80 IFT80-L siRNA Gene expression Gene suppression Cell differentiation
a b s t r a c t Intraflagellar transport (IFT) proteins are evolutionarily conserved throughout all ciliated organisms and are essential for the assembly and maintenance of cilia. IFT80, a component of the IFT complex, was linked recently to a human developmental disorder, Jeune asphyxiating thoracic dystrophy. We report here identification and characterization of a human IFT80 long isoform (namely IFT80-L), the carboxyl terminus of which shares the protein sequence of IFT80. Sequence analysis indicates that IFT80-L is likely an evolutionarily merged product of genes IFT80 and TRIM59, a RING finger gene we reported previously. Expression analysis of IFT80-L demonstrates that IFT80-L is ubiquitously expressed in humans. By using the nerve growth factor-induced cell differentiation assays, we reveal that IFT80-L is highly expressed in the rapidly proliferating cells but not in differentiated cells, which withdraw from the cell cycle. Our findings suggest that IFT80-L, like other IFT proteins, plays an important role in cell proliferation and differentiation. Ó 2008 Published by Elsevier Inc.
Intraflagellar transport (IFT) is an example of the microtubulebased transport machinery, essential for the assembly and maintenance of all eukaryotic motile cilia and flagella and nonmotile sensory cilia [1,2]. The IFT motility drives materials extensively from the cell body to the tips of cilia and flagella and returns them to the cell body, modeling cilia and flagella dynamically. Also, IFT motility localizes receptors and ion channels on cilia or flagella, which sample the extracellular milieu to generate intracellular signaling cascades and to regulate gene expression and guide cell responses [3,4]. In addition, IFT motility has recently been found to play a direct role in transmitting signals in cilia and flagella [5,6]. All of these findings not only reflect the various roles of cilia and flagella in motility, sensory reception, and signaling [7,8], but also manifest the function of IFT in the control of gene regulation and expression, cell proliferation and differentiation, and animal development and behavior [3,4]. Intraflagellar transport was discovered originally in the flagella of Chlamydomonas reinhardtii [9]. Bidirectional IFT motility is powered by kinesins for anterograde transport and by dyneins for retrograde movement along the outer-doublet microtubules of ciliary axonemes [1,2]. The large particles of IFT are composed of more than 17 distinct non-membrane-bound proteins that can be separated into two complexes known as A and B. Complex A consists of six subunits ranging from 43 to 144 kDa, whereas complex B con-
* Corresponding author. Address: University of Virginia, Department of Psychiatry and Neurobehavioral Sciences, Section of Neurobiology, 1670 Discovery Drive, Suite 110, Charlottesville, VA 22911, USA. Fax: +1 434 973 7031. E-mail address: [email protected] (M.D. Li). 0006-291X/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.bbrc.2008.06.085
tains at least 11 subunits ranging from 20 to 172 kDa [10]. All IFT proteins identified to date, along with IFT motor proteins, are evolutionarily conserved in ciliated organisms, but not in non-ciliated organisms such as yeast or higher plants [11]. These proteins are essential for the assembly and maintenance of cilia and flagella, and mutations cause the loss or severe reduction of cilia and flagella in various organisms [12–14]. Ciliary defects can lead to numerous human diseases [15]. Previously, we reported molecular cloning and characterization of a mouse RING finger gene [16], lately termed tripartite motifcontaining 59 (Trim59) in genome annotations. In searching for its homolog in a human brain cDNA library, we identified several transcripts that are merged from TRIM59 (NM_173084) and IFT80 (NM_020800), two genes annotated in human genome databases, in addition to the transcripts for TRIM59 alone. Protein IFT80, termed KIAA1374 or WDR56 previously, contains a motif of WD40 repeats and is a component of IFT complex B [10]. Our novel identified gene, named IFT80-L, encodes a long isoform of IFT80. The carboxyl terminus of IFT80-L shares a protein sequence with IFT80 and is thus inferred to be an IFT component. Recently, Beales et al. revealed in a human genetic study that mutations in IFT80 underlie a subset of Jeune asphyxiating thoracic dystrophy (JATD) cases [17], establishing an association of IFT80/IFT80-L with a human disease. Materials and methods cDNA library screening. A human brain cDNA library constructed in the kTriplEx2 vector (Clontech) was screened by radioactive
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hybridization in accordance with the manufacturer’s instructions. On the basis of the sequence of the previously cloned mouse RING finger gene Trim59 [16], we prepared a hybridization probe using the polymerase chain reaction (PCR) method with a pair of primers, 50 -ggaaatgcacaattttgaggaaga-30 (forward) and 50 -actggagaac ggcttccttatcg-30 (reverse), and incorporation of [a-32P]dCTP (Amersham). All positive colonies were converted from the kTriplEx2 construct into the pTriplEx vector. The isolated plasmids were sequenced, and all cDNA sequences were mapped onto the human genome sequence with BLAST searching. Cells and cell culture. The human embryonic kidney cell line HEK 293 and rat adrenal pheochromocytoma cell line PC12 were purchased from the American Type Culture Collection. Mouse embryonic stem cell (ESC) line AB 1.1 was provided by Dr. P. E. Hasty of The University of Texas Health Science Center at San Antonio. The HEK 293 cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). The PC12 cells were maintained in F12K medium with 15% horse serum and 2.5% FBS. The AB1.1 ESCs were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder cell layer in Knockout-qualified DMEM medium with 15% FBS. All media, sera, and antibiotics were purchased from Invitrogen. Gene suppression assay. Two siRNAs (5siRNA and 3siRNA) targeting the 50 and 30 ends of human IFT80-L (or TRIM59 and IFT-80), respectively, were synthesized and purified by Dharmacon. The targeting sequences were 50 -aacattcttcaggcatctggt-30 and 50 -aacc caacactggcagcatat-30 , respectively. Transient transfection was carried out with LipoFectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Triple transfections were performed for each siRNA. Cells were analyzed 48 h after transfection. Gene expression assay. For quantitative real-time reverse transcriptase (RT)–PCR analysis, the TaqMan method was applied in the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Briefly, total RNA was isolated from cells with Trizol (Invitrogen) and reverse-transcribed into cDNA with SuperScript II RNase H- and random hexamer (Invitrogen). The quantitative real-time PCR was then conducted with a set of TaqMan probes. Specifically, four sets of probes were designed for both the 50 and 30 ends of human IFT80-L and mouse/rat Ift80-L: 50 -gagacctttac gaattccactcaagt-30 (forward), 50 -tgccagttggagcaatttca-30 (reverse) and FAM-ccctaattgcagaagtatt-MGB (probe) for human 5Probe; 50 cgccaacgtgtgcagatg-30 (forward), 50 -tgcttactcgaggataaatttcaaca-30 (reverse) and FAM-aaacagagaccacttcc-MGB (probe) for mouse/rat 5Probe; 50 -aatgggaagatgctgtgagacttt-30 (forward), 50 -agctagacaagc ccacatggtt-30 (reverse) and VIC-tcgctttgttaaggagc-MGB (probe) for human 3Probe; 50 -agcaggcaaacagctcatca-30 (forward), 50 -tcatg ggctttccactgtaaaa-30 (reverse) and VIC-ctcttcaaccaaatgcta-MGB (probe) for mouse/rat 3Probe. A standard PCR amplification protocol was used: 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 25 s at 95 °C and 1 min at 60 °C. All gene expression levels were normalized to an intrinsic control of 18s ribosome RNA. Mutiple-tissue cDNA panel assay. Human multiple-tissue cDNA panel was purchased from Clontech. A pair of primers, 50 -gggatgcttagatcaactttagctc-30 (forward) and 50 -agatcaaatataattggtgtgttc-30 (reverse), was designed to bridge two exons in the 30 end of IFT80-L, as well as in IFT80, and was used for the PCR amplification. The control gene, encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified for 26 cycles for a product of 983 bp with a pair of primers supplied by the manufacturer (Clontech). Gene(s) IFT80-L and IFT80 were amplified for 35 cycles for a product of 704 bp. The PCR procedure was as follows: 2 min at 94 °C followed by cycles of 30 s at 94 °C, 15 s at 55 °C, and 1 min at 72 °C. Cell differentiation. Rat PC12 cells were cultured on collagen IVcoated plates or flasks (BD Biosciences) for rapid proliferation. For differentiation induction, PC12 cells were transferred to collagen
I-coated plates and treated for 5 days with nerve growth factor (NGF; Clontech) at a final concentration of 100 ng/ml in medium containing only 0.5% FBS. Control PC12 cells were maintained on collagen I-coated plates in a medium with 10% FBS and without NGF. Media were replaced every other day. To eliminate the feeder cells, mouse AB1.1 ESCs were cultured with ESGRO LIF (Chemicon) 1000 U/ml and basic fibroblast growth factor (bFGF; Invitrogen) 100 ng/ml in Knockout-qualified DMEM medium with 15% FBS. For differentiation induction, ESCs were first allowed to aggregate for 1 day in Petri dishes with no ESGRO LIF or bFGF added, and then induced with NGF 100 ng/ml for 2 days. The embryoid bodies that formed were further dissociated and transferred to tissue culture plates, and the cells were induced with NGF 100 ng/ml for another 6 days in medium containing only 0.5% FBS. Media were replaced every other day.
