Cell. Mol. Life Sci. DOI 10.1007/s00018-015-1843-0
Cellular and Molecular Life Sciences
REVIEW
Nonsense suppression therapies in ocular genetic diseases Xia Wang • Cheryl Y. Gregory-Evans
Received: 1 October 2014 / Revised: 23 December 2014 / Accepted: 23 January 2015 Ó Springer Basel 2015
Abstract Premature termination codons (PTCs) are caused by nonsense mutations and this leads to either degradation of the mutant mRNA template by nonsensemediated decay (NMD) or the production of a non-functional, truncated polypeptide. PTCs contribute significantly to inherited human diseases including ocular disorders. Nonsense suppression therapy allows readthrough of PTCs, thereby rescuing the production of a full-length functional protein. In this review, we highlight the mechanisms that are involved in discriminating normal translation termination from premature termination codons; the current understanding of nonsense-mediated mRNA decay models (NMD); the association and crosstalk between PTC and the underlying dynamic NMD process; and the suppression therapies that have been employed in nonsense-medicated ocular disease models. Defining the mechanistic complexity of PTC and NMD will be important to improve treatments of the numerous genetic disorders caused by PTC mutations. Keywords Nonsense suppression Nonsense-mediated decay Ocular disease Therapy
Introduction In the past decade, remarkable advances in the molecular mechanism governing nonsense suppression and nonsensemediated decay (NMD) activation have been made, aiming X. Wang C. Y. Gregory-Evans (&) Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC V5Z 3N9, Canada e-mail: [email protected] X. Wang e-mail: [email protected]
to treat nonsense-related genetic diseases. Approximately 11 % of all inherited diseases are caused by nonsense mutations, suggesting that targeting patients with this type of mutation could be relevant to many patients. We provide only a brief overview of the mechanisms, focusing on interactions between premature termination codons (PTC) and NMD, and the therapies in ocular genetic disorders. Our purpose is to underscore and gain insight into the principles of nonsense suppression therapy for ocular diseases in the future.
Eukaryotic translation termination and premature termination codon In organisms, including humans, translation termination occurs when one of the three stop codons (UAA, UGA, or UAG) enters the ribosomal A site [1]. Two translation termination factors are involved in regulating eukaryotic translation termination. Eukaryotic release factor I (eRF1) directly recognizes one of the three stop codons (UAA, UAG, and UGA) [2]. The translation termination efficiency depends on competition between stop codon recognition by eRF1 and decoding of the stop codon by a near-cognate tRNA that is paired using two of the three bases. This decoding process in which an amino acid is incorporated in place of the stop codon, leading to natural suppression, is called ‘readthrough’ [1]. The second termination release factor is eRF3, a GTPase that binds to eRF1 and facilitates in the termination process. When an initial stop codon enters the ribosomal site A and is recognized by eRF1, GTP hydrolysis by eRF3 binds and induces conformational changes in eRF1 [3]. The subsequent adaptation of domain 2 of eRF1 positions its GGQ motif into the peptidyl transferase center of the ribosome [4], which finalizes stop
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codon recognition. This allows eRF1 to stimulate hydrolysis of the ester bond of the peptidyl-tRNA, thus facilitating the release of the completed polypeptide chain [2]. The recognition process signals sampling to identify the appropriate binding partners to form the pre-termination complex, including both aminoacyl-tRNAs and release factor eRF1. However, the molecular details of stop codon recognition by eukaryotic release factors remain poorly understood [2]. The premature translation codon (PTC) is caused by nonsense mutations, resulting in the occurrence of UAA, UAG, or UGA codons in the protein-coding region of the mRNA template, leading to either the production of a truncated polypeptide product or more frequently, to mRNA destabilization through nonsense-mediated mRNA decay (NMD) [5, 6]. It has been indicated that the termination suppression at naturally occurring stop codons occurs at a frequency of \0.1 % [7, 8]. However, the termination suppression at PTCs occurs at a higher frequency of \1 % [9, 10]. This difference may be due to the localization of the stop codon at distinct regions of higher-order structure of the mRNAs, and different protein–factor interactions that occur within the messenger ribonucleoprotein (mRNP) complex [2].
Nonsense-mediated mRNA decay (NMD) NMD is an evolutionarily conserved cellular surveillance and quality control process that is present in all eukaryotic cells. It functions to selectively target mRNA transcripts that harbor a PTC for rapid degradation, thus reducing the synthesis of truncated proteins with potentially harmful dominant-negative effects (for detailed reviews, see [11– 18]). PTCs can be introduced as a result of genetic or somatic mutations, errors in transcription and splicing, or programmed gene rearrangements. As the critical function of mRNA is associated with the primary sequence of its protein-coding region, this NMD is subject to inspection by the molecular signaling in the cell [19]. For both defective mRNAs and proteins, the polymers must first be recognized, subsequently characterized for degradation, and finally destroyed [20]. Creation of a PTC generally activates the NMD pathways [19]. The mechanisms and the regulation of NMD have been extensively studied [14, 19, 21–24]. Over the past decade, however, it has become clear that the role of NMD goes beyond its originally defined function as a quality control mechanism that degrades PTCrelated aberrant mRNAs. NMD is involved in regulating normal development [18, 25], and it itself can be developmentally regulated [14]. It is recently considered as a fundamental developmental regulator of transcriptome
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[19]. Genome-wide analysis of cell lines depleted of NMD factors and NMD-deficient mice have demonstrated that 3–20 % of the transcriptome is deregulated as a consequence of compromised NMD [26–31]. Although it is not yet clear which factors and mechanisms are direct NMD downstream targets in these NMD-deficient models, they have added more complexity to the role of NMD [19].
