Human Molecular Genetics, 2000, Vol. 9, No. 14 2085-2093
© 2000 Oxford University Press
Identification of a novel protein interacting with RPGR
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Received 4 May 2000; Revised and Accepted 6 July 2000.
DDBJ/EMBL/GenBank accession no. AF260257.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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A novel protein, called RPGRIP, has been identified as interacting with the RPGR protein, which is mutated in a severe form of human retinal degeneration, X-linked retinitis pigmentosa (RP3 type). The bovine RPGRIP was identified initially by screening for RPGR-interacting proteins with a bovine retina cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by co-immunoprecipitation of in vitro translated protein and using RPGR mutants. The human RPGRIP gene was isolated and shown to be expressed in retina and testis. Human RPGRIP spans a genomic interval of 34 kb, and consists of 15 exons, some of which are alternatively spliced. It was mapped using monochromosomal and radiation hybrid cell lines to chromosomal region 14q11. The function of RPGRIP is unknown; it shows no homology to proteins of known function, although it is predicted to form two coiled-coil domains at the N-terminus. RPGRIP is a strong candidate gene for causing human retinal degeneration.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations which affects 1 in 4000 of the general population (1,2). Over 30 different genes have been implicated in the disease, of which about half have been identified (http://www.sph.uth.tmc.edu/RetNet/disease.htm ). X-linked RP (xlRP) affects 10–20% of patients in most populations and is one of the most severe forms of RP, with a prevalence in the region of 1 in 25 000 (3,4). xlRP has been genetically mapped to six loci, RP2 (5), RP3 (6), RP6 (7), RP15 (8), RP23 (9) and RP24 (10). The RPGR gene is mutated in the RP3 form of xlRP, which accounts for 70–80% of patients (11). The N-terminal half of RPGR shows homology to RCC1, a guanine nucleotide exchange factor (GEF) for the small nuclear GTPase Ran, which is concerned with nucleocytoplasmic transport and cell cycle control (6,12–13). RPGR shows a complex pattern of alternative splicing, although disease-causing mutations are confined to a single low abundance transcript, consisting of the RCC1-like domain and a novel acidic domain of unknown function (11). The RCC1 domain occurs in several other proteins, including p532 (14), which appears to act as a GEF for Rab and Arf GTPases, suggesting that this domain may generally indicate GEF activity. The crystal structure of RCC1 shows a seven-bladed ß-propeller structure, which is similar to the predicted structure of RPGR (15).
The yeast two-hybrid system (16,17) allows the identification of interacting partners for a gene of interest by exploiting the modular nature of transcription factors. Two potentially interacting proteins are expressed in Saccharomyces cerevisiae, one of which contains the protein of interest (bait) fused to the DNA-binding domain of a yeast transcription factor such as GAL4, whereas the other is fused to the corresponding activation domain. Binding of the bait to an interacting partner leads to activation of the DNA-binding domain and creates a functional transcription factor, which activates downstream reporter genes.
Recently, the yeast two-hybrid system was used to screen a mouse embryo cDNA library using the first 392 amino acids (RCC1-like domain, RLD) of RPGR as bait (18). The 
-subunit of rod cGMP phosphodiesterase (PDED) was identified as an RPGR-interactor in this way. Rod cGMP phosphodiesterase is a component of the visual transduction cascade of vertebrate photoreceptor cells (19). PDED is an abundant, ubiquitous and highly conserved protein which is proposed to have a role in the solubilization of specific membrane proteins (20). The role of RPGR in this interaction is unknown, but it is interesting that PDED also interacts with small GTPases Rab13 and Arl3 (21).
Here, a bovine retina cDNA library has been screened with the RCC1-like domain of RPGR (RPGRRLD) as bait in the yeast two-hybrid system, in order to identify RPGR-interacting proteins. A novel bovine protein was identified which bears no homology to any characterized protein. The human homologue was identified and characterized and its product shown to interact with RPGR in a specific manner, using mutant constructs and in vitro co-immunoprecipitation. The human gene is expressed in a subset of tissues, including testis and retina, and was mapped to chromosomal region 14q11.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Yeast two-hybrid library screening
A GAL4-based yeast two-hybrid system was used to search for proteins that interact with RPGR. A bovine retina cDNA library (22) containing 2 x 106 independent clones was screened using the first 12 exons of RPGR, including the whole of the RCC1-like domain (RPGRRLD), as bait. The majority of the published RP3 mutations are found in this domain, suggesting an important functional role for this part of the protein (23). Selection for interaction was performed in the Y190 strain, which has conditional lacZ and HIS3 reporter genes. Screening 2 x 106 clones led to the identification of 10 clones showing ß-galactosidase activity and histidine prototrophy. Library plasmids were isolated from 8 of the 10 yeast colonies (the remaining 2 were resistant to plasmid recovery) and re-tested for the ability to interact with RPGR. Seven of the eight gave strong positive results and were sequenced. Six of them were found to contain the same insert of 804 bp.