Results In screening a human brain cDNA library for homolog of the mouse RING finger gene, Trim59 [16], we identified multiple positive clones. Sequence analysis revealed five transcripts for human homolog TRIM59 and an additional three transcripts that were distinct from TRIM59 at the 30 end. After mapping these transcripts onto the human genome, we found that all their introns had the consensus splicing donor and acceptor nucleotides, with a ‘‘GT” at the 50 end and an ‘‘AG” at the 30 end, just as documented for other eukaryotic genes. In addition, we found that the three cDNA clones differing from TRIM59 at the 30 end represent a fusion of two genes, TRIM59 (NM_173084) and IFT80 (NM_020800), as annotated in human genome databases. The clones contained the 50 exons from TRIM59 and the 30 exons from IFT80. In an open reading frame analysis, we found that these cDNA sequences encode a predicted protein consisting of a RING finger, a B-box, and a coiled-coil motif (RBCC motif or tripartite motif; TRIM) at the N-terminus and seven WD-40 repeats at the C-terminus (Fig. 1). In this report, we name this newly identified gene IFT80-L, encoding the long isoform of IFT80. In Fig. 1, we provide the repertoire view of the genomic structures of TRIM59, IFT80, and IFT80-L and the predicted motifs for their corresponding encoded proteins. The complete cDNA sequence of IFT80-L is approximately 3.8 kb, maps to human chromosome 3q26.1, and spans more than 200 kb with 21 or 22 exons. The predicted protein, IFT80-L, consists of 1080 amino acid residues. The genomic structures of TRIM59, IFT80, and IFT80-L indicate that TRIM59 and IFT80-L probably are regulated by the same promoter, whereas IFT80 may have its own promoter, notably because there are some exons at the 50 end that cannot be found in transcripts of IFT80-L. Moreover, in BLAST searching of human genome databases, we found two cDNA clones, BC033011 and BC068444, that also contain partial sequences from both TRIM59 and IFT80, similar to our IFT80-L but lacking three exons in the middle of the sequence. Their resultant predicted protein consists of 948 amino acid residues but has only five WD-40 repeats at the C-terminus, likely representing a truncated protein of IFT80-L. The C-terminus of IFT80-L shares the protein sequence of IFT80. Like other IFT proteins, IFT80 is conserved in all ciliated organisms, and its orthologs have been identified in lower organisms such as green algae, nematodes, fruit flies, and zebrafish, as well as higher organisms such as mouse, rat, and human [11]. Demonstrated in Fig. 2 is an alignment of the protein sequences of IFT80 orthologs in human, mouse, rat, nematode, and fruit fly. The sequence analysis reveals that human IFT80 has 41% identity and 55% similarity to Che-2 in Caenorhabditis elegans and 37% identity and 50% similarity to Oseg5 in Drosophila melanogaster, both of which are recognized as one of IFT components essential for the assembly and
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WD40 repeats
TRIM59 (403 aa)
TRIM59 (NM_173084) ATG
IFT80 (777 aa)
IFT80 (NM_020800) ATG
TGA
TAA
Genomic DNA
220 kb
5siRNA 5Probe
IFT80-L cDNA
ATG
TAA 3siRNA
3.8 kb
3Probe
TRIM
IFT80
WD40 repeats
Predicted protein IFT80-L
1080 aa TRIM
318
IFT80
1080
Fig. 1. Genomic structures of IFT80-L and its relevant genes, TRIM59 and IFT80. In middle, exons of IFT80-L, TRIM59, and IFT80 are illustrated approximately in position on human chromosome 3, spanning a region of more than 200 kb. Genomic structure of novel IFT80-L is demonstrated at bottom, whereas those of previously annotated TRIM59 (NM_173084) and IFT80 (NM_020800) are shown at top. Filled regions in cDNA sequences represent the open reading frames. Motifs of proteins are predicted from the SMART protein sequence analysis tool (http://smart.embl-heidelberg.de/). Target sequences of siRNAs for gene suppression and TaqMan probes for gene detection are marked approximately on IFT80-L cDNA sequence. Also defined are the N-terminal TRIM module (amino acids 1–318) and the C-terminal IFT80 module (amino acids 319–1080) of IFT80-L.