Recognition-crosstalk between PTC and NMD One of the first crucial steps in NMD is to recognize that a stop codon at the RNA template is premature, then characterizing the PTC-containing mRNA transcript as aberrant, and finally targeting it for degradation. A circular mRNA structure known as the closed-loop complex is associated with and formed by three proteins in the mRNP complex: the cap-binding protein (eIF4E) is bound to the 50 -cap structure of the mRNA; poly(A)-binding protein (PABP) is bound to the mRNA poly(A) tail; and eIF4G simultaneously binds both eIF4E and PABP, resulting in circularization of the mRNP complex and formation of the closed-loop complex [32–34]. Although this fundamental molecular recognition process has been the subject of much study and some core NMD components are conserved across species, the precise mechanisms of PTC recognition vary largely between cells, species, and mRNA transcripts [13, 14, 24]. NMD models have revealed crosstalk between the transcription and translation machineries, where alternation of the components and factors of mRNP complexes during transcription has an impact on the differential outcome of translation [35]. Four different mechanistic NMD models have been proposed to make a distinction between a normal and premature termination codon (Fig. 1) [18]: (i) The faux 30 UTR model: the recognition depends on the distance between the binding ribosome and the poly (A) tail. Premature codon termination prevents the eRF3 from interacting with PABPC1, which is the characterized normal translation termination interaction, by artificially increasing the length of the 30 UTR and the physical distance between the two proteins [18]. (ii) Exonjunction-complex-dependent (EJC) model: this recognition requires splicing-dependent deposition of a specific protein complex, the core EJC, 20 to 24 nt upstream of exon–exon junctions, and a pioneer round of translation. In normal translation termination, when the translating ribosome reaches the normal stop codon in the last exon, it traverses the entire length of the mRNA during the pioneer round of translation and displaces all the EJCs that it encounters. The communication of eRF3 with PABPC1 subsequently signals normal termination. However, premature translation termination is featured by the presence of a core EJC
Nonsense suppression therapies in ocular genetic diseases
Fig. 1 Mechanic models of nonsense-mediated mRNA decay. a Normal termination process. b Faux 30 UTR model. c EJC model. d Unified model. e Ribosome release model
downstream of the stop codon. This remaining EJC recruits the NMD effectors (UPF2 and UPF3) and the SURF protein complex (SMG1, UPF1, and eRF1 and eRF3), leading to the EJC–SURF interaction and inducing phosphorylation of UPF1 by SMG1, which initiates degradation of the PTC-containing transcript [18]. (iii) The unified model: this model incorporates factors from both the EJC and faux 30 UTR models. Recognition of a premature stop codon involves competition between UPF1 in the SURF complex and eRF3 on the terminating ribosome for interaction with PABPC1. Instead of interaction of eRF3 with PABPC1 in the normal termination event, in a premature termination event, eRF3 and PABPC1 are unable to bind, making interaction of UPF1 with PABPC1 possible. In this model, the EJC is not required for PTC recognition, but it functions as an enhancer at the mRNA template [18]. (iv) The ribosome release model: Translating ribosomes normally protect the mRNA from
degradation as they traverse the transcript. However, in the presence of a PTC, the interaction of UPF1 with eIF3 stimulates ribosome release. This early ribosome release exposes the downstream unprotected mRNA to degradation by nucleases [18]. It is clear that no matter the exact mechanism applied to identify a PTC, the cell can distinguish between a PTC and a natural termination codon [19]. This cellular discrimination serves as a foundation for the concept that it should be possible to develop small molecules that can promote readthrough at a PTC without affecting normal translational termination [19].
