news and views
npg
© 2014 Nature America, Inc. All rights reserved.
Upon assembly, stress granules sequester and inactivate proteins involved in translation of certain mRNAs, promoting a translational shift toward proteins that protect the cell12. Studies have shown that anomalous regulation of the ubiquitin-proteasome system can lead to the formation of RNA stress granules and that these can disturb ubiquitin-proteasome system homeostasis13,14. Notably, anomalous regulation of the ubiquitin-proteasome system and subsequent formation of protein aggregates is found in the hypothalamus of obese mice with diet-induced hypothalamic inflammation15. This process contributes to the long-term deterioration of the neuronal network that maintains whole-body energy homeostasis. Thus, hypothetically, the atypical mechanism
of diet- and aging-induced activation of NF-κB through TGF-β1–induced RNA stress granule formation identified by Yan et al.5 may result in defective regulation of the ubiquitinproteasome system, which could explain the formation of protein aggregates in the hypothalamus in animal models of obesity. To our knowledge, this is the first evidence for an independent hypothalamic mechanism leading to a prodiabetic condition. This places TGF-β1 as a promising potential pharmacological target for the treatment of diabetes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Lam, C.K., Chari, M. & Lam, T.K. Physiology (Bethesda) 24, 159–170 (2009).
2. De Souza, C.T. et al. Endocrinology 146, 4192–4199 (2005). 3. Zhang, X. et al. Cell 135, 61–73 (2008). 4. Zhang, G. et al. Nature 497, 211–216 (2013). 5. Yan, J. et al. Nat. Med. 1001–1008 (2014). 6. Akhurst, R.J. & Hata, A. Nat. Rev. Drug Discov. 11, 790–811 (2012). 7. Wan, Y.Y. & Flavell, R.A. Immunol. Rev. 220, 199–213 (2007). 8. Shull, M.M. et al. Nature 359, 693–699 (1992). 9. Ziyadeh, F.N. et al. Proc. Natl. Acad. Sci. USA 97, 8015–8020 (2000). 10. Yadav, H. et al. Cell Metab. 14, 67–79 (2011). 11. Herder, C. et al. Diabetes Care 32, 1921–1923 (2009). 12. Anderson, P. & Kedersha, N. Trends Biochem. Sci. 33, 141–150 (2008). 13. Takahashi, M. et al. Mol. Cell. Biol. 33, 815–829 (2013). 14. Mazroui, R., Di Marco, S., Kaufman, R.J. & Gallouzi, I.E. Mol. Biol. Cell 18, 2603–2618 (2007). 15. Ignacio-Souza, L.M. et al. Endocrinology 155, 2831–2844 (2014).
Activating internal ribosome entry to treat Duchenne muscular dystrophy Shireen R Lamandé & Kathryn N North Mutations in the DMD gene, encoding dystrophin, cause the most common forms of muscular dystrophy. A new study shows that forcing translation of DMD from an internal ribosome entry site can alleviate Duchenne muscular dystrophy symptoms in a mouse model. Duchenne muscular dystrophy (DMD) is characterized by complete or almost complete loss of dystrophin, an intracellular muscle structural protein that is a crucial component of the major connection between the internal cytoskeleton and the extracellular basement membrane. Children with DMD present in early childhood with progressive muscle weakness leading to loss of ambulation and early death due to respiratory or cardiac failure. In most patients, genotype-phenotype relationships follow the ‘reading frame rule’1, where mutations that disrupt the translational reading frame and introduce premature translation termination codons result in little or no protein production and cause DMD. Mutations in DMD that preserve the open reading frame and protein synthesis usually result in Becker muscular dystrophy (BMD), a less severe disorder in which a truncated form of the protein is produced. Restoration of the reading frame is the principle underlying the exon-skipping approach to DMD therapy, currently in clinical trials2, which aims to produce a truncated Shireen R. Lamandé and Kathryn N. North are in the Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne, Victoria, Australia. e-mail: [email protected]
functional dystrophin protein and convert the DMD phenotype to a BMD phenotype. Interestingly, premature termination codons in DMD exons 1 and 2 (such as p.Trp3X (ref. 3), p.Glu5ValfsX3 (ref. 3) and p.Gln17X (ref. 4)) result in a very mild clinical phenotype, with affected individuals retaining the ability to walk well into adulthood. Instead of triggering nonsense-mediated mRNA decay and therefore complete loss of dystrophin protein, the exon 1 mutation p.Trp3X is thought to initiate translation at two AUG codons in exon 6, producing a protein that lacks the amino acids encoded by exons 1–5 (ref. 3). Although this was proposed to be the mechanism behind the mild phenotype of patients with truncating mutations in the first two DMD exons, it was not known how this downstream translation initiation occurred. In this issue of Nature Medicine, Wein et al.5 demonstrate that DMD exon 6 translation initiation is driven by an internal ribosome entry site (IRES) in exon 5, allowing translation of a truncated form of dystrophin. This finding presents a new avenue for therapy in patients with DMD with truncating mutations in early DMD exons, in which translation beginning in exon 6 could be promoted. Wein et al.5 were puzzled as to why a deletion of exon 2, which would result in a translation
nature medicine volume 20 | number 9 | september 2014