maintenance of cilia [10,18,19]. The identities of rodent orthologs of human IFT80 exceed 94%. In comparative genomic and proteomic analyses, IFT80, Che-2, and Oseg5, along with other IFT proteins, were included in the flagellar and basal body proteome [20,21]. Although our identified cDNA clones already provide evidence that IFT80-L probably does not represent chimeric clones resulting from experimental contamination, we still wanted to determine whether IFT80-L exists in cells as a truly single gene. To tackle this issue, we employed the double small interfering RNA (siRNA) suppression technique. We designed two siRNAs (5siRNA and 3siRNA) targeting the 50 and 30 ends of IFT80-L (or TRIM59 and IFT80 two genes), respectively (Fig. 1). After introducing the two siRNAs separately into human embryonic kidney cells (HEK 293), we measured changes in gene expression with two TaqMan probes (5Probe and 3Probe) targeting as well the 50 and 30 ends of IFT80L (or TRIM59 and IFT80), respectively (Fig. 1). The 5siRNA targeting TRIM59 suppressed both TRIM59 and IFT80 expression, as did the 3siRNA targeting IFT80 (Fig. 3A). Such coordinated expression changes in the siRNA suppression assays demonstrate that IFT80L is indeed a merger of TRIM59 and IFT80 and exists in HEK 293 cells. To establish the expression pattern of IFT80-L in humans, we applied a PCR assay to a commercially available multiple-tissue cDNA panel. We used a pair of primers bridging two exons in the C-terminus of IFT80-L as well as IFT80. Our expression analysis revealed that IFT80-L or IFT80 is expressed in almost all human tissues examined, most abundantly in kidney, pancreas, thymus, testis, and ovary, but only sparsely in skeletal muscle (Fig. 3B). Such an
expression pattern is similar to that reported previously for IFT80 (KIAA1374) based on Northern blot analysis [22], except that the PCR analysis we used is more sensitive so that we detected expression in more tissues. Transcription factor RFX regulates the expression of most IFT proteins [23,24]. It has been shown that IFT proteins (Ift88 and Ift52), IFT motor proteins (dynein 2 heavy chain and light intermediate chain 3), and transcription factor Rfx3 are expressed preferentially in the ependymal layer along the ventricular or subventricular zones [25–27], where neural progenitors or precursor cells exist with proliferation and differentiation. We thus suspected that IFT proteins might be involved in the process of cell proliferation and differentiation. To test our speculation, we induced differentiation with NGF in rat PC12 cells, a widely used model of in vitro irreversible neuronal differentiation. Examining with TaqMan probes for the 50 and 30 ends of Ift80-L, we found that expression of Ift80-L was significantly attenuated after PC12 cells differentiated into neuron-like cells (Fig. 3C). Because pluripotent ESCs and progenitor cells of the central nervous system origin are conserved in the induced promotion of neural cells, we further induced mouse ESCs AB1.1 into neurotypic cells with NGF. Similar to rat PC12 cells, mouse ESCs showed a decrease in Ift80-L expression after they experienced embryoid body formation and NGF-induced neuronal differentiation (Fig. 3C). Differentiation in both rat PC12 cells and mouse ESCs is preceded by withdrawal from the cell cycle. Thus, we conclude that Ift80-L may play a role in cell proliferation and differentiation. Because we used both the 50 and 30 end probes to detect expression changes and the results demonstrated a similar trend in expression changes for both the 50 and 30 ends of
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W. Huang et al. / Biochemical and Biophysical Research Communications 373 (2008) 653–658 WD-40 repeat 1
WD-40 repeat 2
mouse rat human nematode fruit fly
1 1 1 1 1
~~~~~~~~~~~~~~MRLKISLSKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETSLIVKLPDDIYPIDLHWF...PKSLGIKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MRLKISLLKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETSLIVKLPDDIYPIDLHWF...PKSLGIKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MRLKISLLKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETTQIVKLPDDIYPIDFHWF...PKSLGVKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MKLKLSASRKTRHTEMVCGVGWIGTEAILSAADDHVFLLTNTATNESQQILNMPETFFPTSLHIF...PRSQ..TKGGQNDVFAVSTSDGKINILSRNGKVENMVDAHNGAALCARWNSDGTGLLS MKLKTRICKKNSIRNSEKVEESSKSTTNCPLSCVDWSSNEEIYFVSDDHQIFKWSDVSRDSVEVAKLPDDFVPTDMHWLLLGGRSSGGGKG..SDTLLICSNDGRFVILNKSARVERSISAHAAAISSGRWSPDGAGLLT
mouse rat human nematode fruit fly
124 124 124 122 139
VGEDGQVKIWSKTGMLRSTLAQQGTPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRVLYGSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA VGEDGQVKIWSKTGMLRSTLAQQGIPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRVLYGSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA VGEDGQIKIWSKTGMLRSTLAQQGTPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRPLYNSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA SGEDGFVKMWSRSGMLRSVLAQFATAVYCVAWDSTSSNVLYCNADHCYIKSLKMQVAPIKWKAHDGIILCCDWNPTSDLIVTGGEDLKFKVWDGFGQILFNSSVHDYPITSISWNTDGTLFAVGSHNILRLCDKSGWSHS AGEDGVIKIWSRSGMLRSTVVQNEESIRCARWAPNSNSIVFCQGGHISIKPLAANSKIIRWRAHDGLVLSLSWSTQSNIIASGGEDFRFKIWDAQGANLFTSAAEEYAITSVAFNPEKD.YLLWSYNTAR..........
WD-40 repeat 3
WD-40 repeat 4
WD-40 repeat 5
WD-40 repeat 6 mouse rat human nematode fruit fly
264 264 264 262 268
LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQRWEWKNFQVTLTKRRTMQVRNVLND.AVDLLEFRDRVIKASLNHAHLVVSTSLQCYVFSTKNWNTPLIFDLKEGTVSLILQAERHFLLVDGGGIYLHSYEGR LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQRWEWKNFQVTLTKRRTMQVRNVLND.AVDLLEFRDRVIKASLNHAHLVVSTSLQCYVFSTKNWNTPLIFDLKEGTVSLILQAERHFLLVDGGGIYLHSYEGR LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQHWEWKNFQVTLTKRRAMQVRNVLND.AVDLLEFRDRVIKASLNYAHLVVSTSLQCYVFSTKNWNTPIIFDLKEGTVSLILQAERHFLLVDGSSIYLYSYEGR LEKMNAGSVMALSWSPDGTQLAVGTAAGLVFHAHIIDKRLTYEEFEIVQTQKTVIEVRDVSSEVSRETLETKERISKIAILYKYLIVVTSSHIYIYSSKNWNTPTMIEYNERTVNIIVQCEKIFLVSDGMTITIFTYEGR FSSPRVGSIFNLSWSADGTQATCGTSTGQLIVAYAIEQQLVSGNLKATSKSRKSITLKDIAT.GTQDILDFPQRVVSFGLGYGHLVVATTNQVHIYNEKYINTPIIIDGRNDT.RVIEVGKKYFMILDALSIWVYTYTGR
mouse rat human nematode fruit fly
403 403 403 402 406
FISSPKFPGMRTDI..LNAQTVSLSNDTIAIKDKADEKIIFLFEASTGKP.LGDGKLLSHKNEISEIALDQKGLTNDRKIAFIDKNRDLYITSVKRFGKE..EQIIKLGTMVHTLAWCDTCNILCGIQDTRFTVWYYPNT FISSPKFPGMRTDI..LNAQTVSLSNDTIAIKDKADEKIIFLFEASTGKP.LGDGKLLSHKNEISEVALDQKGLTNDRKIAFIDKNRDLYITSVKRFGKE..EQIIKLGTMVHTLAWCDTCNILCGLQDTRFTVWYYPNA FISSPKFPGMRTDI..LNAQTVSLSNDTIAIRDKADEKIIFLFEASTGKP.LGDGKFLSHKNEILEIALDQKGLTNDRKIAFIDKNRDLCITSVKRFGKE..EQIIKLGTMVHTLAWNDTCNILCGLQDTRFIVWYYPNT KLINLNPPGQVMAL..LDERKIDLANDTLVVRDRADNKVLHFFDPTTGKA.QGDGN.LKHEHDIVELTVNQCGPLNDRNVAFRDQIGAVHIAMVKTFGVS..QRMVKIGSLVEQLVFNDVTNMLCGISEGKIAVWPLPNV LHLNPRYPGSQAQIPLLTWRSLSLGLDVLAIRDNSDPTVLHLFDLIPGASRQYDPHSLRAKQQLVQIAACRAGSPEDQFVAFIDSNRELFVSESRNLNSERIDEIYKIGTQLTAIMWASETNILVGVHDSCYSIWYCPGE
mouse rat human nematode fruit fly
538 538 538 536 546
IYVDRDILPKTLYERDASEYSKNPHIVSFVGNQVTIRRADGSLVHISISPYPAILHEYVSSSKWEEAVRLCRFVKEQSMWACLAAMAVANRDMVTAEIAYAAVGEIDKVRYINAIKDL.PSRESKMAHILMFSGNIQEAE VYVDRDILPKTLYERDASEYSKNPHIVSFVGNQVTVRRADGSLVHISISPYPAILHEYVSSSKWEDAVRLCRFVKEQSLWACLAAMAVANRDMVTAEIAYAAVGEIDKVRYINAMKDL.PSRESKMAHILMFSGNIQEAE VYVDRDILPKTLYERDASEFSKNPHIVSFVGNQVTIRRADGSLVHISITPYPAILHEYVSSSKWEDAVRLCRFVKEQTMWACLAAMAVANRDMTTAEIAYAAIGEIDKVQYINSIKNL.PSKESKMAHILLFSGNIQEAE AFHDRNLLQKSLIQKNIGSVGKFPQLANFAGNTIVIRKSDGCLLPTGILPFYGTLITMASQSKWDQAIRLCRSIGNDTMWATFAGLAVLHKNMIVMEIAYAALEDDEKVSLINEIKDK.TDKETRQAMQVVLTGKLADAD GASDPTIIALTTITLDTTEFGKHITIESFEESVLTFRSA.GALLPVNVNMYCEILHRALLEGQWQQALKICRMGQHSSLWATLAAVATRKHQLQISEEAYSAALQIDKVSYLQHLKALTPSSAEQMAENSLMLGRMLEAE
mouse rat human nematode fruit fly
677 677 677 675 685
TVLLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLDTFGKQETNKRYLQYAEGLQIDWEKIKAKIEMEITKERDRSSSGQSSKSVGLKH TILLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLETFGKQETNKRYLQYAEGLQIDWEKIKAKIEMEITKERDRSSSGQSSKNTGLKP IVLLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLETFGKQETNKRYLHYAEGLQIDWEKIKAKIEMEITKEREQSSSSQSSKSIGLKP VLLERSGLSFRSLMLNIQMFKWKRALELGLKNKQW....LEIVMGYREKYLKNCGQKETDPLFLKHMSEVEIDWVHIRELIAAEKAKGNN~~~~~~~~~~~~~~~ TILLHGKKIEQAVGLALRMHNWRRALEISQKHKGEQPELVPRVLQERRKYLKALQREEWDPLYLPLVAKEEADNTSE~~~~~~~~~~~~~~~~~~~~~~~~~~~~
WD-40 repeat 7
777 777 777 760 761
[94.7%] [94.3%] [100%] [41.3%] [37.2%]
(NP_080917) (NP_001013933) (NP_065851) (NP_508106) (NP_610064)
Fig. 2. Alignment of protein sequences of IFT80 orthologs. Human IFT80 (NP_065851) is compared with mouse (NP_080917) and rat (NP_001013933) orthologs, as well as Che-2 in C. elegans (NP_508106) and Oseg5 in D. melanogaster (NP_610064). Also illustrated are seven WD-40 repeats predicted on the basis of human IFT80 protein sequence.