Nonsense mutation therapy strategies Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame
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nonsense mutations (also commonly known as PTCs). This approach utilizes compounds that suppress translation termination at PTCs, allowing translation to continue, so that partial levels of deficient protein function to be restored [36] (Fig. 2). The primary prerequisite for a therapeutic benefit of PTC suppression is the presence of a nonsense mutation-containing translatable mRNA that is not efficiently degraded by NMD [1]. A recent meta-analysis showed that there are more than 7,500 nonsense mutations in 995 different genes that cause inherited diseases. This result revealed that approximately *11 % of all inherited diseases are caused by nonsense mutations, suggesting that targeting patients with this type of mutation could be relevant to many patients with a broad range of genetic diseases [37]. PTC suppression as a potential therapy was first described in 1996 for diseases caused by nonsense mutations in the CFTR gene (mutations in which result in the common genetic disease CF) [38]. The study showed that the aminoglycoside G418 suppresses nonsense mutations in the CFTR gene and restored significant levels of both CFTR protein and function in cultured cells. Since then, approximately 100 studies have been carried out, and the effectiveness of nonsense suppression as a possible treatment has been investigated in nearly 40 different diseases [35]. However, as discussed in a recent study in cystic fibrosis patients who received ataluren treatment in a phase 3 clinical trial, ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients. This may suggest that the factors such as the ages and the choice of a population of patients could be the limitations and affect the therapeutic effects in clinical trials [39].
Fig. 2 The addition of a nonsense suppression drug such as an aminoglycoside allows readthrough to take place when the ribosome encounters the PTC, leading to the production of full-length proteins
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Nonsense mutation-mediated ocular disease therapy Pharmaceutical therapies result in the introduction of amino acids into nonsense mutation sites in order to restore or compensate for full-length protein products, and have been carried out for almost two decades [40]. PTC-mediated ocular diseases that have been studied are summarized and listed in Table 1. The choroideremia mutant zebrafish (chmru848) has a recessive Q32X mutation in the second exon of rep1. The no isthmus (noitu29a) has a recessive nonsense mutation in exon 7.1 of pax2.1 (R139X). The second zebrafish model of ocular coloboma is the grumpy (gupm189) mutant that has a recessive nonsense mutation in lamb1 [41]. Overexpression of firefly luciferase construct (pGL3), with mutant chm, noi, or gup gene products fused to the N-terminus, in COS-7 cells treated with gentamicin and paromomycin revealed luciferase activity readthrough efficiency: paromomycin [ gentamicin; R139X (UGA A) [ Q524X (UAG U) [ Q32X (UAA A). Gentamicin and paromomycin (100 lM) was used to treat choroideremia mutant zebrafish from 10 hpf to 6 or 9 dpf. Western-blot analysis revealed full-length rep1 protein from aminoglycoside treatment. Retinal sections appeared similar to WT fish. Apoptotic activity was greatly reduced throughout the larvae and within the retina with aminoglycoside treatment [41]. In ocular coloboma zebrafish models, although the phenotype did not reach WT levels, retinal histology and gross morphology of noi mutants was improved from aminoglycoside treatment. In grumpy (gupm189) mutant fish, retinal histology showed the closure of the optic fissure from aminoglycoside treatment, however rescue of the lens was not successful. Treated gup remained short in
Nonsense suppression therapies in ocular genetic diseases Table 1 Overview of nonsense suppression experiments in cellular and animal models of ocular diseases Disease
Gene
Mutation Stop codon
Used drug
Retinitis pigmentosa
RP2 (X-linked)
R120X
UGA G Gentamicin UGA C Ataluren
In vitro cell culture
Yes
Rho (autosomal dominant)
S334X
UAA G Gentamicin, G418
In vitro cell culture
Yes
In vivo transgenic mouse
Yes
Rpe65 (autosomal R44X recessive)
UGA U Gentamicin
Choroideremia
REP1 (X-linked recessive)
Q32X
Ocular coloboma
Pax2 (autosomal dominant)
R139X
Lamb1 (autosomal R524 dominant) Usher syndrome type 1 USH1C
R31X
R3X
Pax6
Ex vivo
No
In vitro cell culture
No No
UAA A Gentamicin, paromomycin
In vitro cell culture In vivo zebrafish
Yes Yes
UGA A Gentamicin, paromomycin
In vitro cell culture
Yes
In vivo zebrafish
Yes
UAG U
In vitro cell culture
Yes
UGA A G418, Gentamicin, paromomycin, NB30
[42] [40]
Yes Yes
[44]
In vivo, ex vivo
Yes
[44, 45]
Yes
[45–47]
R643X R929X G194X
UGA A UGA A PTC124
Yes In vitro cell culture, in vivo Yes transgenic mice
length compared with WT [41]. Using an in vitro cell culture study model of retinitis pigmentosa, overexpression of a luciferase reporter (p2luc) stop codon vector and overexpression of a luciferase (pGL3) construct with N-terminus fusion of a short Rho sequence with S334X were introduced in CHO cells and COS-7 cells, respectively. Luciferase assays revealed an increase in readthrough with drug treatment [42, 43]. In the S334X-4 heterozygous transgenic rat model, ERG responses showed an increase in both a- and b-wave amplitudes from gentamicin treatment. Preservation of the photoreceptor cell count was seen at P19 for both G418 and gentamicin. However, G418 showed more systemic toxicity and no animal survived beyond P20. Functional electroretinographic studies showed significant retention of ERG a- and b-waves from drug treatment [44]. In a USH1 mouse model, cDNA overexpression of mutant murine harmonin a1-GFP or mRFP fusion protein was subretinally injected into the newborn mice. Fluorescence microscopy analysis revealed an increase in GFP expression from PTC124 treatment. Western-blot analysis displayed full-length harmonin a1 expression in PTC124-treated retinas [45–48]. Our lab recently treated and analyzed the efficacy of nonsense read-through therapy in the Pax6-deficient mouse model of aniridia (Pax6Sey?/-). For this study, the ‘‘START’’ eye drops containing 1 % ataluren were applied
