A Human Homolog of Yeast Pre-mRNA Splicing Gene, PRP31, Underlies Autosomal Dominant Retinitis Pigmentosa on Chromosome 19q13.4 (RP11)

Molecular Cell, Vol. 8, 375–381, August, 2001, Copyright 2001 by Cell Press

A Human Homolog of Yeast Pre-mRNA Splicing Gene, PRP31, Underlies Autosomal Dominant Retinitis Pigmentosa on Chromosome 19q13.4 (RP11) Eranga N. Vithana,1 Leen Abu-Safieh,1 Maxine J. Allen,2 Alisoun Carey,2 Myrto Papaioannou,1 Christina Chakarova,1 Mai Al-Maghtheh,1 Neil D. Ebenezer,1 Catherine Willis,3 Anthony T. Moore,1,3 Alan C. Bird,1,3 David M. Hunt,1 and Shomi S. Bhattacharya1,4 1 Department of Molecular Genetics Institute of Ophthalmology University College London London, ECIV 9EL 2 Oxagen Limited Abingdon, Oxon, OX14 4RY 3 Moorfields Eye Hospital London, ECIV 2PD United Kingdom

Summary We report mutations in a gene (PRPF31) homologous to Saccharomyces cerevisiae pre-mRNA splicing gene PRP31 in families with autosomal dominant retinitis pigmentosa linked to chromosome 19q13.4 (RP11; MIM 600138). A positional cloning approach supported by bioinformatics identified PRPF31 comprising 14 exons and encoding a protein of 499 amino acids. The level of sequence identity to the yeast PRP31 gene indicates that PRPF31 is also likely to be involved in pre-mRNA splicing. Mutations that include missense substitutions, deletions, and insertions have been identified in four RP11-linked families and three sporadic RP cases. The identification of mutations in a pre-mRNA splicing gene implicates defects in the splicing process as a novel mechanism of photoreceptor degeneration. Introduction Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal dystrophies that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. RP can be inherited as an autosomal dominant, autosomal recessive, or X-linked trait. The autosomal dominant form of RP (adRP) can be caused by mutations in five genes and a further six loci for which the genes remain to be identified. The adRP locus on 19q13.4 (RP11; MIM 600138) was first identified by linkage analysis in a large British family (Al-Maghtheh et al., 1994). Subsequent reports of British (Al-Maghtheh et al., 1996; Vithana et al., 1998), Japanese (Xu et al., 1995), and American families (McGee et al., 1997) linked to this locus implicate RP11 as a major cause of adRP. Affected members from all these pedigrees have type II/regional form of RP. Uniquely, they also show bimodal expressivity defined by the presence of completely asymptomatic 4

Correspondence: [email protected]

individuals who have both affected parents and affected children. This “all or none” form of incomplete penetrance of RP11 differs from the partial penetrance phenotype seen in the RP9 locus, where gene carriers exhibit a range of phenotypes from fully asymptomatic to severe (Kim et al., 1995). A detailed clinical description of RP11 families has been reported previously (Moore et al., 1993; Evans et al., 1995).

