Despite recent fundamental research performed in yeast or standard laboratory cell lines, the molecular link between RP-PRPF proteins and the pathogenesis of retinitis pigmentosa has not been fully clarified. By quantification of major and minor snRNAs, we unexpectedly found that the retina normally expresses up to 7-fold more major snRNAs and approximately twice as many of the minor snRNAs compared with other human tissues. The higher level of snRNAs in the retina is a likely consequence of another surprising result; i.e. this tissue contains the highest volume of processed pre-mRNAs within the whole body, as measured by the amount of spliced genes that were common to 31 human tissues. These results suggest that a deficiency in spliceosome components would be more deleterious for the retina than for other human tissues.
To explore the nature of this potential defect leading to RP, we used lymphoblast cell lines derived from patients with RP with 10 different mutations in all 3 RP-PRPF
genes. Glycerol gradient centrifugation of nuclear extracts revealed alterations in the stoichiometry of the snRNAs, as well as altered tri-snRNP protein composition, in cells carrying RP-PRPF
mutations. Relative to U1 snRNA, U2 snRNA was reduced by 13–18%, while tri-snRNAs were decreased up to 50%. Minor snRNAs were also decreased but only for U4atac
in RP-PRPF8 cells. Quantitative assessment of tri-snRNP protein composition by immunoprecipitation indicated that cells from patients with PRPF31
mutations assembled roughly half of these particles with respect to controls. These data are in agreement with previous work showing that PRPF31
alleles causing RP result in short-lived or absent mRNAs and therefore in no mature protein (26
). Consequently, in these cells, only the wild-type allele can provide functional protein. Although the observed defects are less pronounced compared with complete RNAi-mediated depletion of PRPF31 in HeLa cells (29
), they are in agreement with the described essential role of PRPF31 in tri-snRNP assembly and spliceosome formation (29
Unlike defects in the PRPF31
mutations are predicted to yield stable mutant proteins that are co-expressed in heterozygotes with their wild-type counterparts (2
). Our results support this idea: comparable amounts of PRPF3 and PRPF8 were identified in nuclear extracts from patient and control lymphoblast cells. In addition, glycerol gradient fractionation and immunoprecipitation experiments showed that both mutant and wild-type PRPF8 and PRPF3 proteins were stably associated with tri-snRNPs in the corresponding cell lines. Tri-snRNPs bearing the PRPF3 and PRPF8 mutant proteins did assemble and had similar molecular weights to those of controls, but contained reduced amounts of hSnu114 and hBrr2. Since the two latter proteins are involved in U4/U6 and U4atac
snRNA unwinding (32
), our results suggest that RP-PRPF3 and RP-PRPF8 mutations could affect this process, necessary for the catalytic activation of the spliceosome. In agreement with our results, it has been demonstrated that the yeast homolog of PRPF8-R2310G (one of the mutant PRPF8 forms analyzed here) impairs U4/U6 snRNA unwinding by Brr2, resulting in the disregulation of catalytic activation of the spliceosome (36
). Impairment of the tri-snRNP assembly has been observed by Boon et al
) in a yeast strain carrying the same mutation. The observed alterations in the stoichiometry of U4, U5 and U6 snRNAs in human cells with PRPF8-R2310G could indicate that a similar dysfunction may also be happening in these cells, although at steady-state we observe most of the U4 and U6 (or their minor spliceosome counterparts) in the tri-snRNP-containing portions of the gradient.
Tri-snRNPs containing mutant PRPF3 proteins are also partially devoid of PRPF4 (~50%), whereas those containing mutant PRPF8 partially lack PRPF6 (~75%). Both PRPF4 and PRPF6 play a role in the tri-snRNP assembly (29
) and have been shown to have an additional role in the catalytic activation of the spliceosome (14
To investigate whether alterations in the stoichiometry of spliceosome components elicited defects in its formation, we performed in vitro assembly assays. Our analysis in nuclear extracts from patient lymphoblasts showed a substantial delay in the formation of the splicing complex A. This may be the consequence of a slower formation of complex A due to lower U2 snRNA and, indirectly, U2 snRNP amounts. Alternatively, a reduced conversion rate of complex A into complex B and downstream structures could occur because of the observed tri-snRNP defects. In addition, we cannot exclude that the rate of some proof-reading mechanism that dissociates or degrades abnormal B or C complex assemblies is faster than that of full spliceosome formation, preventing the accumulation of early complexes.
Together, our results from glycerol gradient fractionation, immunoprecipitation and in vitro
spliceosome assembly show that all RP-PRPF
mutations included in this study delay spliceosome assembly, but may act through different molecular mechanisms. While PRPF31
mutations likely affect tri-snRNP formation directly, PRPF8
mutations could indirectly impair both U4/U6.U5 assembly and the catalytic activation of the spliceosome. However, in cell lines from patients, these defects did not lead to aberrant localization of RP-PRPF proteins and their interacting partners (Supplementary Material, Figs S6 and S7
) or to spliceosome destabilization due to putative protein decay (Supplementary Material, Fig. S3
), in contrast to what has been observed after exogenous overexpression of mutant PRPF31 protein in HeLa or PRPF3 in photoreceptor-derived murine cells (43
). The observed defects are also less pronounced compared with the data obtained using thermo-sensitive (haploid) mutant yeast or siRNA-silenced cells (29
), likely because patients are heterozygotes and carry one fully functional copy of RP-PRPF that prevents the complete block of the spliceosome.