[43]
In vitro TNT, cell culture, Ex vivo In vitro TNT, cell culture
UGA A
[42, 50]
In vivo zebrafish
UGA C G418, NB30, gentamicin, paromomycin UGA C
R245X
Aniridia
Response References
In vivo transgenic mouse
PTC124, gentamicin PCDH15
Model
Yes Yes [48]
to mouse eyes twice daily. This topical administration of ‘‘START’’ therapy on Pax6Sey?/- mouse eyes represented the most promising results. The therapy reversed corneal, lens, and retinal malformation, and restored electrophysiological function of the retina, preventing disease from progressing [49]. This study suggests that the eye retains significant developmental plasticity into the post-natal period and stays susceptible to molecular remodeling, providing a window of opportunity to treat childhood blindness [49]. More recently, a study has demonstrated that treatment of R120X fibroblasts with G418 caused an increase in 40 % of RP2 mRNA and 20 % of protein levels, while PTC124 can restore up to 13 % of RP2 protein levels, but did not significantly increase RP2 mRNA levels. The stored RP2 protein was functional in reversing the cellular phenotypes in cells lacking RP2 proteins [50].
Mechanism of PTC124 activity in cell-line luciferase assays A recent study compared the efficacy of two read-through agents, PTC124 and aminoglycoside G418 with PTC reporter assays using b-galactosidase and the two kinds of luciferase in cell lines, including transient transfection, stable cell lines, and plate-based functional enzyme assays.
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In each assay, G418 appeared to promote translation of proteins with premature stop codons, while PTC124 had no effect except in the presence of the firefly luciferase. Additionally, the researchers assessed the effects of G418 and PTC124 on different types of stop codon sequence context and on stop codons followed by various downstream nucleotides. They found that only G418 promoted readthrough. In all cases, G418 stimulated protein production in varying extents depending upon the sequence, whereas PTC124 had no effect [51]. This suggested that cDNA constructs made for reporter assays lack relevance for the potential absence of mRNA regulatory processes in genes, which could be the mechanic elements through which PTC124 exerts its read-through activity. An earlier study in 2011 reported that PTC124 did not show read-through activity on skin cells from patients with a type of nonsense mutation-driven peroxisome biogenesis disorder [52]. The researchers then argued that PTC124 was not promoting read-through, but rather binding and stabilizing the firefly luciferase proteins that managed to ‘‘leak’’ through the translation process despite their nonsense mutations [53]. However, our group has assessed dose-dependent readthrough of the naturally occurring Gly194X stop codon mutation (UGA) in the mouse Pax6 gene using an in vitro luciferase reporter assay. Maximal read-through of 22.7 ± 2.3 % was achieved at a concentration of 3 lM of PTC124 (data not published). PTC1240 s mechanism remains unresolved. A better understanding of this mechanistic discrepancy should be important for the future read-through drug development.
Future perspectives The strong and intricate relationship between PTC and PTC-bearing mRNA availability leads to the idea that the combination of NMD-inhibiting molecules with PTC-suppressing drugs would more likely enhance readthrough efficiency. Inhibiting NMD should increase the abundance of nonsense-containing mRNA, which can then be targeted by nonsense-suppression drugs to upregulate the production of full-length proteins during translation [35]. The combinatorial drug therapy could involve the regulation of both transcriptional and translational processes. Cocktails of aminoglycosides, non-aminoglycosides, or a mixture of both may generate synergistic effects on the level of nonsense-suppression therapy. Moreover, targeting assessory proteins may be an alternative strategy for improving nonsense-suppression therapies. In addition to the reported nonsense mutations in a variety of ocular diseases (Table 1), a number of other recent reports indicate that nonsense suppression could be relevant to other eye diseases. For instance, a novel mutation of
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MIP (p.R113X) was found in a Chinese cataract family. This is the first nonsense mutation of MIP identified in cataract patients [54]. In another report, two homozygous nonsense mutations [c.565C [ T (p.Glu189*)] and [c.409C [ T (p.Arg137*)] have been identified in RAB28 in a German and Moroccan Jewish families, respectively. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases and truncated RAB28 protein has been found in autosomal-recessive cone-rod dystrophy (CRD) [55]. These nonsense mutations identify other ocular diseases that could benefit nonsensesuppression treatment. Significant resources need to be directed towards making knock-in nonsense mutation animal models so that nonsense suppression can be tested in vivo.