The positive clones were tested for the ability to activate the Y190 reporter genes in the absence of the RPGR bait plasmid and also for the ability to interact with RCC1, Ran and SNF1 (an unrelated protein) (Fig. 1A). The results of these tests were negative, suggesting that the original positive result was specific for and dependent on the presence of RPGR.
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Isolation of human RPGRIP
A search of the expressed sequence tag (EST) databases for sequences similar to the bovine interactor identified nine human and seven mouse previously uncharacterized homologues, which showed 86% (human) and 76% (mouse) nucleotide identity to the bovine sequence. A human IMAGE EST clone was obtained (accession no. r93221), and the 678 bp insert, which contained an open reading frame (ORF), was subcloned into the pACTII vector. This human clone was tested as a candidate RPGR-interactor in the yeast two-hybrid system (Fig. 1A). It was found to interact with the N-terminal, RCC1-like domain but not with the C-terminal domain of RPGR (the last seven exons), or with RCC1, Ran, PDED or SNF1. In addition, it did not activate the Y190 reporter genes in the absence of a bait construct. The new RPGR-interacting protein was given the name RPGRIP (RPGR-interacting protein).
A partial human RPGRIP (hRPGRIP) cDNA was obtained by PCR amplification of a human testis cDNA using primers derived from overlapping ESTs. 5' and 3' rapid amplification of cDNA ends (RACE) experiments were carried out to extend the sequence. Using a primer complementary to the 5' end of human EST r93221, a 1.2 kb 5' RACE product was amplified from a human testis Marathon Ready cDNA pool. This was sequenced on both strands and extended the ORF to 1758 bp. The nucleotide context of the first ATG triplet conforms well to the Kozak consensus (24) (Fig. 2). 5' RACE experiments using human retina cDNA identified a transcript starting 12 bp downstream of the testis transcript. Like the testis 5' RACE product, the retina transcript contains in-frame stop codons upstream of the translation initiation codon, consistent with it containing the first exon.
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Similarly, 3' RACE experiments identified a transcript extending 84 bp beyond the end of the ORF. Neither of the two most commonly used hexameric polyadenylation signals (AATAAA and ATTAAA) is present at the 3' end of the cDNA; however, 31 bp upstream of the 3' end of the RACE product is a hexamer that differs from the canonical AATAAA by a single nucleotide (AGTAAA), which according to Graber et al. (25) is the fifth most commonly used hexameric polyadenylation signal. In addition, eight of the nine human ESTs located at the 3' end (ai964059, ai655818, ai632512, ai05922, aa782963, aw681763, aa928161 and w26173) terminate 81–83 bp 3' to the end of the termination codon, consistent with this being the correct polyadenylation signal, since each EST was oligo(dT) primed. The remaining EST (aa476670) is also oligo(dT) primed but ends 315 bp 3' to the end of the termination codon. Immediately 3' to the end of this EST is an A-rich region (17/20), suggesting that an extended transcript has false-primed within this region. The length of the cDNA from the start of the 5'-untranslated region (UTR) to the major polyadenylation signal is 1909 bp, of which 1758 bp are an ORF, sufficient to code for a 586 amino acid protein with a predicted size of 67 kDa (Fig. 2).
Genomic structure of hRPGRIP
The genomic structure of the hRPGRIP gene was obtained from a 196 kb sequence submission (GenBank accession no. AL135744) from a chromosome 14 contig, which includes the entire hRPGRIP gene sequence. The exon–intron boundaries and intron sizes are shown in Table 1. Twelve exons were detected initially. The boundaries conform to the GT/AG rule (26), with the exception of the splice donor site at the end of exon 4. The sequence at the exon 4–intron 4 junction was checked repeatedly and confirmed as CTG
gcaagt. It is rare to observe a GC instead of the conserved GT present at most splice donor sites, but other examples of functional splice sites with this sequence have been reported (27).
