PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa

Abstract

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.

Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing

“…Prp8-S2197F was unable to perform the second step at all. Together these data show that the Prp8-RP decrease in splicing efficiency revealed through microarrays, RT-qPCR, and in the ACT1-CUP1 reporters is due not only to problems with tri-snRNP stability and spliceosome activation as previously thought (Boon et al 2007;Maeder et al 2009;Tanackovic et al 2011;, but also due to decreased efficiency during splicing catalysis, particularly at the second catalytic step.…”

“…We found the decreased second step catalytic efficiency revealed by our ACT1-CUP1 and in vitro bimolecular splicing assays to be of particular interest because it indicates that proper control of Brr2 by Prp8 is disrupted by the Prp8-RP mutants at this later step, not simply during splicing assembly as has been previously demonstrated (Boon et al 2007;Maeder et al 2009;Tanackovic et al 2011;. Prp8, which directly regulates Brr2 activity (Maeder et al 2009;Nielsen and Staley 2012;Mozaffari-Jovin et al 2013;Nguyen et al 2013), has also been implicated in 3 ′ SS selection Guthrie 1995a,b, 1996;Siatecka et al 1999) as well as the transition between the first and second catalytic steps Query and Konarska 2004;Liu et al 2007).…”

“…Previously, Tanackovic et al (2011) demonstrated a decrease in splicing efficiency of over 50 housekeeping genes in lymphocytes isolated from humans carrying Prp8-RP mutants. This is consistent with previous data showing that distinct Prp8 mutants affect different pools of pre-mRNA transcripts in both yeast (Pleiss et al 2007) and humans (Ivings et al 2008).…”

“…The defect in tri-snRNP assembly and stability results from decreased Prp8 protein stability caused by the Prp8-RP mutants (Pena et al 2007;Maeder et al 2009;Mozaffari-Jovin et al 2013) as well as U5 maturation defects (Boon et al 2007) that make it less likely that intact trisnRNPs will form. Cells with Prp8-RP mutations also show decreased levels of other tri-snRNP components, including Snu114 and Brr2 (Tanackovic et al 2011), further decreasing tri-snRNP levels. The well-documented spliceosome activation defect (Boon et al 2007;Tanackovic et al 2011;Mozaffari-Jovin et al 2013) seen in the presence of Prp8-RP mutants, commonly attributed to the negative effects on Brr2 helicase and ATPase coupling activity caused by Prp8-RP mutants (Pena et al 2007;Maeder et al 2009) and reflected in the decreased efficiency of both catalytic steps in our in vitro splicing assays (Fig.…”

L’épissage des ARN pré-messagers : quand le splicéosome perd pied

“…Le second exemple porte sur la rétinite pigmentaire, une maladie géné-tique fréquente (avec une prévalence de 1/4 000) conduisant à une cécité progressive due à de nombreuses mutations distinctes avec une ségrégation essentiellement mendélienne, autosomique ou liée à l'X, récessive ou dominante. Certaines des mutations dominantes touchent des protéines du splicéosome, comme PRPF (pre-mRNA processing factor) 31, PRPF8, PRPF3 et hBRR2 (une hélicase essentielle pour l'épissage des pré-ARNm) en particulier ( Figure 4B) [12]. Ces protéines sont impliquées dans l'assemblage ou le désassemblage du complexe tri-snRNP U4/U6.U5.…”