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Cellular and Molecular Life Sciences
REVIEW
Nonsense suppression therapies in ocular genetic diseases Xia Wang • Cheryl Y. Gregory-Evans
Received: 1 October 2014 / Revised: 23 December 2014 / Accepted: 23 January 2015 Ó Springer Basel 2015
Abstract Premature termination codons (PTCs) are caused by nonsense mutations and this leads to either degradation of the mutant mRNA template by nonsensemediated decay (NMD) or the production of a non-functional, truncated polypeptide. PTCs contribute significantly to inherited human diseases including ocular disorders. Nonsense suppression therapy allows readthrough of PTCs, thereby rescuing the production of a full-length functional protein. In this review, we highlight the mechanisms that are involved in discriminating normal translation termination from premature termination codons; the current understanding of nonsense-mediated mRNA decay models (NMD); the association and crosstalk between PTC and the underlying dynamic NMD process; and the suppression therapies that have been employed in nonsense-medicated ocular disease models. Defining the mechanistic complexity of PTC and NMD will be important to improve treatments of the numerous genetic disorders caused by PTC mutations. Keywords Nonsense suppression Nonsense-mediated decay Ocular disease Therapy
Introduction In the past decade, remarkable advances in the molecular mechanism governing nonsense suppression and nonsensemediated decay (NMD) activation have been made, aiming X. Wang C. Y. Gregory-Evans (&) Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC V5Z 3N9, Canada e-mail: [email protected] X. Wang e-mail: [email protected]
to treat nonsense-related genetic diseases. Approximately 11 % of all inherited diseases are caused by nonsense mutations, suggesting that targeting patients with this type of mutation could be relevant to many patients. We provide only a brief overview of the mechanisms, focusing on interactions between premature termination codons (PTC) and NMD, and the therapies in ocular genetic disorders. Our purpose is to underscore and gain insight into the principles of nonsense suppression therapy for ocular diseases in the future.
Eukaryotic translation termination and premature termination codon In organisms, including humans, translation termination occurs when one of the three stop codons (UAA, UGA, or UAG) enters the ribosomal A site [1]. Two translation termination factors are involved in regulating eukaryotic translation termination. Eukaryotic release factor I (eRF1) directly recognizes one of the three stop codons (UAA, UAG, and UGA) [2]. The translation termination efficiency depends on competition between stop codon recognition by eRF1 and decoding of the stop codon by a near-cognate tRNA that is paired using two of the three bases. This decoding process in which an amino acid is incorporated in place of the stop codon, leading to natural suppression, is called ‘readthrough’ [1]. The second termination release factor is eRF3, a GTPase that binds to eRF1 and facilitates in the termination process. When an initial stop codon enters the ribosomal site A and is recognized by eRF1, GTP hydrolysis by eRF3 binds and induces conformational changes in eRF1 [3]. The subsequent adaptation of domain 2 of eRF1 positions its GGQ motif into the peptidyl transferase center of the ribosome [4], which finalizes stop
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codon recognition. This allows eRF1 to stimulate hydrolysis of the ester bond of the peptidyl-tRNA, thus facilitating the release of the completed polypeptide chain [2]. The recognition process signals sampling to identify the appropriate binding partners to form the pre-termination complex, including both aminoacyl-tRNAs and release factor eRF1. However, the molecular details of stop codon recognition by eukaryotic release factors remain poorly understood [2]. The premature translation codon (PTC) is caused by nonsense mutations, resulting in the occurrence of UAA, UAG, or UGA codons in the protein-coding region of the mRNA template, leading to either the production of a truncated polypeptide product or more frequently, to mRNA destabilization through nonsense-mediated mRNA decay (NMD) [5, 6]. It has been indicated that the termination suppression at naturally occurring stop codons occurs at a frequency of \0.1 % [7, 8]. However, the termination suppression at PTCs occurs at a higher frequency of \1 % [9, 10]. This difference may be due to the localization of the stop codon at distinct regions of higher-order structure of the mRNAs, and different protein–factor interactions that occur within the messenger ribonucleoprotein (mRNP) complex [2].
Nonsense-mediated mRNA decay (NMD) NMD is an evolutionarily conserved cellular surveillance and quality control process that is present in all eukaryotic cells. It functions to selectively target mRNA transcripts that harbor a PTC for rapid degradation, thus reducing the synthesis of truncated proteins with potentially harmful dominant-negative effects (for detailed reviews, see [11– 18]). PTCs can be introduced as a result of genetic or somatic mutations, errors in transcription and splicing, or programmed gene rearrangements. As the critical function of mRNA is associated with the primary sequence of its protein-coding region, this NMD is subject to inspection by the molecular signaling in the cell [19]. For both defective mRNAs and proteins, the polymers must first be recognized, subsequently characterized for degradation, and finally destroyed [20]. Creation of a PTC generally activates the NMD pathways [19]. The mechanisms and the regulation of NMD have been extensively studied [14, 19, 21–24]. Over the past decade, however, it has become clear that the role of NMD goes beyond its originally defined function as a quality control mechanism that degrades PTCrelated aberrant mRNAs. NMD is involved in regulating normal development [18, 25], and it itself can be developmentally regulated [14]. It is recently considered as a fundamental developmental regulator of transcriptome
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[19]. Genome-wide analysis of cell lines depleted of NMD factors and NMD-deficient mice have demonstrated that 3–20 % of the transcriptome is deregulated as a consequence of compromised NMD [26–31]. Although it is not yet clear which factors and mechanisms are direct NMD downstream targets in these NMD-deficient models, they have added more complexity to the role of NMD [19].
