Daniel Mordes, Liya Yuan, Lili Xu, Mariko Kawada, Robert S. Molday, Jane Y. Wu |National Library of Medicine | 2007 March 9 | Vol. 26, Issue 2 | 291–300 | doi:10.1016/j.nbd.2006.08.026
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 websites: 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, 2001) and PRPF3 (or HPRP3; for RP18, Chakarova, 2002). Another adRP gene, PAP1 (for RP9), has also been implicated in pre-mRNA splicing (Maita et al., 2004 and 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 is crucial for the formation of the catalytically active center in the spliceosome. A number of proteins, including PRPF3, PRPF8 or PRPF31, play important roles in the formation 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, 1997). Mammalian PRPF3, PRPF8 or 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 complex 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. Co-transfection 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.
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