Ift80-L in differentiation, we suspect that Ift80-L exists in rat PC12 cells and mouse AB1.1 ESCs. Discussion We report here the identification of a long isoform of IFT80 protein encoded by IFT80-L. In human genome databases, IFT80-L was previously annotated as two separate genes, TRIM59 and IFT80 (Fig. 1). We demonstrate in this report that IFT80-L is in fact a single gene that represents a merger of TRIM59 and IFT80. Not only did we screen out the complete cDNAs from a human brain cDNA library, but we confirmed it with double siRNA suppression (Fig. 3A). To some extent, the presence of BC033011 and BC068444, two cDNAs sequences, in human genome databases provided additional evidence that IFT80-L was not generated from chimeric clones. Moreover, in mouse ESCs and rat PC12 cells, expression of both the 50 and 30 ends of Ift80-L was reduced at the similar rate during NGF-induced cell differentiation (Fig. 3C). Thus, evidence is piling up that IFT80-L exists in human beings as well as in rodents. Protein IFT80-L probably is an evolved Rosetta stone protein, converged from TRIM59 and IFT80. IFT80 is highly homologous to Che-2 in C. elegans, Oseg5 in D. melanogaster, TtIft80 in Tetrahymena thermophila, and ift80 in zebrafish [11,12,17,18] and is evolutionarily conserved, as are other IFT proteins in ciliated organisms [11]. However, we could not find any evidence of an IFT80-L ortholog in lower organisms. In view of this, we postulate that IFT80-L is present only in higher organisms such as rodents and humans, whereas IFT80 represents the remnant of evolution. Interestingly, we cannot find a canonical X-box sequence, a conserved cis-element for the binding of transcription factor RFX, in the promoter region of IFT80-L or IFT80. In contrast, well-defined X boxes have been identified in the promoters of Che-2 and Oseg5 [23,24]. This
finding also indicates the evolutionary difference between lower and higher organisms in expression regulation of IFT80 orthologs. Protein IFT80/IFT-L is one of the IFT components and is important for the assembly and maintenance of cilia [17–19]. The C. elegans mutants of che-2 have extremely short cilia in most ciliated sensory neurons and show defects in various behaviors such as chemotaxis and osmotic avoidance [19]. Disruption of TtIft80 in T. thermophila results in short, aberrant, or absent cilia; multiple nuclei and oral apparatuses; disorganized cortical ciliary rows; and cytokinesis defects [17]. In zebrafish, knockdown of ift80 produces large cystic kidneys accompanied by pericardial edema [17], a phenotype similar to the results of mutations of other IFT components [13]. Thus far, most IFT80 orthologs, including Che-2 [19], TtIft80 [17], and Oseg5 [18], have been characterized to localize to the cilium, basal body, and/or centrosome, just as do other IFT proteins [1,12,18]; and Che-2 has been shown to move along the ciliary axoneme at the same rate as other IFT proteins and IFT motor proteins [28,29]. The IFT motility is important for cilia formation and animal development. Disruptions of IFT proteins block sonic hedgehog developmental signaling in the neural tube, to cause defects of motile and nonmotile cilia on the ventral node of mouse embryo, and further, to lead to errors in embryo development [30]. The importance of IFT80/IFT80-L in development is recently evidenced by a finding that recessive mutations in IFT80/IFT80-L are linked to human disease JATD, an early development disorder that often leads to death in infancy [17]. We demonstrate in this report that Ift80-L is highly expressed in rapidly proliferating neural progenitor cells, but is attenuated after cell differentiation (Fig. 3C), and propose that IFT-L plays a role in early animal development. In summary, we identify here a long isoform, IFT80-L, in humans. Both IFT80 and IFT80-L have the same C-terminal sequence, with seven WD-40 domains, and are a human homolog of an IFT
W. Huang et al. / Biochemical and Biophysical Research Communications 373 (2008) 653–658
IFT80-L mRNA Expression (%)
A
657
Control 5probe 3probe
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Fig. 3. (A) Existence of IFT80-L in HEK 293 cells. Cells were transiently transfected with 5siRNA and 3siRNA, respectively. Gene expressions were detected by two TaqMan human probes using quantitative real-time RT-PCR. (B) Expression profile of IFT80-L and/or IFT80 in human tissues. A pair of primers bridging two exons in the 30 end of IFT80L as well as in IFT80 was used for PCR amplification of a human multiple-tissue cDNA panel. Gene GAPDH was used as a control. (C) Ift80-L is down-regulated after cell differentiation. Rat PC12 cells and mouse AB1.1 embryo stem cells were induced with NGF for neurotypic differentiation. Expressions of Ift80-L were detected by two TaqMan mouse/rat probes using the quantitative real-time RT-PCR. Data in (A) and (C) are shown as means ± SD.
component. We also demonstrate that IFT80/IFT80-L is expressed in many human tissues and potentially involved in cell proliferation and differentiation. Although IFT80-L/IFT80 has been associated with IFT motility and has been linked with a human disease, its function in mammals is still largely unknown. It will be of considerable interest to investigate the effects of complete loss of Ift80-L or Ift80 in a mouse model. As Ift80-L and Ift80 might be essential in cell differentiation and animal early development, we speculate that a conditional knockout of Ift80-L or Ift80 might be more feasible and fruitful. Acknowledgments We are grateful to Dr. Paul E. Hasty of the University of Texas Health Science Center at San Antonio for providing AB1.1 ESCs and assistance in ESC culture. This project was in part supported by NIH grants DA-12844 and DA-13783 to M.D.L.