Results and Discussion Physical Mapping and Screening of Positional Candidate Genes Haplotype analysis in the five previously reported British RP11-linked families (Al-Maghtheh et al., 1994, 1996; Vithana et al., 1998) showed no evidence for a founder effect, but enabled linkage refinement to a 3 cM interval defined by proximal marker D19S927 and distal marker D19S781.2 (GenBank accession number AF069627; Vithana et al., 1999). The latter marker was isolated in our laboratory from a cosmid clone mapped to the region by Lawrence Livermore National Laboratories (LLNL; http://www.llnl.gov/). By incorporating the refinement data into the sequence generated by the LLNL of 19q13.4, the RP11 interval between D19S927 and D19S781.2 was estimated to be approximately 600 kb. We have established that inserts of five BAC clones, all currently being sequenced by LLNL, cover the entire RP11 candidate region (Figure 1). The analysis of these BAC sequences by the nucleotide identification package NIX (http://www.hgmp.mrc.ac.uk) identified several predicted genes and two known genes, PRKCG and RPS9, both of which have been excluded from involvement in RP11 (Al-Maghtheh et al., 1998; Vithana et al., 1999). Screening of all the NIX-predicted genes in the affected members of RP11-linked families by DHPLC and genomic sequencing led to the identification of mutations in a gene (PRPF31) that encodes a protein with homology to yeast pre-mRNA splicing factor Prp31p (Weidenhammer et al., 1996, 1997). The genomic organization of PRPF31 (precursor RNA processing factor 31) was determined by the comparison of the cDNA (GenBank accession number AL050369) and genomic sequence obtained from the BAC clones CTD-3093M3 (GenBank accession number AC012314) and RP11-402N14 (GenBank accession number AC009968). The PRPF31 gene comprises 14 exons, spans approximately 18 kb of DNA, and encodes a protein of 499 amino acids. PCR analysis of cDNAs from a variety of normal tissues including retina with a primer pair that amplifies exons 13 and 14 of PRPF31 as a single amplicon produced the expected 350-bp product in all tissues (Figure 2). This result is in agreement with the EST database (http://www.ncbi.nlm.nih. gov/UniGene/), which shows PRPF31 gene to be expressed in a wide range of tissues including the neural tissues, brain, and retina.

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Figure 1. Physical Map of the RP11 Interval and the Genomic Organization of PRPF31 STSs, ESTs, and genes mapping within the refined interval D19S927 and D19S781.2 were used to construct the physical map based on PAC and BAC clones. Only a few of the clones of the contig are presented; ones being sequenced by LLNL are numbered in red. The genes excluded from RP causation, including NIX-predicted genes (WI-17997, CACNG5, L-CACNG, KIAA0691, FB1, BB1, and A006I07), are shown in red. An enlarged view of the PRPF31 gene is shown. It consists of 14 coding exons between 61 and 185 bp in length, with the initiation and stop codons (ATG and TAG) present in exons 2 and 14, respectively. The first intron is within the 5⬘ UTR. All exons follow the GT/AG rule (Breathnach and Chambon, 1981). Sizes of exons and introns are drawn to scale.

Spectrum of Mutations in PRPF31 Gene To date, we have identified putative pathogenic mutations in four of our RP11 families and in three individuals

Figure 2. Expression of PRPF31 cDNA expression profile for exons 13 and 14 of PRPF31 showing amplification of the 350-bp product from all tissues including retina. A ubiquitously expressed gene, PGM1 (Whitehouse et al., 1992), was used as a control; its 400-bp fragment was also observed in all tissues.

in a cohort of 50 sporadic adRP cases from the UK (Table 1). The mutations in the RP11-linked families cosegregated with the disease in all cases, and two examples are shown in Figure 3. None of the mutations were present in 100 control subjects, thereby excluding the possibility that the identified mutations were rare polymorphisms. In the family AD5, an 11-bp deletion (1115– 1125 del) in exon 11 results in an aberrant truncated protein of 469 residues. In family AD29 (Al-Maghtheh et al., 1996), the missense mutation results in an A216P substitution in a residue conserved across homologous genes in S. cerevisiae, S. pombe, D. melanogaster, and A. thaliana. The mutation identified in family RP1907 (AlMaghtheh et al., 1996) affects the third base in the donor site of intron 6 (IVS6⫹3A⬎G). A neural network computer program for recognizing splice sites (available at http:// www.fruitfly.org/seq_tools/splice.html) predicted a reduction in the likelihood of splicing at this site (probability reduced from 0.99 to 0.68). Retention of intron 6 would result in a truncated protein of 186 amino acids. A similar result would be expected from the mutation in the previously unreported RP11-linked family RP677 where a deletion in intron 6 (IVS6-3 to -45 del) removes

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Table 1. Mutations Detected in PRPF31 Family/Individual

Exon/Intron

Nucleotide alteration*

Protein alteration/Predicted change

RP677 RP1907 AD29 AD5 SP14 SP42 SP117

Intron 6 Intron 6 Exon 7 Exon 11 Exon 7 Exon 7 Exon 8

IVS6-3 to -45 del IVS6⫹3A⬎G 646G⬎C 1115–1125 del 580–581 dup 33 bp 581C⬎A 769–770 ins A