Recognition-crosstalk between PTC and NMD One of the first crucial steps in NMD is to recognize that a stop codon at the RNA template is premature, then characterizing the PTC-containing mRNA transcript as aberrant, and finally targeting it for degradation. A circular mRNA structure known as the closed-loop complex is associated with and formed by three proteins in the mRNP complex: the cap-binding protein (eIF4E) is bound to the 50 -cap structure of the mRNA; poly(A)-binding protein (PABP) is bound to the mRNA poly(A) tail; and eIF4G simultaneously binds both eIF4E and PABP, resulting in circularization of the mRNP complex and formation of the closed-loop complex [32–34]. Although this fundamental molecular recognition process has been the subject of much study and some core NMD components are conserved across species, the precise mechanisms of PTC recognition vary largely between cells, species, and mRNA transcripts [13, 14, 24]. NMD models have revealed crosstalk between the transcription and translation machineries, where alternation of the components and factors of mRNP complexes during transcription has an impact on the differential outcome of translation [35]. Four different mechanistic NMD models have been proposed to make a distinction between a normal and premature termination codon (Fig. 1) [18]: (i) The faux 30 UTR model: the recognition depends on the distance between the binding ribosome and the poly (A) tail. Premature codon termination prevents the eRF3 from interacting with PABPC1, which is the characterized normal translation termination interaction, by artificially increasing the length of the 30 UTR and the physical distance between the two proteins [18]. (ii) Exonjunction-complex-dependent (EJC) model: this recognition requires splicing-dependent deposition of a specific protein complex, the core EJC, 20 to 24 nt upstream of exon–exon junctions, and a pioneer round of translation. In normal translation termination, when the translating ribosome reaches the normal stop codon in the last exon, it traverses the entire length of the mRNA during the pioneer round of translation and displaces all the EJCs that it encounters. The communication of eRF3 with PABPC1 subsequently signals normal termination. However, premature translation termination is featured by the presence of a core EJC
Nonsense suppression therapies in ocular genetic diseases
Fig. 1 Mechanic models of nonsense-mediated mRNA decay. a Normal termination process. b Faux 30 UTR model. c EJC model. d Unified model. e Ribosome release model
downstream of the stop codon. This remaining EJC recruits the NMD effectors (UPF2 and UPF3) and the SURF protein complex (SMG1, UPF1, and eRF1 and eRF3), leading to the EJC–SURF interaction and inducing phosphorylation of UPF1 by SMG1, which initiates degradation of the PTC-containing transcript [18]. (iii) The unified model: this model incorporates factors from both the EJC and faux 30 UTR models. Recognition of a premature stop codon involves competition between UPF1 in the SURF complex and eRF3 on the terminating ribosome for interaction with PABPC1. Instead of interaction of eRF3 with PABPC1 in the normal termination event, in a premature termination event, eRF3 and PABPC1 are unable to bind, making interaction of UPF1 with PABPC1 possible. In this model, the EJC is not required for PTC recognition, but it functions as an enhancer at the mRNA template [18]. (iv) The ribosome release model: Translating ribosomes normally protect the mRNA from
degradation as they traverse the transcript. However, in the presence of a PTC, the interaction of UPF1 with eIF3 stimulates ribosome release. This early ribosome release exposes the downstream unprotected mRNA to degradation by nucleases [18]. It is clear that no matter the exact mechanism applied to identify a PTC, the cell can distinguish between a PTC and a natural termination codon [19]. This cellular discrimination serves as a foundation for the concept that it should be possible to develop small molecules that can promote readthrough at a PTC without affecting normal translational termination [19].