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A panel of bioinformatics programs was applied to the genomic sequence containing RPGRIP. The GENSCAN program (28) strongly predicted three extra exons in the middle of intron 4 that had not been detected in cDNAs or RACE products using primers flanking this region. Twelve exons were also predicted to lie upstream of the first detected exon. Primers were designed to anneal to these exons and were used in conjunction with primers from downstream exons. Human retina-derived cDNA was used as the template source in attempts to amplify these exons by PCR. The three exons predicted to lie in intron 4 were detected and maintained the ORF of the shorter transcript, but the 12 upstream exons were not detected. The boundaries of the new exons were sequenced at the genomic level and found to have canonical splice site boundaries (Table 1), as predicted by GENSCAN. The extra exons extend the cDNA from 1946 to 2894 bp and the ORF to 2706 bp, predicting a protein of 902 amino acids and a size of 97 kDa.
The full-length hRPGRIP cDNA and intron–exon boundaries are shown in Figure 2A and a schematic representation of the two detected major transcripts in Figure 2B. The arrow in Figure 2B indicates the position of the non-canonical splice site at the exon 3–exon 4 junction. It is interesting to note that the three exons immediately 3' of this splice site are absent from PCR-amplified RPGRIP transcripts.
Interaction of hRPGRIP with mutant RPGR peptides
An experiment was carried out to determine whether the interaction between hRPGRIP and RPGR is disrupted when point mutations associated with xlRP are present in the RPGR coding sequence. Eight mutant RPGRRLD constructs were tested in the yeast two-hybrid system. The wild-type RPGRRLD was found to interact with RPGRIP, but the mutants showed either absent (V36F, G60V, H98Q, F130C, G215V, P235S and C250R) or slightly decreased (G215V and G275S) reporter gene activity (Fig. 1B). All of the disease-causing mutations tested, with the exception of F130C, are situated within conserved residues of the RPGRRLD (15), and are therefore likely to be important for correct folding and function.
Co-immunoprecipitation of RPGR and RPGRIP
The interaction between RPGR and the RPGRIP was confirmed by in vitro co-immunoprecipitation. In vitro transcribed/translated and 35S-labelled epitope-tagged RPGR, RPGRIP, Max and lamin proteins were prepared. From a mixture of RPGR-myc and RPGRIP-haemagglutinin (HA), it was possible to co-immunoprecipitate both proteins using either anti-myc or anti-HA antibodies (Fig. 3). From a mixture of RPGR-myc and Max-HA, only RPGR-myc was immunoprecipitated by anti-myc. Similarly, from a mixture of RPGRIP-HA and lamin-myc, only RPGRIP-HA was immunoprecipitated by anti-HA. These results suggest that the interaction between RPGR and RPGRIP is specific and not an artefact of the yeast two-hybrid system. The bands obtained with anti-c-myc mouse monoclonal antibody (lanes 1 and 3) are weaker than those with anti-HA rabbit polyclonal antibody (lanes 2 and 4), either because protein G, used to capture antigen–antibody complexes, binds more strongly to rabbit than to mouse antibodies or because the polyclonal antibody binds with higher avidity than the monoclonal (29).
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Expression of RPGRIP
A human multiple tissue northern blot (MTN2) was probed using radiolabelled RPGRIP cDNA. Two different transcripts were detected in the testis lane, a strong band of 2.0 kb and a weak band of 3.1 kb, but no signal was detected in spleen, thymus, prostate, ovary, small intestine, colon or peripheral leukocytes (Fig. 4). A second human multiple tissue northern blot (MTN1) was probed in the same way, and only a very weak 6.2 kb band was identified in the lane containing skeletal muscle RNA, which is presumed to be non-specific, but no signal was obtained in other tissues, including pancreas, kidney, liver, lung, placenta, brain and heart (data not shown). In addition, no signal was obtained with a northern blot containing human retina (data not shown). RPGRIP ESTs were identified from a limited range of human tissues, namely fetal liver/spleen (r93221), retina (w28191 and w26173), testis (ai015922 and aa782963), mixed fetal lung, testis and B-cell (aa928161 and aw081763) and pooled germ cell tumour (ai964059, ai655818 and ai632512).
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In order to carry out a more sensitive screen for low level RPGRIP expression, samples of human and bovine total RNA were used to amplify bovine RPGRIP by a single round of RT–PCR. Transcripts were amplified from human retina and testis RNA after one round of amplification (35 cycles) but not from adrenal, brain, heart, kidney, liver, lung or spleen RNAs (Fig. 5A). The lower retina band was sequenced and found to lack exon 12. One round of amplification was sufficient to produce a band of the correct size from the majority of the bovine samples, suggesting that the transcript is more widely expressed (Fig. 5B). When a second round of amplification was carried out (using a single nested primer), a signal was detected in most human tissues (data not shown), as in the case of bovine tissues, but this may be detecting illegitimate transcription products (30).