Inactivation of splice acceptor site Inactivation of donor splice site Ala216Pro Frameshift, 98 novel aa then STOP Frameshift and insertion of 11 aa inframe Ala194Glu Frameshift, 20 novel aa then STOP

* Nucleotide designation commencing 1 at position 36 of GenBank accession number AL050369.

the essential polypyrimidine tract located between the intron branchpoint and the splice acceptor site. No mutations have so far been identified in the coding or flanking intronic regions of PRPF31 in RP11 families AD2 (AlMaghtheh et al., 1996) and AD11 (Vithana et al., 1998), but large rearrangements, mutations within the promoter or intronic sequences, are currently being investigated in these families. The three mutations identified in sporadic RP individuals include a missense and two insertion mutations (Table 1; Figure 4). In individual SP42, a 581C⬎A transversion causes the nonconservative amino acid change A194E. In individual SP14, a 33bp insertion (580–581 dup 33 bp) into codon 194, the same codon mutated in SP42, duplicates residues EL ERLEEACDM, producing a slightly longer protein of 510 residues. In individual SP117, a single bp insertion (769– 770 ins A) produces a truncated protein of 277 residues. All the above mutations showed distinct elution profiles on DHPLC that were not found in 100 control subjects, confirming that these mutations are also not common polymorphisms. Asymptomatics and Symptomatic Individuals Have Different Wild-Type Alleles An allelic effect has been suggested as the possible mechanism for the nonpenetrance of mutations at the RP11 locus (McGee et al., 1997). Sib-pair analysis has shown a statistically significant correlation between the inheritance of the wild-type allele from the noncarrier/ normal parent and the presence of disease in carrier offspring. We have typed five polymorphic SNPs identified within PRPF31 gene in sibships from AD5 and AD29 and demonstrated that asymptomatic patients inherit a different wild-type allele to the one inherited by symptomatic patients (data not shown). Function of the PRPF31 Protein A bioinformatics approach was used to assess the likely function of the PRPF31 protein (GenBank accession number NP_056444). Homology screening of the protein databases revealed homologous proteins in D. melanogaster (GenBank accession number AAF49655) and A. thaliana (GenBank accession number T02269), as well as the two previously mentioned PRP31 proteins from S. cerevisiae (GenBank accession number NP_011605) and S. pombe (GenBank accession number CAA17928; Weidenhammer et al., 1996, 1997; Bishop et al., 2000). Database screening did not, however, reveal any homologs of PRPF31 in humans, implying that PRPF31 is very

likely to be present as a single copy gene and is therefore the functional equivalent of yeast PRP31 in the human. Although the overall sequence identities between PRPF31 and the two yeast proteins are modest (20% with S. cerevisiae Prp31p and 38% with S. pombe prp31⫹), these are considered significant for comparison across such divergent species. In addition, certain domains within the protein share a much higher percentage identity (up to 47%), suggesting a common functionality (Figure 4). In particular, all the above proteins contain a region highly homologous to Nop, a putative snoRNA binding domain (Pfam; Bateman et al., 2000; GenBank accession number PF01798). This domain is present in various pre-RNA processing ribonucleoproteins such as Nop5p (Wu et al., 1998) and Nop56p (Gautier et al., 1997). In Prp31p, the Nop domain is believed to mediate protein-RNA interactions required for spliceosome assembly. Further evidence for a nuclear role for PRPF31 is the presence of putative nuclear localization signals in the medial region of the protein (Figure 4). All this is suggestive of a role for PRPF31 in pre-mRNA splicing. Physiological Implications Removal of intron sequences by splicing occurs by two sequential trans-esterification reaction steps that are catalyzed by the components of a large RNA-protein complex, termed the spliceosome (Kramer, 1996; Burge et al., 1999). The formation of the major spliceosome involves the stepwise assembly of four small nuclear ribonucleoprotein particles (snRNP U1, U2, U4/U6, and U5) and many non-snRNP splicing factors on a premRNA. In splicing, the U1 snRNA first base pairs with the 5⬘ splice site to produce a commitment complex. The U2 snRNP then interacts with the branchpoint sequence in an ATP-dependent fashion to form the prespliceosome. The U4/U6 and U5 snRNPs associate with this complex as a single tri-snRNP particle to form the spliceosome. Conformational rearrangements then occur, allowing sequential cleavage at the 5⬘ and 3⬘ splice sites followed by ligation of the two exons to form the mature mRNA species. Studies carried out in S. cerevisiae have revealed that more than 40 protein factors are essential for spliceosome formation and the in vivo splicing reaction. A number of these factors have been identified in screens of temperature-sensitive strains for those exhibiting a splicing defect at the nonpermissive temperature. These are referred to as PRP genes, for precursor RNA processing. The PRP31 gene in yeast was iden-