Nonsense mutation therapy strategies Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame
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nonsense mutations (also commonly known as PTCs). This approach utilizes compounds that suppress translation termination at PTCs, allowing translation to continue, so that partial levels of deficient protein function to be restored [36] (Fig. 2). The primary prerequisite for a therapeutic benefit of PTC suppression is the presence of a nonsense mutation-containing translatable mRNA that is not efficiently degraded by NMD [1]. A recent meta-analysis showed that there are more than 7,500 nonsense mutations in 995 different genes that cause inherited diseases. This result revealed that approximately *11 % of all inherited diseases are caused by nonsense mutations, suggesting that targeting patients with this type of mutation could be relevant to many patients with a broad range of genetic diseases [37]. PTC suppression as a potential therapy was first described in 1996 for diseases caused by nonsense mutations in the CFTR gene (mutations in which result in the common genetic disease CF) [38]. The study showed that the aminoglycoside G418 suppresses nonsense mutations in the CFTR gene and restored significant levels of both CFTR protein and function in cultured cells. Since then, approximately 100 studies have been carried out, and the effectiveness of nonsense suppression as a possible treatment has been investigated in nearly 40 different diseases [35]. However, as discussed in a recent study in cystic fibrosis patients who received ataluren treatment in a phase 3 clinical trial, ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients. This may suggest that the factors such as the ages and the choice of a population of patients could be the limitations and affect the therapeutic effects in clinical trials [39].
Fig. 2 The addition of a nonsense suppression drug such as an aminoglycoside allows readthrough to take place when the ribosome encounters the PTC, leading to the production of full-length proteins
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Nonsense mutation-mediated ocular disease therapy Pharmaceutical therapies result in the introduction of amino acids into nonsense mutation sites in order to restore or compensate for full-length protein products, and have been carried out for almost two decades [40]. PTC-mediated ocular diseases that have been studied are summarized and listed in Table 1. The choroideremia mutant zebrafish (chmru848) has a recessive Q32X mutation in the second exon of rep1. The no isthmus (noitu29a) has a recessive nonsense mutation in exon 7.1 of pax2.1 (R139X). The second zebrafish model of ocular coloboma is the grumpy (gupm189) mutant that has a recessive nonsense mutation in lamb1 [41]. Overexpression of firefly luciferase construct (pGL3), with mutant chm, noi, or gup gene products fused to the N-terminus, in COS-7 cells treated with gentamicin and paromomycin revealed luciferase activity readthrough efficiency: paromomycin [ gentamicin; R139X (UGA A) [ Q524X (UAG U) [ Q32X (UAA A). Gentamicin and paromomycin (100 lM) was used to treat choroideremia mutant zebrafish from 10 hpf to 6 or 9 dpf. Western-blot analysis revealed full-length rep1 protein from aminoglycoside treatment. Retinal sections appeared similar to WT fish. Apoptotic activity was greatly reduced throughout the larvae and within the retina with aminoglycoside treatment [41]. In ocular coloboma zebrafish models, although the phenotype did not reach WT levels, retinal histology and gross morphology of noi mutants was improved from aminoglycoside treatment. In grumpy (gupm189) mutant fish, retinal histology showed the closure of the optic fissure from aminoglycoside treatment, however rescue of the lens was not successful. Treated gup remained short in
Nonsense suppression therapies in ocular genetic diseases Table 1 Overview of nonsense suppression experiments in cellular and animal models of ocular diseases Disease
Gene
Mutation Stop codon
Used drug
Retinitis pigmentosa
RP2 (X-linked)
R120X
UGA G Gentamicin UGA C Ataluren
In vitro cell culture
Yes
Rho (autosomal dominant)
S334X
UAA G Gentamicin, G418
In vitro cell culture
Yes
In vivo transgenic mouse
Yes
Rpe65 (autosomal R44X recessive)
UGA U Gentamicin
Choroideremia
REP1 (X-linked recessive)
Q32X
Ocular coloboma
Pax2 (autosomal dominant)
R139X
Lamb1 (autosomal R524 dominant) Usher syndrome type 1 USH1C
R31X
R3X
Pax6
Ex vivo
No
In vitro cell culture
No No
UAA A Gentamicin, paromomycin
In vitro cell culture In vivo zebrafish
Yes Yes
UGA A Gentamicin, paromomycin
In vitro cell culture
Yes
In vivo zebrafish
Yes
UAG U
In vitro cell culture
Yes
UGA A G418, Gentamicin, paromomycin, NB30
[42] [40]
Yes Yes
[44]
In vivo, ex vivo
Yes
[44, 45]
Yes
[45–47]
R643X R929X G194X
UGA A UGA A PTC124
Yes In vitro cell culture, in vivo Yes transgenic mice
length compared with WT [41]. Using an in vitro cell culture study model of retinitis pigmentosa, overexpression of a luciferase reporter (p2luc) stop codon vector and overexpression of a luciferase (pGL3) construct with N-terminus fusion of a short Rho sequence with S334X were introduced in CHO cells and COS-7 cells, respectively. Luciferase assays revealed an increase in readthrough with drug treatment [42, 43]. In the S334X-4 heterozygous transgenic rat model, ERG responses showed an increase in both a- and b-wave amplitudes from gentamicin treatment. Preservation of the photoreceptor cell count was seen at P19 for both G418 and gentamicin. However, G418 showed more systemic toxicity and no animal survived beyond P20. Functional electroretinographic studies showed significant retention of ERG a- and b-waves from drug treatment [44]. In a USH1 mouse model, cDNA overexpression of mutant murine harmonin a1-GFP or mRFP fusion protein was subretinally injected into the newborn mice. Fluorescence microscopy analysis revealed an increase in GFP expression from PTC124 treatment. Western-blot analysis displayed full-length harmonin a1 expression in PTC124-treated retinas [45–48]. Our lab recently treated and analyzed the efficacy of nonsense read-through therapy in the Pax6-deficient mouse model of aniridia (Pax6Sey?/-). For this study, the ‘‘START’’ eye drops containing 1 % ataluren were applied