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Identification and characterization of a long isoform of human IFT80, IFT80-L Weihua Huang, Justin K. Kane, Ming D. Li * Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, VA 22911, USA
a r t i c l e
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Article history: Received 12 June 2008 Available online 2 July 2008
Keywords: IFT IFT80 IFT80-L siRNA Gene expression Gene suppression Cell differentiation
a b s t r a c t Intraflagellar transport (IFT) proteins are evolutionarily conserved throughout all ciliated organisms and are essential for the assembly and maintenance of cilia. IFT80, a component of the IFT complex, was linked recently to a human developmental disorder, Jeune asphyxiating thoracic dystrophy. We report here identification and characterization of a human IFT80 long isoform (namely IFT80-L), the carboxyl terminus of which shares the protein sequence of IFT80. Sequence analysis indicates that IFT80-L is likely an evolutionarily merged product of genes IFT80 and TRIM59, a RING finger gene we reported previously. Expression analysis of IFT80-L demonstrates that IFT80-L is ubiquitously expressed in humans. By using the nerve growth factor-induced cell differentiation assays, we reveal that IFT80-L is highly expressed in the rapidly proliferating cells but not in differentiated cells, which withdraw from the cell cycle. Our findings suggest that IFT80-L, like other IFT proteins, plays an important role in cell proliferation and differentiation. Ó 2008 Published by Elsevier Inc.
Intraflagellar transport (IFT) is an example of the microtubulebased transport machinery, essential for the assembly and maintenance of all eukaryotic motile cilia and flagella and nonmotile sensory cilia [1,2]. The IFT motility drives materials extensively from the cell body to the tips of cilia and flagella and returns them to the cell body, modeling cilia and flagella dynamically. Also, IFT motility localizes receptors and ion channels on cilia or flagella, which sample the extracellular milieu to generate intracellular signaling cascades and to regulate gene expression and guide cell responses [3,4]. In addition, IFT motility has recently been found to play a direct role in transmitting signals in cilia and flagella [5,6]. All of these findings not only reflect the various roles of cilia and flagella in motility, sensory reception, and signaling [7,8], but also manifest the function of IFT in the control of gene regulation and expression, cell proliferation and differentiation, and animal development and behavior [3,4]. Intraflagellar transport was discovered originally in the flagella of Chlamydomonas reinhardtii [9]. Bidirectional IFT motility is powered by kinesins for anterograde transport and by dyneins for retrograde movement along the outer-doublet microtubules of ciliary axonemes [1,2]. The large particles of IFT are composed of more than 17 distinct non-membrane-bound proteins that can be separated into two complexes known as A and B. Complex A consists of six subunits ranging from 43 to 144 kDa, whereas complex B con-
* Corresponding author. Address: University of Virginia, Department of Psychiatry and Neurobehavioral Sciences, Section of Neurobiology, 1670 Discovery Drive, Suite 110, Charlottesville, VA 22911, USA. Fax: +1 434 973 7031. E-mail address: [email protected] (M.D. Li). 0006-291X/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.bbrc.2008.06.085
tains at least 11 subunits ranging from 20 to 172 kDa [10]. All IFT proteins identified to date, along with IFT motor proteins, are evolutionarily conserved in ciliated organisms, but not in non-ciliated organisms such as yeast or higher plants [11]. These proteins are essential for the assembly and maintenance of cilia and flagella, and mutations cause the loss or severe reduction of cilia and flagella in various organisms [12–14]. Ciliary defects can lead to numerous human diseases [15]. Previously, we reported molecular cloning and characterization of a mouse RING finger gene [16], lately termed tripartite motifcontaining 59 (Trim59) in genome annotations. In searching for its homolog in a human brain cDNA library, we identified several transcripts that are merged from TRIM59 (NM_173084) and IFT80 (NM_020800), two genes annotated in human genome databases, in addition to the transcripts for TRIM59 alone. Protein IFT80, termed KIAA1374 or WDR56 previously, contains a motif of WD40 repeats and is a component of IFT complex B [10]. Our novel identified gene, named IFT80-L, encodes a long isoform of IFT80. The carboxyl terminus of IFT80-L shares a protein sequence with IFT80 and is thus inferred to be an IFT component. Recently, Beales et al. revealed in a human genetic study that mutations in IFT80 underlie a subset of Jeune asphyxiating thoracic dystrophy (JATD) cases [17], establishing an association of IFT80/IFT80-L with a human disease. Materials and methods cDNA library screening. A human brain cDNA library constructed in the kTriplEx2 vector (Clontech) was screened by radioactive
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hybridization in accordance with the manufacturer’s instructions. On the basis of the sequence of the previously cloned mouse RING finger gene Trim59 [16], we prepared a hybridization probe using the polymerase chain reaction (PCR) method with a pair of primers, 50 -ggaaatgcacaattttgaggaaga-30 (forward) and 50 -actggagaac ggcttccttatcg-30 (reverse), and incorporation of [a-32P]dCTP (Amersham). All positive colonies were converted from the kTriplEx2 construct into the pTriplEx vector. The isolated plasmids were sequenced, and all cDNA sequences were mapped onto the human genome sequence with BLAST searching. Cells and cell culture. The human embryonic kidney cell line HEK 293 and rat adrenal pheochromocytoma cell line PC12 were purchased from the American Type Culture Collection. Mouse embryonic stem cell (ESC) line AB 1.1 was provided by Dr. P. E. Hasty of The University of Texas Health Science Center at San Antonio. The HEK 293 cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). The PC12 cells were maintained in F12K medium with 15% horse serum and 2.5% FBS. The AB1.1 ESCs were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder cell layer in Knockout-qualified DMEM medium with 15% FBS. All media, sera, and antibiotics were purchased from Invitrogen. Gene suppression assay. Two siRNAs (5siRNA and 3siRNA) targeting the 50 and 30 ends of human IFT80-L (or TRIM59 and IFT-80), respectively, were synthesized and purified by Dharmacon. The targeting sequences were 50 -aacattcttcaggcatctggt-30 and 50 -aacc caacactggcagcatat-30 , respectively. Transient transfection was carried out with LipoFectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Triple transfections were performed for each siRNA. Cells were analyzed 48 h after transfection. Gene expression assay. For quantitative real-time reverse transcriptase (RT)–PCR analysis, the TaqMan method was applied in the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Briefly, total RNA was isolated from cells with Trizol (Invitrogen) and reverse-transcribed into cDNA with SuperScript II RNase H- and random hexamer (Invitrogen). The quantitative real-time PCR was then conducted with a set of TaqMan probes. Specifically, four sets of probes were designed for both the 50 and 30 ends of human IFT80-L and mouse/rat Ift80-L: 50 -gagacctttac gaattccactcaagt-30 (forward), 50 -tgccagttggagcaatttca-30 (reverse) and FAM-ccctaattgcagaagtatt-MGB (probe) for human 5Probe; 50 cgccaacgtgtgcagatg-30 (forward), 50 -tgcttactcgaggataaatttcaaca-30 (reverse) and FAM-aaacagagaccacttcc-MGB (probe) for mouse/rat 5Probe; 50 -aatgggaagatgctgtgagacttt-30 (forward), 50 -agctagacaagc ccacatggtt-30 (reverse) and VIC-tcgctttgttaaggagc-MGB (probe) for human 3Probe; 50 -agcaggcaaacagctcatca-30 (forward), 50 -tcatg ggctttccactgtaaaa-30 (reverse) and VIC-ctcttcaaccaaatgcta-MGB (probe) for mouse/rat 3Probe. A standard PCR amplification protocol was used: 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 25 s at 95 °C and 1 min at 60 °C. All gene expression levels were normalized to an intrinsic control of 18s ribosome RNA. Mutiple-tissue cDNA panel assay. Human multiple-tissue cDNA panel was purchased from Clontech. A pair of primers, 50 -gggatgcttagatcaactttagctc-30 (forward) and 50 -agatcaaatataattggtgtgttc-30 (reverse), was designed to bridge two exons in the 30 end of IFT80-L, as well as in IFT80, and was used for the PCR amplification. The control gene, encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified for 26 cycles for a product of 983 bp with a pair of primers supplied by the manufacturer (Clontech). Gene(s) IFT80-L and IFT80 were amplified for 35 cycles for a product of 704 bp. The PCR procedure was as follows: 2 min at 94 °C followed by cycles of 30 s at 94 °C, 15 s at 55 °C, and 1 min at 72 °C. Cell differentiation. Rat PC12 cells were cultured on collagen IVcoated plates or flasks (BD Biosciences) for rapid proliferation. For differentiation induction, PC12 cells were transferred to collagen
I-coated plates and treated for 5 days with nerve growth factor (NGF; Clontech) at a final concentration of 100 ng/ml in medium containing only 0.5% FBS. Control PC12 cells were maintained on collagen I-coated plates in a medium with 10% FBS and without NGF. Media were replaced every other day. To eliminate the feeder cells, mouse AB1.1 ESCs were cultured with ESGRO LIF (Chemicon) 1000 U/ml and basic fibroblast growth factor (bFGF; Invitrogen) 100 ng/ml in Knockout-qualified DMEM medium with 15% FBS. For differentiation induction, ESCs were first allowed to aggregate for 1 day in Petri dishes with no ESGRO LIF or bFGF added, and then induced with NGF 100 ng/ml for 2 days. The embryoid bodies that formed were further dissociated and transferred to tissue culture plates, and the cells were induced with NGF 100 ng/ml for another 6 days in medium containing only 0.5% FBS. Media were replaced every other day.