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Figure 3. Cosegregation Analysis of the Mutations (A) Segregation of the 43-bp deletion mutation (IVS6-3 to -45 del) in pedigree RP677 as shown by agarose gel electrophoresis. Lane assignment (1–10) corresponds to samples of the respective individuals in the pedigree. Individual 7 in the pedigree is an asymptomatic obligate carrier and demonstrates the incomplete penetrance in this pedigree. All affected individuals exhibit the deleted allele. (B) Segregation of the 11-bp deletion mutation (1115–1125 del) in pedigree AD5. Only a branch of AD5 pedigree is shown along with the haplotypes for the RP11 markers; the asterisk denotes individual II-2 in the original pedigree (Al-Maghtheh et al., 1994). Lane assignment (1–22) corresponds to samples of the respective individuals in the pedigree. Individuals 12 and 15 are asymptomatic obligate carriers, while individuals 3, 9, 14, and 22 denote asymptomatic disease haplotype carriers. All affected haplotype carriers exhibit the deleted allele.

tified in such a screen. According to studies in S. cerevisiae, Prp31p is involved in recruiting the U4/U6.U5 tri-snRNP to the prespliceosome complex and/or in stabilizing these interactions to form the mature spliceosomal complex (Weidenhammer et al., 1997). Its role is thought to be essentially one of tethering the tri-snRNP to the spliceosome complex rather than in forming or maintaining individual snRNP complexes. The identification of mutations in four out of six RP11-

linked families and three additional mutations in sporadic RP patients provides convincing evidence that PRPF31 is the disease gene in this form of dominant RP. The involvement of factors associated with splicing in disease has been reported in one other case. SMNI, the gene mutated in spinal muscular atrophy, is thought to play a critical role in spliceosomal snRNP assembly and in pre-mRNA splicing (Pellizzoni et al., 1999). Interestingly, both PRPF31 and SMN1 splicing factors, al-

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Figure 4. Protein Alignment of Human PRPF31 (DKFZP566JI53 Protein, Residues 95 to 383), PRP31-like Proteins from Other Species, and Nop, a Putative snoRNA Binding Domain PRPF31 protein shows highest homologies over its full-length to the CG6876 gene product of D. melanogaster (60% over the entire protein), A. thaliana hypothetical protein T13D8.1 (45% from residues 2 to 490), prp31⫹ from S. pombe (38% from residues 1 to 477), and Prp31p from S. cerevisiae (31% from residues 97 to 426). Amino acids, which exhibit complete identity across species, are boxed, whereas amino acids identical to those of the Nop snoRNA domain are shown in red. The region between residues 351 and 364 of PRPF31 (highlighted in blue) with the consensus sequence 351(R/K)(R/K)(R/K) R XX(R/K)(R/K)XRKXKE364 harbors three putative nuclear localization signals (NLS) in agreement with the proposed consensus sequence, K(K/R)X(K/R), for monopartite NLSs (Chelsky et al., 1989). Red circles indicate the most likely signal sequence of the three according to PSORT, a program that identifies subcellular localization signals. Blue diamonds indicate the amino acid residues 194 and 216 mutated in sporadic case 42 (SP42) and AD29, respectively. In individual SP14, the underlined residues ELERLEEACDM are duplicated due to mutation (580–581 dup 33 bp). In individual SP117, the insertion mutation (769–770 ins A) adds 20 novel amino acids after codon 256 followed by a premature stop in codon 278; the novel residues of this protein are shown in mauve. In AD5, the deletion mutation (1115–1125 del) adds 98 novel amino acids after codon 371 followed by a premature stop in codon 470; the first 13 novel residues of the aberrant protein are shown in mauve.

