The vast majority of PRPF31 mutations that cause RP result in the creation of termination codons before the natural stop of the reading frame. In the present study, we investigated 6 different PRPF31 mutations that recapitulate this common situation using lymphoblastoid cell lines derived from 9 patients with autosomal dominant RP. Initially we demonstrated that there is a striking difference in mRNA expression levels between mutant and nonmutant alleles. We then showed that this difference could not be attributed to a modification in transcriptional activity of the mutant allele or to instability of the mutant mRNA transcript. We further demonstrated that all mutations resulting in PTCs located before the last exon result in mRNA alleles that are actively degraded by NMD. Of the small proportion of mutant transcripts that are still detectable, all fail to be translated into proteins, at least to a level detectable by highly sensitive and quantitative immunoblotting. Patient cell lines were also shown to contain a decreased level of full-length PRPF31 protein, the subnuclear localization of which cannot be distinguished from that of controls and is perhaps indicative of its normal functioning. Altogether, these data indicate that NMD plays a central role in the degradation of the mutant mRNA containing PTCs, which protects cells from potentially negative effects of these transcripts and their encoded products. Furthermore, we concluded that all of the PRPF31 mutations analyzed result in null alleles and that heterozygous patients who carry them can be considered functional hemizygotes.
We investigated dominant mutations in PRPF31
, a pre-mRNA splicing factor that is essential for cell survival, yet causes a tissue-specific phenotype such as nonsyndromic retinal degeneration. We adopted this system as an extreme example of the elevated genetic and allelic heterogeneity displayed by RP, for which a similar phenotype may correspond to a high number of mutations encoded by many different genes (5
). For this kind of investigation and for medical research in general, access to suitable material for molecular and functional studies of diseases is of paramount importance. The ex vivo cell cultures used in this study naturally express PRPF31
and represent, we believe, the best possible experimental model for studying PRPF31
molecular genetics, because retinal biopsies are obviously not practicable, transgenic animals with similar splicing factor mutations have thus far failed to show any phenotype (41
), and cell-based transfections of mutant protein constructs do not recapitulate the natural situation of a partial lack of PRPF31.
Quantification of PRPF31
mRNA extracted from whole cells from all patient-derived cell lines showed a significant underrepresentation of mRNA molecules originating from the mutant DNA allele, in agreement with previous results based on the measurement of allelic or total transcripts (28
). The magnitude of this decrease showed some variation and was possibly mutation-dependent; however, the reduction was consistent in all cells analyzed (Figure ). Total mRNA at steady state is influenced by a number of processes, including the rate of transcription and the stability of transcripts. By quantifying allelic expression levels of pre-mRNA in the nucleus, we excluded the possibility that mutant PRPF31
RNA is transcribed at a reduced rate (Figure A) or has a reduced intrinsic stability as compared with nonmutant transcripts (Figure , B and C). We therefore conclude that posttranscriptional mechanisms are central to the observed cellular phenotype.
Despite in silico predictions, mutant PRPF31
mRNA degradation by NMD has never been proven. Inhibiting NMD with chemical treatments results in an increase of all PRPF31
mRNA alleles that encode PTCs located before the last exon, which indicates that this cellular mechanism is indeed responsible for their active degradation and for the overall reduction in PRPF31
expression (Figure ). An exception to this trend is seen with the mutation c.177+1delG, which also displays reduced amounts of mutant transcripts but seems to be insensitive to NMD inhibition. This mutation is peculiar, because it causes the skipping of exon 2, which contains the natural start codon of the reading frame (28
), and results in the likely use of an alternative AUG. Multiple out-of-frame AUG codons exist in the PRPF31
sequence, which potentially leads to PTC-containing, NMD-sensitive mRNA transcripts. However, in silico analysis of possible PRPF31
AUG codons matching the features of natural start sites (46
) indicated that all 14 potential translation start sites are in-frame with the PRPF31
natural stop codon, the first 3 of which lie within exon 2. It has been shown that the loss of multiple in-frame AUG codons at the beginning of the coding sequence results in a substantial decrease in mRNA expression (47
). Therefore, the reduced expression level of this NMD-insensitive mutation could be explained by this phenomenon. Furthermore, we excluded, on the basis of real-time PCR experiments using primers that are located outside the rearrangement (28
), the possibility that the c.177+1delG change could result in a leaky mutation, producing only occasionally the skipping of exon 2 but overall resulting in a regular amount of spliced mRNA. Although we do not have enough data to support a definitive genotype-phenotype correlation, it is of clinical interest that an individual who carries this mutation exhibits a milder phenotype by ERG than do individuals with other mutations (Table ).