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Chromosomal mapping of hRPGRIP
Database searching identified a sequence-tagged site (STS h14a1407, GenBank accession no. G35988) with homology to RPGRIP. The STS contains 320 bp of RPGRIP genomic sequence covering the intron 12–exon 13 boundary and had been mapped to chromosomal region 14q11 in the course of HAPPY mapping of human chromosome 14 by random fragmentation of haploid cells (31).
A panel of human monochromosomal somatic cell hybrid DNAs was also screened by PCR to determine the chromosome in which RPGRIP is located. Human chromosome 14 was found to provide a template for amplification of a genomic fragment of RPGRIP, whereas none of the other panel samples, including the rodent control, produced fragments of the expected size.
In order to determine a more precise chromosomal location for RPGRIP, a PCR analysis was performed on the HGMP-RC subset of the Genebridge 4 radiation hybrid panel (32). The results were submitted to the Whitehead Institute and showed that RPGRIP is located between markers D14S264 and D14S275 (6.94 cRays from D14S264), which are located in 14q11.
Amino acid sequence analysis
The predicted RPGRIP amino acid sequence was analysed using the HGMP-RC PIX assembly of bioinformatics programs in an attempt to identify possible secondary or tertiary structural features. RPGRIP is predicted to be a soluble protein by the SOSUI system, which analyses proteins on the basis of the physicochemical properties of the amino acid sequence (33). The protein was analysed with the Coils program (34), which predicted RPGRIP to have two coiled-coil domains at the N-terminus, the first encompassing residues 14–78, and the second encompassing residues 150–200 (Fig. 2). BLAST and FASTA searches of dbSPTR identified only a single protein, KIAA1005 (accession no. AB023222), as homologous to RPGRIP. This is an uncharacterized protein of 1055 amino acids, which is 34% identical and 53% similar to RPGRIP (35). No other predicted motifs or domains were identified.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The yeast two-hybrid system is a widely used method for detecting interacting proteins and gaining insights into protein function and pathways (36). This is an in vivo method for detecting protein–protein interactions and requires confirmation with one or more independent methods, but provides a powerful means of screening large numbers of cDNA clones for interacting products (16,17). The function of the RPGR protein is unknown, although Linari et al. (18) previously showed by screening an embryonic mouse cDNA library using the yeast two-hybrid system that it interacts with PDED. This interaction was confirmed by in vitro protein binding (pull-down) assays and affinity measurements (KD = 90 nM), which was consistent with a physiological interaction. Although the function of PDED is still unclear, it has been shown to interact with small GTPases Rab13 and Arl3 (21) and may have a role in the solubilization of Rab13 from membranes (20). It has been postulated that RPGR also interacts with a small GTPase on the basis of its homology to RCC1, a GEF for the Ran GTPase; however, this has yet to be demonstrated.
It is not clear why PDED was not detected on two-hybrid screening of the bovine retina library, since it is known to be expressed in this tissue (37). Possible reasons include differences in the RPGR ‘bait’, a species difference or the exhaustiveness of library screening. Linari et al. (18) used an RPGR bait containing amino acids 1–392, whereas here a bait consisting of amino acids 1–502 was used (the RLD consists of residues 39–365). It seems unlikely that this difference would account for the failure to identify PDED. A second possibility is a species difference, since the RPGR–PDED interaction was detected by screening a mouse library with human RPGR, whereas a bovine library was used in this study. Human, mouse and bovine PDE are unusually well conserved, with 98% of amino acids identical between the three species (38). Only one conservative substitution of 150 residues separates bovine from human (Thr68Ala, human:mouse) and three substitutions separate mouse from human (Thr68Ala, Glu10Asp and Arg144Lys). This also seems unlikely, leaving the third possibility, namely a difference in transcript abundance between the mouse embryo and bovine retina libraries. An estimated 2 x 106 bovine retina clones were screened, but it remains a possibility that further screening would identify PDED.