though ubiquitously expressed, only cause degeneration in a restricted set of neurones, namely the rod photoreceptor cells and the motor neurone cells, respectively. The preponderance of protein truncation mutations in RP11 suggests that the usual pathophysiological basis of adRP at this locus is the functional loss of one allele resulting in haploinsufficiency, although it is possible that the missense mutations may have a dominant-negative effect. Ultimately, only direct functional studies of the mutant and wild-type proteins would distinguish the effects of different mutations on the function of the protein and also the impact of the mutant on the wild-type protein. The incomplete penetrance phenotype of RP11 remains unexplained. One possible explanation is the existence of a common high-expression wild-type allele that is able to compensate for the mutant allele. Although the PRPF31 gene is widely expressed and would be expected to play a central role in mRNA processing in most tissues, it is only within the rod photoreceptors that mutations result in a disease phenotype. The disk membranes in the outer segments of rods are continuously renewed, and it has been shown that in vertebrate retinae, the level of mRNA for opsin, the most highly abundant protein (Hamm and Bownds, 1986) in disks, fluctuates with a daily rhythm. The highest level of mRNA for rod opsins has been observed before light onset prior to disk shedding and during disk assembly (Korenbrot and Fernald, 1989; von Schantz et al., 1999).

There is therefore a very high demand for rod opsin synthesis at these times and any defect in mRNA splicing may severely compromise the cell, such that the required levels of rod opsin production for the maintenance of rod outer segment ultrastructure and phototransduction is not achieved. The fact that low levels of rhodopsin causes retinal degeneration is evident in mice carrying rhodopsin gene knockouts, where a rapid retinal degeneration is shown in homozygous mice and a slow degeneration in heterozygous mice (Humphries et al., 1997; Lem et al., 1999). Alternatively, mutations in PRPF31 may lead to incomplete intron removal and the production of aberrant proteins. Here again, the high rate of rod opsin production may make the rod photoreceptors particularly susceptible to such a mutant effect. Relevant to both these interpretations is the observation that adRP linked to chromosome 17p is caused by mutations in PRPC8 (McKie et al., 2001), the human ortholog of yeast splicing factor Prp8p (Brown and Beggs, 1992). In conclusion, we have shown that mutations in PRPF31, the putative human ortholog of yeast premRNA splicing factor, are responsible for dominant retinitis pigmentosa on 19q13.4. We therefore expect the process underlying RP11 pathogenesis to be distinct from those operating in other forms of autosomal dominant RP where mutations have previously been identified in structural proteins and transcription factors (Dryja et al., 1990; Farrar et al., 1991; Bessant et al., 1999). PRPF31 mutations may trigger a novel mechanism of