The mutation c.1115_1125del removes an exonic splicing enhancer in exon 11 and produces 2 distinct mRNA forms. A long form that displays a regular splicing pattern, carries this 11-bp deletion, and is NMD-insensitive, and a short form that exhibits an exon 10–exon 12 splicing rearrangement creates an NMD-sensitive PTC (Figure ) (30
). A comparison of our results for pre-mRNA with those for steady state RNA measurements indicated that PRPF31
DNA alleles with the c.1115_1125del are mostly spliced into the short, NMD-sensitive RNA form (~86%), whereas the long, NMD-insensitive mRNA represents the remaining ~14% of the mutant allele (Figure , Supplemental Table 1). The reduction in expression of the long mRNA mutant form is thus due to a preferential bias in favor of the short form during splicing, rather than to degradation via the NMD pathway. Indeed, the long form is untouched by the NMD process, remains constant in nuclear and cytoplasmic extracts (Figure ), and is potentially available as a template for protein synthesis. It is of interest to note that, although within the same range, the values relative to the percentage of the mutant long mRNA form with respect to the wild-type mRNA are slightly different when measured from total RNA and by semiquantitative RT-PCR (~14%; Supplemental Table 1) and when quantified after cellular fractionation by allele-specific real-time PCR (~25%; Figure ). Although we have no proven explanation for this discrepancy, it is likely that the different methods used may have been responsible.
To study the subsequent effect of PRPF31
mutations at the protein level, we developed an anti-PRPF31 antibody specific to the first 15 N-terminal amino acids. This antibody is potentially able to recognize all PTC-containing mutations (except for c.177+1delG), in contrast with all currently available antibodies that recognize only C-terminal epitopes. Quantitative Western blotting showed that the decrease in total PRPF31
mRNA expression was reflected in the reduced abundance of PRPF31 protein in lymphoblastoid cell lines from all patients with PRPF31
mutations, mostly as a direct consequence of NMD targeting of mutant transcripts. However, whereas all are heterozygotes, these cell lines appeared to produce more than 50% of PRPF31 full-length protein (~69% on average; Figure ), with respect to controls. A similar tendency was previously observed at the mRNA level, as measured by real-time PCR and microarray hybridization, in a comparison of patients with PRPF31
mutations and healthy controls (28
). These values are potentially explained by a feedback compensatory mechanism that could be triggered by the lack of functional PRPF31, a mechanism worthy of further investigation.
Interestingly, although we were able to detect quantitative differences in full-length PRPF31 in these Western blots, we were unable to detect any truncated PRPF31 proteins resulting from the translation of any mutant alleles (Figure ). This finding is significant because we demonstrated that this detection system was sensitive enough to identify PRPF31 variants as rare as 3%–4% of the full-length form and that the amount of mutant PRPF31
mRNA that escapes degradation was on average 14% of that of the wild-type transcripts. These transcripts may represent mRNA that has been marked for NMD and not yet degraded or mRNA that is not effectively translated. Alternatively, the truncated proteins derived from this mRNA could be quickly degraded or intrinsically unstable. Indeed, there are a number of examples in patient-derived cell-based systems where protein products from mutant alleles cannot be detected and are suggested to be unstable (48
). Meanwhile, the detection of truncated proteins after inhibition of NMD remains atypical (39
). The discrepancy between the presence of mutant transcripts and the complete lack of mutant proteins is particularly relevant for the long mRNA form originating from the c.1115_1125del mutation, which amounts to 14% of the wild-type mRNA, is not targeted for NMD, and nonetheless does not seem to result in any truncated protein. Altogether, these data suggest the presence of an additional control mechanism that prevents the formation of truncated proteins, possibly one that is connected to NMD and/or to the splicing process.
We addressed this concept by treating cell lines containing PRPF31
mutations with wortmannin, which inhibits NMD while preserving protein synthesis, and observed a strong increase in the production of mutant mRNA. However, most interestingly, there was no complementary detection of truncated proteins, even at mRNA levels that were equal to those of the wild-type allele (Figure ). Similar observations have been reported for one specific example, where expression of 20 BRCA1
mutant alleles and 1 CHK2
mutant allele was elevated in the presence of wortmannin (39
). When treated in conjunction with the lysosome inhibitor chloroquine, 2 truncated BRCA1 proteins were detected, which suggests that these mutant proteins were unstable and rapidly degraded; however, no such truncated bands were detected for any of the other mutations tested. Using a similar approach, we supplemented the culture medium of all our cell lines with wortmannin, chloroquine, and the proteasome inhibitor MG132. Despite this treatment, we could not rescue the expression of any mutant PRPF31 protein forms, which suggests that lysosomal and proteasomal degradation pathways have little or no impact on the abundance of PRPF31 truncated proteins in vivo and that, perhaps, the mRNA encoding these mutations is not translated.