Here, we identify a second interacting protein, RPGRIP, by screening a bovine retinal cDNA library with a clone containing the first 12 exons of human RPGR (RCC1-like domain) using the yeast two-hybrid system. Six of seven strongly positive clones were shown to contain fragments of the bovine RPGRIP gene, and the interaction was confirmed with human RPGRIP. Two full-length human cDNAs of 1.95 and 2.89 kb were identified, which appeared to be alternatively spliced orthologues of the bovine gene. The predicted products would yield proteins of 586 and 902 amino acids, respectively, with molecular masses of 67 and 97 kDa.
Further confirmation of the specificity of this interaction came from showing that six of eight xlRP disease-associated RPGR mutations abolish the interaction in yeast two-hybrid experiments. The failure of the G215V and G275S mutants to abolish this interaction, while both abolished interaction with PDED (18), is interesting. Conversely, the V36F variant, which is associated with X-linked congenital stationary night blindness (T. Meitinger, personal communication), does abolish the interaction with RPGRIP, which it does not for PDED (18). These results suggest that different RPGR subdomains within the RLD are likely to be involved in interaction with RPGRIP and PDED, and that the latter may be more important for full expression of the xlRP disease.
Further evidence relating to the specificity of the RPGR–RPGRIP interaction comes from expression studies. RPGR shows multiple alternatively spliced products in human, mouse and bovine tissues, most of which are widely expressed, but one of which (exon ORF14/15 transcript) is expressed exclusively in bovine retina and testis (11). Human retinal RPGR transcripts have only been found using RT–PCR, and show a complex pattern of expression, with two major and several minor transcripts (11). In northern analyses of multiple human tissue RNA samples, RPGRIP is only detectable in testis, although it is also readily detected in retina using more sensitive RT–PCR analyses (Fig. 5A). In bovine tissues, RPGRIP expression is detectable by RT–PCR in a wide range of tissues (Fig. 5B). This difference between human and bovine tissues may reflect a genuine species difference or a difference in mRNA quality. Human retina RNA could only be obtained 24 h post-mortem compared with <30 min for bovine tissues. The observation that RPGRIP could be detected in a wide range of human tissues following two rounds of RT–PCR amplification supports the latter possibility, although there is a danger of detecting illegitimate transcription products in this way (30). The predicted full-length RPGRIP cDNAs are 2.89 kb, which includes exons 5–7, and 1.95 kb if these exons are absent, consistent with the observed bands of 2.0 and 3.1 kb detected in testis by northern analysis. Failure to detect a signal in retina by northern analysis may again be related to mRNA quality.
The human RPGRIP gene is located on the long arm of chromosome 14 in band 14q11, a region containing no known retinal disease genes; however, only a minority of retinitis pigmentosa patients are accounted for by known or mapped genes (approximately one-third; http://www.sph.uth.tmc.edu/RetNet/disease.htm ). The amino acid sequence of RPGRIP gives little indication of its function. Sequence analysis indicates a soluble protein with two coiled-coil domains at the N-terminus of the protein and identifies an uncharacterized human homologue, KIAA1005. Further work will be necessary to elucidate its function, but RPGRIP remains a strong candidate gene for human retinal degenerations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Two-hybrid screening
A bovine retina cDNA library, kindly provided by Dr C.H. Sung, was used in the GAL4 yeast two-hybrid activation domain vector pACTII as previously described (22). The first 12 exons of RPGR were cloned into the two-hybrid bait vector pAS1 (39). The bait construct and the bovine retina library were then co-transfected into S.cerevisiae strain Y190 (40). Y190 (Trp–, Leu–, His–, gal4–, gal80–) contains two genomically integrated conditional reporter genes, HIS3 and LacZ, which are under the control of GAL4-responsive upstream activation sites. Transformants were plated onto synthetic complete medium lacking histidine, leucine and tryptophan, and grown at 30°C in the presence of 25 mM 3-aminotriazole. Putative positive colonies were selected by growth on medium lacking histidine and by expression of ß-galactosidase activity, as described previously (41). Plasmids were recovered from yeast colonies by glass bead homogenization and phenol–chloroform extraction (42). Library plasmids (containing a gene encoding leucine prototrophy) were amplified selectively in the leucine auxotrophic bacterial strain HB101 (Gibco BRL, Paisley, UK) grown on medium lacking leucine. Mutant RPGR constructs were obtained from Dr J. Becker (Max Planck Institut für Molekulare Physiologie, Dortmund, Germany) and the inserts subcloned into pAS1. These were then tested in the yeast two-hybrid system for their ability to interact with RPGRIP as described above. Ran and RCC1 were amplified by PCR from a cDNA library, subcloned into pAS1 and sequenced. As the positive control for yeast two-hybrid experiments, Y190 cells were transfected with pAS1.SNF1 and pACTII.SNF4. SNF1 and SNF4 are two yeast proteins known to interact (16).