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photoreceptor degeneration, and this would suggest that PRPF31 plays a critical role in the maintenance of normal rod function. Experimental Procedures Patients, Families, and Samples Patients with RP who agreed to participate in this study underwent clinical evaluation at Moorfields Eye Hospital. The subjects included individuals from families that had been previously shown to link to the RP11 locus for dominant RP on chromosome 19q13.4. Blood samples were collected and DNA was extracted using standard protocols. Patients gave fully informed consent and the study protocol was approved by the hospital ethics committee. Construction of RP11 Physical Map and Isolation of Novel Markers The DNA contig spanning the RP11 interval is composed mainly of BAC and PAC clones. The PAC clones were isolated by screening the RPC11 PAC library (Ioannou and de Jong, 1996) with all the relevant sequence-tagged sites (STSs) from all high-resolution genetic and physical maps of 19q. The information for BAC and cosmid clones mapping to the RP11 interval were obtained from high-resolution physical maps at LLNL (http://bbrp.llnl.gov/bbrp/genome/ genome.html) and were STS content mapped by PCR to test their integrity. The STS content of the region was increased by the isolation of novel microsatellite markers (e.g., D19S785, D19S781.1, D19S781.2, and D19S781.5) and nonpolymorphic STSs generated by sequence sampling of PAC and cosmid clones. After obtaining DNA using the alkaline lysis method, PAC and cosmid clones were direct sequenced using T7 and Sp6/T3 primers to obtain end sequences. To isolate the microsatellite markers, cosmid clones from the RP11 interval were digested by Sau3AI and HaeIII, size fractionated by agarose gel electrophoresis, and Southern blotted. A G4(GT)13 oligomer was then used to probe the filter to identify clones with CA repeats. pBS subclones of cosmids positive for CA repeats were isolated and sequenced using T3 and T7 primers and Big Dye terminator chemistry (Perkin Elmer). The microsatellite marker D19S781.2 (observed heterozygosity, 0.65) isolated from cosmid 30712 was used to refine the RP11 interval in pedigree AD2 that originally showed the distal crossover with marker D19S418 (AlMaghtheh et al., 1996). Mutation Screening and Cosegregation Analysis For mutation analysis of the PRPF31 gene, we amplified coding exons from patient genomic DNA using primers located in flanking intron and UTR sequences. PCR reactions were carried out in 25 ␮l reaction volumes containing 10 mM Tris-HCl (pH 8.9), 50 mM KCl, 1.5 mM MgCl2, 12.5 pmol of each primer, 200 ␮M each dNTP, 50–100 ng of patient genomic DNA, and 0.25 units of BioTaqTM thermostable DNA polymerase (Bioline). Cycling parameters were 3 min at 94⬚C, followed by 35 cycles of 30 s at 94⬚C, 30 s at the melting temperature (Tm) of the primers (54⬚C–65⬚C), and 30 s at 72⬚C with a final 5 min extension at 72⬚C. PCR products were purified using Qiaquick columns (Qiagen). Denaturing high-performance liquid chromatography (DHPLC) and direct sequencing was used for mutation detection. For DHPLC, PCR products were analyzed using the WAVER nucleic acid fragment analysis system (Transgenomic). The buffers used for DHPLC consist of buffer A (0.1 M triethylammonium acetate [TEAA]) and buffer B (0.1 M TEAA with 25% acetonitrile). DNA fragment elution profiles were captured online and visually displayed using the Transgenomic WAVEMAKERTM software. Chromatograms were compared with those of normal controls to detect samples with altered elution profiles. Sequence variations were identified by automated bidirectional sequencing using an ABI377 sequencer (Perkin Elmer) and Big Dye terminator chemistries (Perkin Elmer). Cosegregation analysis and screening of controls was carried out by PCR/2% agarose gel electrophoresis (IVS6-3 to -45 del in pedigree RP677), PCR/heteroduplex analysis (1115–1125 del in pedigree AD5), or by DHPLC. For heteroduplex analysis, PCR products were run overnight on MDE gels and ethidium bromide stained to test for the presence of heteroduplexes.