Results In screening a human brain cDNA library for homolog of the mouse RING finger gene, Trim59 [16], we identified multiple positive clones. Sequence analysis revealed five transcripts for human homolog TRIM59 and an additional three transcripts that were distinct from TRIM59 at the 30 end. After mapping these transcripts onto the human genome, we found that all their introns had the consensus splicing donor and acceptor nucleotides, with a ‘‘GT” at the 50 end and an ‘‘AG” at the 30 end, just as documented for other eukaryotic genes. In addition, we found that the three cDNA clones differing from TRIM59 at the 30 end represent a fusion of two genes, TRIM59 (NM_173084) and IFT80 (NM_020800), as annotated in human genome databases. The clones contained the 50 exons from TRIM59 and the 30 exons from IFT80. In an open reading frame analysis, we found that these cDNA sequences encode a predicted protein consisting of a RING finger, a B-box, and a coiled-coil motif (RBCC motif or tripartite motif; TRIM) at the N-terminus and seven WD-40 repeats at the C-terminus (Fig. 1). In this report, we name this newly identified gene IFT80-L, encoding the long isoform of IFT80. In Fig. 1, we provide the repertoire view of the genomic structures of TRIM59, IFT80, and IFT80-L and the predicted motifs for their corresponding encoded proteins. The complete cDNA sequence of IFT80-L is approximately 3.8 kb, maps to human chromosome 3q26.1, and spans more than 200 kb with 21 or 22 exons. The predicted protein, IFT80-L, consists of 1080 amino acid residues. The genomic structures of TRIM59, IFT80, and IFT80-L indicate that TRIM59 and IFT80-L probably are regulated by the same promoter, whereas IFT80 may have its own promoter, notably because there are some exons at the 50 end that cannot be found in transcripts of IFT80-L. Moreover, in BLAST searching of human genome databases, we found two cDNA clones, BC033011 and BC068444, that also contain partial sequences from both TRIM59 and IFT80, similar to our IFT80-L but lacking three exons in the middle of the sequence. Their resultant predicted protein consists of 948 amino acid residues but has only five WD-40 repeats at the C-terminus, likely representing a truncated protein of IFT80-L. The C-terminus of IFT80-L shares the protein sequence of IFT80. Like other IFT proteins, IFT80 is conserved in all ciliated organisms, and its orthologs have been identified in lower organisms such as green algae, nematodes, fruit flies, and zebrafish, as well as higher organisms such as mouse, rat, and human [11]. Demonstrated in Fig. 2 is an alignment of the protein sequences of IFT80 orthologs in human, mouse, rat, nematode, and fruit fly. The sequence analysis reveals that human IFT80 has 41% identity and 55% similarity to Che-2 in Caenorhabditis elegans and 37% identity and 50% similarity to Oseg5 in Drosophila melanogaster, both of which are recognized as one of IFT components essential for the assembly and
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Fig. 1. Genomic structures of IFT80-L and its relevant genes, TRIM59 and IFT80. In middle, exons of IFT80-L, TRIM59, and IFT80 are illustrated approximately in position on human chromosome 3, spanning a region of more than 200 kb. Genomic structure of novel IFT80-L is demonstrated at bottom, whereas those of previously annotated TRIM59 (NM_173084) and IFT80 (NM_020800) are shown at top. Filled regions in cDNA sequences represent the open reading frames. Motifs of proteins are predicted from the SMART protein sequence analysis tool (http://smart.embl-heidelberg.de/). Target sequences of siRNAs for gene suppression and TaqMan probes for gene detection are marked approximately on IFT80-L cDNA sequence. Also defined are the N-terminal TRIM module (amino acids 1–318) and the C-terminal IFT80 module (amino acids 319–1080) of IFT80-L.
maintenance of cilia [10,18,19]. The identities of rodent orthologs of human IFT80 exceed 94%. In comparative genomic and proteomic analyses, IFT80, Che-2, and Oseg5, along with other IFT proteins, were included in the flagellar and basal body proteome [20,21]. Although our identified cDNA clones already provide evidence that IFT80-L probably does not represent chimeric clones resulting from experimental contamination, we still wanted to determine whether IFT80-L exists in cells as a truly single gene. To tackle this issue, we employed the double small interfering RNA (siRNA) suppression technique. We designed two siRNAs (5siRNA and 3siRNA) targeting the 50 and 30 ends of IFT80-L (or TRIM59 and IFT80 two genes), respectively (Fig. 1). After introducing the two siRNAs separately into human embryonic kidney cells (HEK 293), we measured changes in gene expression with two TaqMan probes (5Probe and 3Probe) targeting as well the 50 and 30 ends of IFT80L (or TRIM59 and IFT80), respectively (Fig. 1). The 5siRNA targeting TRIM59 suppressed both TRIM59 and IFT80 expression, as did the 3siRNA targeting IFT80 (Fig. 3A). Such coordinated expression changes in the siRNA suppression assays demonstrate that IFT80L is indeed a merger of TRIM59 and IFT80 and exists in HEK 293 cells. To establish the expression pattern of IFT80-L in humans, we applied a PCR assay to a commercially available multiple-tissue cDNA panel. We used a pair of primers bridging two exons in the C-terminus of IFT80-L as well as IFT80. Our expression analysis revealed that IFT80-L or IFT80 is expressed in almost all human tissues examined, most abundantly in kidney, pancreas, thymus, testis, and ovary, but only sparsely in skeletal muscle (Fig. 3B). Such an
