Identification of photoreceptor genes affected by PRPF31 mutations associated with autosomal dominant retinitis pigmentosa

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Abstract

Several ubiquitously expressed genes encoding pre-mRNA splicing factors have been associated with autosomal dominant retinitis pigmentosa (adRP), including PRPF31, PRPF3 and PRPF8. Molecular mechanisms by which defects in pre-mRNA splicing factors cause photoreceptor degeneration are not clear. To investigate the role of pre-mRNA splicing in photoreceptor gene expression and function, we have begun to search for photoreceptor genes whose pre-mRNA splicing is affected by mutations in PRPF31. Using an immunoprecipitation-coupled-microarray method, we identified a number of transcripts associated with PRPF31-containing complexes, including peripherin/RDS, FSCN2 and other photoreceptor-expressed genes. We constructed minigenes to study the effects of PRPF31 mutations on the pre-mRNA splicing of these photoreceptor specific genes. Our experiments demonstrated that mutant PRPF31 significantly inhibited pre-mRNA splicing of RDS and FSCN2. These observations suggest a functional link between ubiquitously expressed and retina-specifically expressed adRP genes. Our results indicate that PRPF31 mutations lead to defective pre-mRNA splicing of photoreceptor-specific genes and that the ubiquitously expressed adRP gene, PRPF31, is critical for pre-mRNA splicing of a subset of photoreceptor genes. Our results provide an explanation for the photoreceptor-specific phenotype of PRPF31 mutations.

Introduction

Retinitis pigmentosa (RP), a common cause of blindness, is a group of inherited diseases characterized by the loss of photoreceptor cells. More than a hundred genetic loci have been associated with retinal degeneration (Baehr and Chen, 2001, Swaroop and Zack, 2002; see Web sites: www.sph.uth.tmc.edu/RetNet and www.uwcm.ac.uk/uwcm/mg). As a genetically heterogeneous disease, RP displays all three modes of Mendelian inheritance: autosomal dominant (adRP), autosomal recessive (arRP) and X-linked (xlRP). Many RP genes are expressed specifically or predominantly in the retina. Recently, four adRP genes have been identified that are ubiquitously expressed in different tissues and associated with RNA processing. Three of these non-retina-specific adRP genes encode proteins essential for pre-mRNA splicing, pre-mRNA processing factors (PRPF), including PRPF31 (or PRP31; for RP11, Vithana et al., 2001), PRPF8 (PRP8 or PRPC8; for RP13, McKie et al., 2001) and PRPF3 (or HPRP3; for RP18, Chakarova et al., 2002). Another adRP gene, PAP1 (for RP9), has also been implicated in pre-mRNA splicing (Maita et al., 2004, Maita et al., 2005). Among these, PRPF31 has been reported as the second most common adRP gene, only second to the rhodopsin gene (Vithana et al., 1998). An interesting question is how mutations in ubiquitously expressed pre-mRNA splicing factor genes such as PRPF31 cause photoreceptor-specific disease.

Most mammalian transcription units contain at least one intron that must be removed by a process known as pre-mRNA splicing to form functional messenger RNA (mRNA). As the most upstream step of post-transcriptional regulation, pre-mRNA splicing is critical for mammalian gene expression. Pre-mRNA splicing employs a two-step transesterification mechanism. The first step involves cleavage at the 5′ splice site and formation of a lariat intermediate. The second step is cleavage at the 3′ splice site with concomitant ligation of the 5′ and 3′ exons. The sites of cleavage and ligation are defined by conserved cis-elements including the 5′ splice site (5′ss), the branch point sequence, the polypyrimidine tract and the 3′ splice site (3′ss) consensus sequence. The splicing reaction occurs in spliceosomes, the large RNA–protein complexes that contain pre-mRNA, five small nuclear ribonucleoprotein (snRNP) particles, U1, U2, U4/U6 and U5, as well as a number of non-snRNP protein factors (Burge et al., 1999, Hastings and Krainer, 2001, Zhou et al., 2002, Wu et al., 2004). Following the initial recognition of splice sites by U1snRNP and U2snRNP together with early-step protein factors, the assembly and incorporation of the U4/U6.U5 tri-snRNP are crucial for the formation of the catalytically active center in the spliceosome. A number of proteins, including PRPF3, PRPF8 and PRPF31, play important roles in the formation of the tri-snRNP and assembly of the mature spliceosome. These splicing factors are highly conserved through evolution, from yeast to mammals. Originally identified in a screen for splicing defects, yeast prp31 is an essential gene encoding a 60 kDa protein. It assists in recruiting the U4/U6.U5 tri-snRNP to prespliceosome complexes and is critical for pre-mRNA splicing (Maddock et al., 1996, Weidenhammer et al., 1996, Weidenhammer et al., 1997). Mammalian PRPF3, PRPF8 and PRPF31 proteins likely play similar roles in pre-mRNA splicing as their yeast counterparts. However, it is not clear how mutations in these splicing factors lead to photoreceptor cell death and retinal degeneration.