Expression Studies Expression of PRPF31 was assessed by PCR amplification of QuickCloneTM human cDNAs (Clontech) from retina, brain, heart, skeletal muscle, kidney, liver, colon, and peripheral blood leukocytes using exonic primers from exon 13 and 14, which amplify a 350-bp product. PCR products were then electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining. The ubiquitously expressed PGM1 gene (Whitehouse et al., 1992) was used as an amplification control. Acknowledgments We thank Claire Dimon, Rosie Nicholls, and Crystal Baker at Oxagen and Louise Ocaka at the Institute of Ophthalmology for their technical assistance. We thank Dr. Sue Wilkie for advice and critical reading of this manuscript. This work was funded by MRC grants G0000011 and G9301094, the Wellcome Trust Grant (043006 and 041275), the Foundation for Fighting Blindness (USA), the British Retinitis Pigmentosa Society, and Fight For Sight. M.P. is supported by a Marie Curie Fellowship and C.C. is funded by the European Union. We would like to especially acknowledge the patients and their families for taking part in this study. Received April 10, 2001; revised June 26, 2001. References Al-Maghtheh, M., Inglehearn, C.F., Keen, T.J., Evans, K., Moore, A.T., Jay, M., Bird, A.C., and Bhattacharya, S.S. (1994). Identification of a sixth locus for autosomal dominant retinitis pigmentosa on chromosome 19. Hum. Mol. Genet. 3, 351–354. Al-Maghtheh, M., Vithana, E., Tarttelin, E., Jay, M., Evans, K., Moore, T., Bhattacharya, S., and Inglehearn, C.F. (1996). Evidence for a major retinitis pigmentosa locus on 19q13.4 (RP11) and association with a unique bimodal expressivity phenotype. Am. J. Hum. Genet. 59, 864–871. Al-Maghtheh, M., Vithana, E.N., Inglehearn, C.F., Moore, T., Bird, A.C., and Bhattacharya, S.S. (1998). Segregation of a PRKCG mutation in two RP11 families. Am. J. Hum. Genet. 62, 1248–1252. Bateman, A., Birney, E., Durbin, R., Eddy, S.R., Howe, K.L., and Sonnhammer, E.L. (2000). The Pfam protein families database. Nucleic Acids Res. 28, 263–266. Bessant, D.A., Payne, A.M., Mitton, K.P., Wang, Q.L., Swain, P.K., Plant, C., Bird, A.C., Zack, D.J., Swaroop, A., and Bhattacharya, S.S. (1999). A mutation in NRL is associated with autosomal dominant retinitis pigmentosa. Nat. Genet. 21, 355–356. Bishop, D.T., McDonald, W.H., Gould, K.L., and Forsburg, S.L. (2000). Isolation of an essential Schizosaccharomyces pombe gene, prp31(⫹), that links splicing and meiosis. Nucleic Acids Res. 28, 2214–2220. Breathnach, R., and Chambon, P. (1981). Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349–383. Brown, J.D., and Beggs, J.D. (1992). Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 11, 3721–3729. Burge, C.B., Tuschl, T., and Sharp, P.A. (1999). Splicing of precursors to mRNAs by the spliceosomes. In The RNA World, II, R.F. Gestland, T.R. Chech, and J.F. Atkins, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 525–560. Chelsky, D., Ralph, R., and Jonak, G. (1989). Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol. Cell. Biol. 9, 2487–2492. Dryja, T.P., McGee, T.L., Hahn, L.B., Cowley, G.S., Olsson, J.E., Reichel, E., Sandberg, M.A., and Berson, E.L. (1990). Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N. Engl. J. Med. 323, 1302–1307. Evans, K., Al-Maghtheh, M., Fitzke, F.W., Moore, A.T., Jay, M., Inglehearn, C.F., Arden, G.B., and Bird, A.C. (1995). Bimodal expressivity in dominant retinitis pigmentosa genetically linked to chromosome 19q. Br. J. Ophthalmol. 79, 841–846. Farrar, G.J., Kenna, P., Jordan, S.A., Kumar-Singh, R., Humphries,

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Wu, P., Brockenbrough, J.S., Metcalfe, A.C., Chen, S., and Aris, J.P. (1998). Nop5p is a small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast. J. Biol. Chem. 273, 16453–16463. Xu, S., Nakazawa, M., Tamai, M., and Gal, A. (1995). Autosomal dominant retinitis pigmentosa locus on chromosome 19q in a Japanese family. J. Med. Genet. 32, 915–916. Accession Numbers D19S781.2, GenBank accession number AF069627; DKFZP566J153 mRNA, GenBank accession number AL050369; DKFZP566J153 protein, GenBank accession number NP_056444; BAC clone CTD3093M3, GenBank accession number AC012314; BAC clone RP11402N14, GenBank accession number AC009968; Saccharomyces cerevisiae pre-mRNA splicing protein (Prp31p), GenBank accession number NP_011605; Schizosaccharomyces pombe prp31⫹, GenBank accession number CAA17928; Arabidopsis thaliana hypothetical protein T13D8.6, GenBank accession number T02269; and Drosophila melanogaster CG6876 gene product, GenBank accession number AAF49655.