Pre-mRNA splicing analyses in extracts from patients showed that the observed defects in spliceosome composition translated into a reduction in splicing activity. Although the splicing process itself was not abolished (as expected, since heterozygous RP-PRPF
mutations are not lethal), its efficiency was reduced. This is probably a direct consequence of observed defects detected in vitro
with extracts bearing PRPF
mutations. While it is difficult to extrapolate these results to an in vivo
context, it is plausible that RP-PRPF
mutations can cause a systemic splicing defect in patients. In support of this view, we showed that 8.8% of the introns from naturally expressed genes (summarizing different assortments of splicing signals) displayed dramatic splicing problems in lymphoblasts from patients. This value may be even higher considering that our experimental setup did not take into consideration the counteracting effect of nonsense-mediated decay, a cellular sentinel eliminating many abnormally spliced transcripts. The mis-spliced transcripts identified in this study, selected mainly because of their intronic splicing motifs, are likely not directly involved in RP pathogenesis. However, they clearly indicate that cells with RP-PRPF mutations display transcript-specific defects, in agreement with data from yeast (45
) and human cells (46
). The nature of the signal, motif or structure that confers responsiveness to defective PRPF proteins remains for the moment unknown.
The analysis of AS in RP-PRPF cells identified alterations in splicing patterns in a subset of the transcripts analyzed. Although all of them seemed to be expressed in the retina according to online databases, it is difficult to find a direct link between these transcripts and RP. It is perhaps worth noting that mutations in the zebrafish homolog of AXIN1
result in dwarfed or absent eye development (47
). However, this ocular phenotype is clinically different from RP, and therefore this finding may have no particular meaning from a mechanistic point of view. The effects of PRPF8 depletion on ASEs were more pronounced compared with the partial RP-PRPF deficiencies displayed by heterozygous cell lines. When the beneficial effects of wild-type PRPF8 were removed, ~10% of the investigated transcripts displayed changes in AS pattern in at least four of the five cell lines depleted of PRPF8.
Although tri-snRNP is a constitutive splicing factor, reduction in its level or its composition may also impact AS. Direct effects on AS may occur because of the observed slowdown of specific U4/U6.U5-dependent steps of spliceosome assembly on competing pairs of splice sites. Similarly, it was demonstrated that mutations in the RNA portion of the constitutive splicing factor U1 snRNP can change its affinity for competing splice sites and alter splice site selection (48
). Likewise, mutations in SMN, a protein component essential for the snRNP maturation machinery, change the stoichiometry of snRNAs and promote defects in the AS of numerous transcripts (23
). Also, down-regulation of constitutive splicing factors U2AF65 and PUF60 in mammalian cells and core spliceosome components in Drosophila
can alter the choice of alternative splice sites (49
). There may, however, be alternate explanations. For example, altered tri-snRNP particles may elicit secondary defects in the expression of one or several splicing regulatory proteins. Secondary effects could also alter the expression of a factor unrelated to splicing that is involved in differential mRNA stability, thereby changing the relative proportion of different splice forms.
Remarkably, changes in AS patterns in RP-PRPF cells and upon PRPF8 knock-down also suggest that late steps of spliceosome function can reprogram splicing decisions. In agreement with this, the U5 snRNP-associated factors PRPF6 and USP39 have recently been shown to affect the splice site choice for several transcripts, including Mcl1
Although patients with RP-PRPF
mutations have splicing defects in their lymphoblasts and presumably in other tissues as well, they show disease only in the retina, likely because of a threshold effect determined by its high pre-mRNA splicing activity. These mutations may affect many tissues at sub-pathological levels, or may not manifest their deleterious effects within a human lifetime, whereas retinal cells could undergo cell death due to an increased accumulation of aberrant mRNAs produced through impaired constitutive and/or AS. Another model, which is not incompatible with the previous one, would rely on mechanisms involving selective mis-splicing of one or a few RP genes (53
), although specific studies investigating this possibility have not analyzed mRNA from non-ocular tissues (55
), and recent results have shown that processing of two transcripts important for photoreceptor function in human cells is not affected by RP-PRPF mutations (57
). At the moment, our data cannot unambiguously favor one model over the other, and the precise mechanisms by which these defects lead to photoreceptor cell death remain to be clarified.
In conclusion, autosomal dominant RP caused by mutations in PRPF
genes seems to be a generalized splicing disease that may more dramatically affect a subset of pre-mRNAs. In agreement with the observation that patients do not suffer from syndromic symptoms and appear to have a normal lifespan (58
), functional splicing defects are globally mild compared with lethal PRPF
knockouts in yeast and mammalian cells. This picture is very similar to that recently shown for another tissue-specific and progressive disorder, spinal muscular atrophy, which is caused by the systemic deficiency of SMN, a protein that is essential for the biogenesis of snRNPs (23
). Our results indicate that future treatment strategies for PRPF-linked RP should be aimed at correcting pre-mRNA splicing impairment.