[43]
In vitro TNT, cell culture, Ex vivo In vitro TNT, cell culture
UGA A
[42, 50]
In vivo zebrafish
UGA C G418, NB30, gentamicin, paromomycin UGA C
R245X
Aniridia
Response References
In vivo transgenic mouse
PTC124, gentamicin PCDH15
Model
Yes Yes [48]
to mouse eyes twice daily. This topical administration of ‘‘START’’ therapy on Pax6Sey?/- mouse eyes represented the most promising results. The therapy reversed corneal, lens, and retinal malformation, and restored electrophysiological function of the retina, preventing disease from progressing [49]. This study suggests that the eye retains significant developmental plasticity into the post-natal period and stays susceptible to molecular remodeling, providing a window of opportunity to treat childhood blindness [49]. More recently, a study has demonstrated that treatment of R120X fibroblasts with G418 caused an increase in 40 % of RP2 mRNA and 20 % of protein levels, while PTC124 can restore up to 13 % of RP2 protein levels, but did not significantly increase RP2 mRNA levels. The stored RP2 protein was functional in reversing the cellular phenotypes in cells lacking RP2 proteins [50].
Mechanism of PTC124 activity in cell-line luciferase assays A recent study compared the efficacy of two read-through agents, PTC124 and aminoglycoside G418 with PTC reporter assays using b-galactosidase and the two kinds of luciferase in cell lines, including transient transfection, stable cell lines, and plate-based functional enzyme assays.
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In each assay, G418 appeared to promote translation of proteins with premature stop codons, while PTC124 had no effect except in the presence of the firefly luciferase. Additionally, the researchers assessed the effects of G418 and PTC124 on different types of stop codon sequence context and on stop codons followed by various downstream nucleotides. They found that only G418 promoted readthrough. In all cases, G418 stimulated protein production in varying extents depending upon the sequence, whereas PTC124 had no effect [51]. This suggested that cDNA constructs made for reporter assays lack relevance for the potential absence of mRNA regulatory processes in genes, which could be the mechanic elements through which PTC124 exerts its read-through activity. An earlier study in 2011 reported that PTC124 did not show read-through activity on skin cells from patients with a type of nonsense mutation-driven peroxisome biogenesis disorder [52]. The researchers then argued that PTC124 was not promoting read-through, but rather binding and stabilizing the firefly luciferase proteins that managed to ‘‘leak’’ through the translation process despite their nonsense mutations [53]. However, our group has assessed dose-dependent readthrough of the naturally occurring Gly194X stop codon mutation (UGA) in the mouse Pax6 gene using an in vitro luciferase reporter assay. Maximal read-through of 22.7 ± 2.3 % was achieved at a concentration of 3 lM of PTC124 (data not published). PTC1240 s mechanism remains unresolved. A better understanding of this mechanistic discrepancy should be important for the future read-through drug development.
Future perspectives The strong and intricate relationship between PTC and PTC-bearing mRNA availability leads to the idea that the combination of NMD-inhibiting molecules with PTC-suppressing drugs would more likely enhance readthrough efficiency. Inhibiting NMD should increase the abundance of nonsense-containing mRNA, which can then be targeted by nonsense-suppression drugs to upregulate the production of full-length proteins during translation [35]. The combinatorial drug therapy could involve the regulation of both transcriptional and translational processes. Cocktails of aminoglycosides, non-aminoglycosides, or a mixture of both may generate synergistic effects on the level of nonsense-suppression therapy. Moreover, targeting assessory proteins may be an alternative strategy for improving nonsense-suppression therapies. In addition to the reported nonsense mutations in a variety of ocular diseases (Table 1), a number of other recent reports indicate that nonsense suppression could be relevant to other eye diseases. For instance, a novel mutation of
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MIP (p.R113X) was found in a Chinese cataract family. This is the first nonsense mutation of MIP identified in cataract patients [54]. In another report, two homozygous nonsense mutations [c.565C [ T (p.Glu189*)] and [c.409C [ T (p.Arg137*)] have been identified in RAB28 in a German and Moroccan Jewish families, respectively. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases and truncated RAB28 protein has been found in autosomal-recessive cone-rod dystrophy (CRD) [55]. These nonsense mutations identify other ocular diseases that could benefit nonsensesuppression treatment. Significant resources need to be directed towards making knock-in nonsense mutation animal models so that nonsense suppression can be tested in vivo.
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