expression pattern is similar to that reported previously for IFT80 (KIAA1374) based on Northern blot analysis [22], except that the PCR analysis we used is more sensitive so that we detected expression in more tissues. Transcription factor RFX regulates the expression of most IFT proteins [23,24]. It has been shown that IFT proteins (Ift88 and Ift52), IFT motor proteins (dynein 2 heavy chain and light intermediate chain 3), and transcription factor Rfx3 are expressed preferentially in the ependymal layer along the ventricular or subventricular zones [25–27], where neural progenitors or precursor cells exist with proliferation and differentiation. We thus suspected that IFT proteins might be involved in the process of cell proliferation and differentiation. To test our speculation, we induced differentiation with NGF in rat PC12 cells, a widely used model of in vitro irreversible neuronal differentiation. Examining with TaqMan probes for the 50 and 30 ends of Ift80-L, we found that expression of Ift80-L was significantly attenuated after PC12 cells differentiated into neuron-like cells (Fig. 3C). Because pluripotent ESCs and progenitor cells of the central nervous system origin are conserved in the induced promotion of neural cells, we further induced mouse ESCs AB1.1 into neurotypic cells with NGF. Similar to rat PC12 cells, mouse ESCs showed a decrease in Ift80-L expression after they experienced embryoid body formation and NGF-induced neuronal differentiation (Fig. 3C). Differentiation in both rat PC12 cells and mouse ESCs is preceded by withdrawal from the cell cycle. Thus, we conclude that Ift80-L may play a role in cell proliferation and differentiation. Because we used both the 50 and 30 end probes to detect expression changes and the results demonstrated a similar trend in expression changes for both the 50 and 30 ends of
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WD-40 repeat 2
mouse rat human nematode fruit fly
1 1 1 1 1
~~~~~~~~~~~~~~MRLKISLSKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETSLIVKLPDDIYPIDLHWF...PKSLGIKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MRLKISLLKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETSLIVKLPDDIYPIDLHWF...PKSLGIKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MRLKISLLKEPKHQELVSCVGWTTAEELYSCSDDHQIVKWNLLTSETTQIVKLPDDIYPIDFHWF...PKSLGVKKQTQAESFVLTSSDGKFHLISKLGRVEKSVEAHCGAVLAGRWNYEGTALVT ~~~~~~~~~~~~~~MKLKLSASRKTRHTEMVCGVGWIGTEAILSAADDHVFLLTNTATNESQQILNMPETFFPTSLHIF...PRSQ..TKGGQNDVFAVSTSDGKINILSRNGKVENMVDAHNGAALCARWNSDGTGLLS MKLKTRICKKNSIRNSEKVEESSKSTTNCPLSCVDWSSNEEIYFVSDDHQIFKWSDVSRDSVEVAKLPDDFVPTDMHWLLLGGRSSGGGKG..SDTLLICSNDGRFVILNKSARVERSISAHAAAISSGRWSPDGAGLLT
mouse rat human nematode fruit fly
124 124 124 122 139
VGEDGQVKIWSKTGMLRSTLAQQGTPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRVLYGSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA VGEDGQVKIWSKTGMLRSTLAQQGIPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRVLYGSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA VGEDGQIKIWSKTGMLRSTLAQQGTPVYSVAWGPDSEKVLYTAGKQLIIKPLQPNAKVLQWKAHDGIILKVDWNSVNDLILSAGEDCKYKVWDSYGRPLYNSQPHEHPITSVAWAPDGELFAVGSFHTLRLCDKTGWSYA SGEDGFVKMWSRSGMLRSVLAQFATAVYCVAWDSTSSNVLYCNADHCYIKSLKMQVAPIKWKAHDGIILCCDWNPTSDLIVTGGEDLKFKVWDGFGQILFNSSVHDYPITSISWNTDGTLFAVGSHNILRLCDKSGWSHS AGEDGVIKIWSRSGMLRSTVVQNEESIRCARWAPNSNSIVFCQGGHISIKPLAANSKIIRWRAHDGLVLSLSWSTQSNIIASGGEDFRFKIWDAQGANLFTSAAEEYAITSVAFNPEKD.YLLWSYNTAR..........
WD-40 repeat 3
WD-40 repeat 4
WD-40 repeat 5
WD-40 repeat 6 mouse rat human nematode fruit fly
264 264 264 262 268
LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQRWEWKNFQVTLTKRRTMQVRNVLND.AVDLLEFRDRVIKASLNHAHLVVSTSLQCYVFSTKNWNTPLIFDLKEGTVSLILQAERHFLLVDGGGIYLHSYEGR LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQRWEWKNFQVTLTKRRTMQVRNVLND.AVDLLEFRDRVIKASLNHAHLVVSTSLQCYVFSTKNWNTPLIFDLKEGTVSLILQAERHFLLVDGGGIYLHSYEGR LEKPNTGSIFNIAWSIDGTQIAGACGNGHVVFAHVVEQHWEWKNFQVTLTKRRAMQVRNVLND.AVDLLEFRDRVIKASLNYAHLVVSTSLQCYVFSTKNWNTPIIFDLKEGTVSLILQAERHFLLVDGSSIYLYSYEGR LEKMNAGSVMALSWSPDGTQLAVGTAAGLVFHAHIIDKRLTYEEFEIVQTQKTVIEVRDVSSEVSRETLETKERISKIAILYKYLIVVTSSHIYIYSSKNWNTPTMIEYNERTVNIIVQCEKIFLVSDGMTITIFTYEGR FSSPRVGSIFNLSWSADGTQATCGTSTGQLIVAYAIEQQLVSGNLKATSKSRKSITLKDIAT.GTQDILDFPQRVVSFGLGYGHLVVATTNQVHIYNEKYINTPIIIDGRNDT.RVIEVGKKYFMILDALSIWVYTYTGR
mouse rat human nematode fruit fly
403 403 403 402 406
FISSPKFPGMRTDI..LNAQTVSLSNDTIAIKDKADEKIIFLFEASTGKP.LGDGKLLSHKNEISEIALDQKGLTNDRKIAFIDKNRDLYITSVKRFGKE..EQIIKLGTMVHTLAWCDTCNILCGIQDTRFTVWYYPNT FISSPKFPGMRTDI..LNAQTVSLSNDTIAIKDKADEKIIFLFEASTGKP.LGDGKLLSHKNEISEVALDQKGLTNDRKIAFIDKNRDLYITSVKRFGKE..EQIIKLGTMVHTLAWCDTCNILCGLQDTRFTVWYYPNA FISSPKFPGMRTDI..LNAQTVSLSNDTIAIRDKADEKIIFLFEASTGKP.LGDGKFLSHKNEILEIALDQKGLTNDRKIAFIDKNRDLCITSVKRFGKE..EQIIKLGTMVHTLAWNDTCNILCGLQDTRFIVWYYPNT KLINLNPPGQVMAL..LDERKIDLANDTLVVRDRADNKVLHFFDPTTGKA.QGDGN.LKHEHDIVELTVNQCGPLNDRNVAFRDQIGAVHIAMVKTFGVS..QRMVKIGSLVEQLVFNDVTNMLCGISEGKIAVWPLPNV LHLNPRYPGSQAQIPLLTWRSLSLGLDVLAIRDNSDPTVLHLFDLIPGASRQYDPHSLRAKQQLVQIAACRAGSPEDQFVAFIDSNRELFVSESRNLNSERIDEIYKIGTQLTAIMWASETNILVGVHDSCYSIWYCPGE
mouse rat human nematode fruit fly
538 538 538 536 546
IYVDRDILPKTLYERDASEYSKNPHIVSFVGNQVTIRRADGSLVHISISPYPAILHEYVSSSKWEEAVRLCRFVKEQSMWACLAAMAVANRDMVTAEIAYAAVGEIDKVRYINAIKDL.PSRESKMAHILMFSGNIQEAE VYVDRDILPKTLYERDASEYSKNPHIVSFVGNQVTVRRADGSLVHISISPYPAILHEYVSSSKWEDAVRLCRFVKEQSLWACLAAMAVANRDMVTAEIAYAAVGEIDKVRYINAMKDL.PSRESKMAHILMFSGNIQEAE VYVDRDILPKTLYERDASEFSKNPHIVSFVGNQVTIRRADGSLVHISITPYPAILHEYVSSSKWEDAVRLCRFVKEQTMWACLAAMAVANRDMTTAEIAYAAIGEIDKVQYINSIKNL.PSKESKMAHILLFSGNIQEAE AFHDRNLLQKSLIQKNIGSVGKFPQLANFAGNTIVIRKSDGCLLPTGILPFYGTLITMASQSKWDQAIRLCRSIGNDTMWATFAGLAVLHKNMIVMEIAYAALEDDEKVSLINEIKDK.TDKETRQAMQVVLTGKLADAD GASDPTIIALTTITLDTTEFGKHITIESFEESVLTFRSA.GALLPVNVNMYCEILHRALLEGQWQQALKICRMGQHSSLWATLAAVATRKHQLQISEEAYSAALQIDKVSYLQHLKALTPSSAEQMAENSLMLGRMLEAE
mouse rat human nematode fruit fly
677 677 677 675 685
TVLLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLDTFGKQETNKRYLQYAEGLQIDWEKIKAKIEMEITKERDRSSSGQSSKSVGLKH TILLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLETFGKQETNKRYLQYAEGLQIDWEKIKAKIEMEITKERDRSSSGQSSKNTGLKP IVLLQAGLVYQAIQININLYNWERALELAVKYKTH....VDTVLAYRQKFLETFGKQETNKRYLHYAEGLQIDWEKIKAKIEMEITKEREQSSSSQSSKSIGLKP VLLERSGLSFRSLMLNIQMFKWKRALELGLKNKQW....LEIVMGYREKYLKNCGQKETDPLFLKHMSEVEIDWVHIRELIAAEKAKGNN~~~~~~~~~~~~~~~ TILLHGKKIEQAVGLALRMHNWRRALEISQKHKGEQPELVPRVLQERRKYLKALQREEWDPLYLPLVAKEEADNTSE~~~~~~~~~~~~~~~~~~~~~~~~~~~~
WD-40 repeat 7
777 777 777 760 761
[94.7%] [94.3%] [100%] [41.3%] [37.2%]
(NP_080917) (NP_001013933) (NP_065851) (NP_508106) (NP_610064)
Fig. 2. Alignment of protein sequences of IFT80 orthologs. Human IFT80 (NP_065851) is compared with mouse (NP_080917) and rat (NP_001013933) orthologs, as well as Che-2 in C. elegans (NP_508106) and Oseg5 in D. melanogaster (NP_610064). Also illustrated are seven WD-40 repeats predicted on the basis of human IFT80 protein sequence.