Co-immunoprecipitation
The inserts from the pAS.RPGR and pACT.RPGRIP constructs were amplified by PCR. One of the primers used in each case contained a non-annealing 5' end encoding an epitope tag (myc in the case of RPGR and HA for RPGRIP). The amplified sequences were used to produce [35S]methionine-labelled proteins by in vitro transcription–translation using a TnT rabbit reticulocyte lysate system (Promega, Southampton, UK). The proteins were mixed and immunoprecipitated by the addition of either anti-myc or anti-HA antibody in the presence of protein G–agarose (Clontech, Basingstoke, UK). Control experiments were performed by mixing RPGR-myc with HA-tagged radiolabelled Max protein and RPGRIP-HA with myc-tagged lamin protein and immunoprecipitating with anti-myc and anti-HA, respectively. The immunoprecipitation products were separated on a 12% polyacrylamide gel, which subsequently was soaked in Amplify Fluorographic Reagent (Amersham Pharmacia Biotech, Little Chalfont, UK). The gel was dried and exposed to Biomax X-ray film (Kodak, Cambridge, UK) overnight.
Northern blot analysis
A PCR fragment encompassing the C-terminal 678 bp of RPGRIP was used to prepare a random primed 32P-labelled probe using the High Prime kit (Boehringer Mannheim, Mannheim, Germany). The probe was hybridized to human RNA blots (MTN1 and MTN2; Clontech) using the Ultrahyb (Ambion, Austin, TX) hybridization solution at 42°C overnight. The blot was washed twice for 5 min in 2x SSC, 0.1% SDS at 42°C and then exposed to a phosphor screen overnight.
Reverse transcriptase–PCR
The Access RT–PCR kit (Promega) was used to amplify human and bovine RPGRIP from samples of total human and bovine RNA. The reactions were carried out in an MJ Research (Waltham, MA) PTC-200 Peltier thermal cycler using the following programme: 48°C for 45 min, 94°C for 2 min, then 35 cycles of 94°C for 1 min, 53°C (human) or 60°C (bovine) for 1 min and 72°C for 2 min, then 72°C for 10 min.
RACE
Human retina and testis Marathon ready cDNAs (Clontech) were used to obtain 5' and 3' RACE products specific for RPGRIP. Two rounds of PCR were carried out using nested primers and the Expand High Fidelity proof-reading polymerase mix (Boehringer Mannheim) in a Perkin Elmer-Cetus (Foster City, CA) DNA thermal cycler. The sequences of the RACE products were determined on both strands.
Chromosomal assignment
A human monochromosomal somatic cell hybrid DNA panel (43) [UK Human Genome Mapping Project Resource Centre (HGMP-RC, Cambridge, UK)] and the HGMP subset of the Genebridge 4 radiation hybrid panel (32) were screened by PCR using a Perkin Elmer-Cetus DNA thermal cycler using the following programme: 94°C for 2 min, then 30 cycles of 94°C for 30 min, 52°C for 1 min, 72°C for 1 min, then 72°C for 7 min.
Protein prediction programs
The PIX suite of protein prediction programs was used (HGMP-RC; http://www.hgmp.mrc.ac.uk/Registered/Webapp/pix/ ) which includes prediction of cell localization [Psort (44)], solubility analysis [SOSUI (33)], BLAST searches against domain databases [SBASE (45) and PRODOM (46)], searches against motif and domain databases [Pfam (47), PRINTS (48), BLOCKS (49) and PROSITE (50)], transmembrane predictions [Tmpred (51), Tmap (52), DAS (53) and Phd (54)] and signal peptide predictions [Sigcleave (55)]. The protein was also analysed with the Coils program, which calculates the probability that a sequence will adopt a coiled-coil conformation (34). The four alternative options were run, i.e. using the MTK scoring matrix weighted and unweighted and using the MTIDK scoring matrix weighted and unweighted.
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ACKNOWLEDGEMENTS |
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We thank C.H. Sung for donating the bovine retina cDNA library, and J. Becker for donating the mutant RPGR constructs. We acknowledge the generous financial support of the Foundation Fighting Blindness, British Retinitis Pigmentosa Society and Guide Dogs for the Blind.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 131 467 8437; Fax: +44 131 343 2620; Email: alan.wright@hgu.mrc.ac.uk
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REFERENCES |
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