Here we describe our efforts to identify downstream “target” genes for PRPF31 using a combined molecular and biochemical approach. Immunoprecipitation of PRPF31-containing ribonucleoprotein complexes followed by microarray led to the identification of 146 RNA transcripts, including several known adRP genes. We focused on RDS and FSCN2, two photoreceptor-specific genes linked to adRP, to further test effects of PRPF31 on splicing. Cotransfection of adRP mutants of PRPF31 with a minigene of RDS or FSCN2 indicated that mutant PRPF31 proteins inhibit the pre-mRNA splicing of RDS and FSCN2 genes. Expression of the mutant PRPF31 proteins led to a significant reduction in RDS expression in cultured retinal cells. These experiments show that mutations in PRPF31 inhibit pre-mRNA splicing of certain genes expressed in photoreceptor cells. Our study reveals a functional relationship between the general splicing factor, PRPF31, and expression of photoreceptor-specific genes, RDS and FSCN2. Taken together, these observations demonstrate that PRPF31 plays an important role in the pre-mRNA splicing of a subset of photoreceptor-specific genes.

Section snippets

Plasmid construction and antibody preparation

The mammalian plasmids expressing either wild-type or mutant PRPF31 proteins were constructed by inserting the corresponding cDNA fragments into pCS2 vector downstream of the cytomegalovirus (CMV) promoter. Anti-PRPF31 polyclonal antibodies were prepared in chicks using synthetic peptides (corresponding to amino acid residue 416–432) (Aves Labs, Inc). Antibodies used in this study include anti-peripherin/RDS monoclonal antibody Per 5H2 (Connell et al., 1991), anti-rhodopsin (Chemicon),

Identification of RNA transcripts associated with PRPF31-containing splicing complexes using an immunoprecipitation-coupled microarray approach

PRPF31 mutations cause photoreceptor loss and retinal degeneration. This finding prompted us to test the hypothesis that PRPF31 may play a critical role for pre-mRNA splicing of retina-specific genes. To begin to search for potential target genes whose pre-mRNA splicing is affected by PRPF31 mutations, we designed an immunoprecipitation-coupled microarray approach to identify RNA transcripts associated with PRPF31-containing splicing complex. Using an affinity-purified polyclonal anti-PRPF31

Discussion

Pre-mRNA splicing, the most upstream post-transcriptional regulatory process, is a critical step in mammalian gene expression. Most mammalian transcription units contain introns that must be precisely removed to form functional mRNAs. Defects in pre-mRNA splicing play an important role in the pathogenesis of many human diseases. Neurodegenerative diseases have been associated with mutations affecting splicing machinery. One example is spinal muscular atrophy (SMA), a motor neuron degenerative

Acknowledgments

We are grateful to members of the Wu laboratory for generous help, insightful suggestions and critical reading of the manuscript. This work was supported by grants to J.Y.W from NIH (EY014576, AG17518, GM07967) and Muscular Dystrophy Association and by vision center core grant (EY08126) and a grant to RSM from NEI (EY02422). J.Y.W. was also supported by a Scholar Award from the Leukemia Society of America.

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