There is strong support for a lack of translation of similar mutant alleles in cancer cell lines by a process known as nonsense-mediated translational repression (NMTR). In a recent study, a number of natural mutations that encode PTCs before the last exon were shown to escape NMD, yet truncated protein transcripts were not detected and their mRNAs were not associated with polysomes (55
), which indicates that the mutant mRNA is not translated. In light of this finding, we suggest that the small amount of mRNA transcripts present in our patient cell lines is likely subject to NMTR, which prevented the synthesis of truncated proteins. Likewise, mutant mRNA that is present at elevated levels, due to the inhibition of NMD, is perhaps also a target for NMTR and hence not translated. Therefore, products of mutant alleles containing PTCs would appear to be degraded or inactivated by 2 complementary mechanisms, NMD and NMTR, which effectively prevent the synthesis and remove all traces of truncated proteins resulting from their transcription and translation.
To date, some PRPF31
mutations have undergone functional analysis. For the p.Ala194Glu and p.Ala216Pro missense changes, ectopic expression of mutant cDNA in standard laboratory cell lines or by in vitro assays demonstrated that these 2 amino acids are essential for the interaction with the spliceosome protein 15.5K and U4 snRNA (56
). Furthermore, plasmids carrying these mutations produce PRPF31 proteins that are unable to translocate into the nucleus with high efficiency and, therefore, have a reduced function in the splicing process (56
). Although these important data shed new light on the functional role of PRPF31 in pre-mRNA splicing, they do not represent the situation commonly found in patients with PRPF31
-linked RP, whereby a majority of the mutations result in PTCs. Other experiments in transfected cells have tested the effect of the expression of 2 truncated forms of the PRPF31 protein, theoretically derived form the c.769_770insA and c.1115_1125del mutations (58
). Unfortunately, these experiments have little to no biological or clinical relevance, considering our main findings on the lack of expression of c.1115_1125del alleles and other PTC-containing alleles under normal conditions and that both cDNA constructs used were incorrectly truncated at the site where the frameshift occurs and not at the site of the PTC. Significant and very interesting data exist on the effects of RNAi-based PRPF31
inactivation, which mimic more closely the situation demonstrated by findings and in patients with large deletions. These experiments showed that a strong PRPF31 depletion leads to the accumulation of essential spliceosome components in Cajal bodies and, therefore, likely to the block of the splicing process (33
). PRPF31 immunodepletion experiments in mammalian cells induced a block in the formation of the catalytic tri-snRNP particles (32
), and complete loss of PRPF31 has also been shown to be lethal in yeast (7
). Our data show that there is an underrepresentation, but not a complete absence, of PRPF31 protein in patient cell lines and that the nonmutant protein is localized normally within these cells. This finding further supports the notion that the PRPF31 protein present in patients functions normally, although this cannot be directly tested, and thus the situation in patients is very different from that observed in knockdown and immunodepletion experiments.
Taken together, our results are particularly important concerning the molecular etiology of RP, because they indicate that PRPF31 mutations very likely cause RP via a haploinsufficiency mechanism rather than through a dominant-negative effect. We cannot completely exclude the presence of small, undetectable amounts of truncated forms of PRPF31 or of tissue-specific NMD (60
) that are potentially active in lymphoblastoid cell lines but not in the retina. However, both of these possibilities are unlikely given that recent genetic screenings have shown that a non-negligible portion of patients are true hemizygotes who carry large deletions in the PRPF31
). The crucial question that remains is how is it possible that PRPF31, an essential component of the spliceosome (a macromolecular machinery that is vital in all eukaryotic organisms) and a protein necessary for cell survival, is implicated in a tissue-specific, non-life-threatening phenotype such as dominant RP. Some hypotheses implicate the alteration of alternative splicing in the retina or of retinal-specific genes; others assume an effect on nearly all photoreceptor transcripts, a cell type in which demand for mRNA and therefore splicing is perhaps elevated. Other explanations take into account a possible alternative “moonlighting” function, unrelated to splicing, which PRPF31 performs specifically in the retina. Whatever the mechanism, what is certain is that a mild reduction in the PRPF31 content somehow results in certain retinal-specific deficiencies. Finally, our finding that haploinsufficiency is the likely mechanism for nearly 90% of all known PRPF31
mutations has important consequences for future gene therapy in an easily accessible tissue, where supplementary expression of a common full-length PRPF31
cDNA construct could be the treatment for many genetically different cases.