Ift80-L in differentiation, we suspect that Ift80-L exists in rat PC12 cells and mouse AB1.1 ESCs. Discussion We report here the identification of a long isoform of IFT80 protein encoded by IFT80-L. In human genome databases, IFT80-L was previously annotated as two separate genes, TRIM59 and IFT80 (Fig. 1). We demonstrate in this report that IFT80-L is in fact a single gene that represents a merger of TRIM59 and IFT80. Not only did we screen out the complete cDNAs from a human brain cDNA library, but we confirmed it with double siRNA suppression (Fig. 3A). To some extent, the presence of BC033011 and BC068444, two cDNAs sequences, in human genome databases provided additional evidence that IFT80-L was not generated from chimeric clones. Moreover, in mouse ESCs and rat PC12 cells, expression of both the 50 and 30 ends of Ift80-L was reduced at the similar rate during NGF-induced cell differentiation (Fig. 3C). Thus, evidence is piling up that IFT80-L exists in human beings as well as in rodents. Protein IFT80-L probably is an evolved Rosetta stone protein, converged from TRIM59 and IFT80. IFT80 is highly homologous to Che-2 in C. elegans, Oseg5 in D. melanogaster, TtIft80 in Tetrahymena thermophila, and ift80 in zebrafish [11,12,17,18] and is evolutionarily conserved, as are other IFT proteins in ciliated organisms [11]. However, we could not find any evidence of an IFT80-L ortholog in lower organisms. In view of this, we postulate that IFT80-L is present only in higher organisms such as rodents and humans, whereas IFT80 represents the remnant of evolution. Interestingly, we cannot find a canonical X-box sequence, a conserved cis-element for the binding of transcription factor RFX, in the promoter region of IFT80-L or IFT80. In contrast, well-defined X boxes have been identified in the promoters of Che-2 and Oseg5 [23,24]. This
finding also indicates the evolutionary difference between lower and higher organisms in expression regulation of IFT80 orthologs. Protein IFT80/IFT-L is one of the IFT components and is important for the assembly and maintenance of cilia [17–19]. The C. elegans mutants of che-2 have extremely short cilia in most ciliated sensory neurons and show defects in various behaviors such as chemotaxis and osmotic avoidance [19]. Disruption of TtIft80 in T. thermophila results in short, aberrant, or absent cilia; multiple nuclei and oral apparatuses; disorganized cortical ciliary rows; and cytokinesis defects [17]. In zebrafish, knockdown of ift80 produces large cystic kidneys accompanied by pericardial edema [17], a phenotype similar to the results of mutations of other IFT components [13]. Thus far, most IFT80 orthologs, including Che-2 [19], TtIft80 [17], and Oseg5 [18], have been characterized to localize to the cilium, basal body, and/or centrosome, just as do other IFT proteins [1,12,18]; and Che-2 has been shown to move along the ciliary axoneme at the same rate as other IFT proteins and IFT motor proteins [28,29]. The IFT motility is important for cilia formation and animal development. Disruptions of IFT proteins block sonic hedgehog developmental signaling in the neural tube, to cause defects of motile and nonmotile cilia on the ventral node of mouse embryo, and further, to lead to errors in embryo development [30]. The importance of IFT80/IFT80-L in development is recently evidenced by a finding that recessive mutations in IFT80/IFT80-L are linked to human disease JATD, an early development disorder that often leads to death in infancy [17]. We demonstrate in this report that Ift80-L is highly expressed in rapidly proliferating neural progenitor cells, but is attenuated after cell differentiation (Fig. 3C), and propose that IFT-L plays a role in early animal development. In summary, we identify here a long isoform, IFT80-L, in humans. Both IFT80 and IFT80-L have the same C-terminal sequence, with seven WD-40 domains, and are a human homolog of an IFT
W. Huang et al. / Biochemical and Biophysical Research Communications 373 (2008) 653–658
IFT80-L mRNA Expression (%)
A
657
Control 5probe 3probe
100
80
60
40
20
0 5-siRNA
3-siRNA
B
IFT80-L
C
IFT80-L mRNA Expression (%)
GAPDH
w/o NGF w/ NGF, 5-probe w/ NGF, 3-probe
100
80
60
40
20
0 PC12
AB1.1
Fig. 3. (A) Existence of IFT80-L in HEK 293 cells. Cells were transiently transfected with 5siRNA and 3siRNA, respectively. Gene expressions were detected by two TaqMan human probes using quantitative real-time RT-PCR. (B) Expression profile of IFT80-L and/or IFT80 in human tissues. A pair of primers bridging two exons in the 30 end of IFT80L as well as in IFT80 was used for PCR amplification of a human multiple-tissue cDNA panel. Gene GAPDH was used as a control. (C) Ift80-L is down-regulated after cell differentiation. Rat PC12 cells and mouse AB1.1 embryo stem cells were induced with NGF for neurotypic differentiation. Expressions of Ift80-L were detected by two TaqMan mouse/rat probes using the quantitative real-time RT-PCR. Data in (A) and (C) are shown as means ± SD.
component. We also demonstrate that IFT80/IFT80-L is expressed in many human tissues and potentially involved in cell proliferation and differentiation. Although IFT80-L/IFT80 has been associated with IFT motility and has been linked with a human disease, its function in mammals is still largely unknown. It will be of considerable interest to investigate the effects of complete loss of Ift80-L or Ift80 in a mouse model. As Ift80-L and Ift80 might be essential in cell differentiation and animal early development, we speculate that a conditional knockout of Ift80-L or Ift80 might be more feasible and fruitful. Acknowledgments We are grateful to Dr. Paul E. Hasty of the University of Texas Health Science Center at San Antonio for providing AB1.1 ESCs and assistance in ESC culture. This project was in part supported by NIH grants DA-12844 and DA-13783 to M.D.L.
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