frameshift and a premature translation termination codon in exon 3, and hence severe Duchenne muscular dystrophy according to the reading frame rule, had never been reported. They hypothesized that this mutation might have mild symptoms and therefore evade detection, which they confirmed when they identified a DMD exon 2 deletion in an entirely asymptomatic individual. The authors carried out mass spectrometry on muscle biopsy tissue from this individual and detected a form of dystrophin with a smaller molecular weight than normal; this form did not have any peptides encoded by exons 1 through 5, consistent with translation initiation of this protein in exon 6. The second subject, with mild BMD, had an exon 2 frameshift mutation and a similar smaller-molecular-weight dystrophin lacking the normal N terminus. Using muscle RNA from this subject, they were able to carry out ribosome profiling to determine the translation efficiency along the DMD mRNA. They found normal levels of exon 1 translation initiation followed by termination in exon 2 and translation again after the AUG translation start codons in exon 6. The authors then carried out in vitro translation assays using a recombinant version of the DMD mRNA that excluded the 5′ translation 987
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Figure 1 Exon 2 skipping in a DMD mouse model with an exon 2 duplication can ameliorate the DMD phenotype. Dmd pre-mRNA and mRNA is shown with coding regions in red and the 5∙ UTR and 3∙ UTR in blue. Duplication of exon 2 in a DMD mouse model (left) results in a premature translation termination codon (PTC) in exon 2 and no translation of Dmd and severe DMD symptoms. Wein et al.5 show that inducing exon 2 skipping in this mouse model using AAV-encoded U7 snRNAs (right) activates a Dmd IRES (red line), produces N-truncated functional dystrophin (green) and restores muscle function. The resulting phenotype is mild.
initiation codon but included sequences from exon 1 to part of exon 6 in frame with a reporter. They showed cap-independent translation in rabbit reticulocyte lysate, which is consistent with IRES activity within DMD exons 1–5. They confirmed this in mouse C2C12 myoblasts and, using deletion constructs, mapped the IRES to 71 nucleotides in exon 5. The IRES was active in two myoblast cell lines but not in HEK293K cells, suggesting the IRES requires muscle-specific factors for activity. Duplication of exon 2 is the most common single-exon duplication in the human DMD gene. It introduces a premature termination codon and results in the severe DMD phenotype. The authors showed in C2C12 myoblasts that the duplication prevents translation from the IRES in exon 5, explaining the severe clinical phenotype associated with this mutation. In the same cell line, they showed that deletion of exon 2 did not disrupt translation from the IRES. This finding suggested the possibility that exon-skipping strategies could be used to delete exon 2 in individuals with exon 2 duplications and activate the IRES to produce functional N-truncated dystrophin to ameliorate the DMD phenotype.
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The authors then infected myoblasts derived from a subject with an exon 2 duplication with adeno-associated virus (AAV) vectors encoding optimized U7 small nuclear RNAs (snRNAs) designed to achieve exon 2 skipping. In this manner exon 2 was skipped and N-truncated dystrophin was synthesized by the myoblasts. To test the potential for a similar therapeutic approach in vivo, they injected the tibialis anterior muscle of a DMD mouse model carrying a Dmd exon 2 duplication with their AAV exon 2–skipping vector. Exon 2 was efficiently skipped and N-truncated dystrophin was expressed in the muscle (Fig. 1). Crucially, exon 2 skipping improved muscle integrity and function and restored the dystrophin-associated glycoprotein complex. This research now opens the way for a new therapy for the up to 6% of patients with DMD that carry the exon duplication; however, some important questions remain. Although patients with exon 2 duplications are obvious candidates for exon 2–skipping therapy, we do not currently know which premature termination mutations trigger nonsense-mediated mRNA decay causing DMD and which ones activate the IRES and ameliorate the phenotype. Some
clues can be found in the Leiden Open Variation Database (www.lovd.nl), which contains 13 different nucleotide substitutions that introduce premature termination codons in exons 1–5. Although the phenotype details for these mutations are incomplete, premature terminations before and including codon 45 (in exon 3) result in BMD, whereas those from codon 60 to 119 produce DMD in most people, suggesting that these termination codons are too far from the initiation codon to escape nonsense-mediated mRNA decay. Thus, in principle, patients with DMD with mutations that introduce premature termination codons in exons 3, 4 and 5 that trigger mRNA decay could also benefit from exon 2–skipping therapies. AAV delivery of U7 snRNAs to induce DMD exon skipping, restore the dystrophin reading frame and produce functional dystrophin with an internal deletion has potential advantages over the antisense oligonucleotide exonskipping approaches currently in human clinical trials. In contrast to antisense oligonucleotide therapies6, AAV vectors produce stable in vivo expression, reducing the need for repeated administration, and can efficiently infect most muscles including cardiac muscle7. Although the existence and activity of vertebrate cellular mRNA IRESs is somewhat controversial8, the evidence presented for an IRES in exon 5 of the DMD gene that is active in vivo is compelling. There are currently only a few known examples of IRES elements in the coding region of genes (rather than in the 5′ untranslated region (UTR)), notably one in the APC gene, associated with colorectal cancer9, and one in CACNA1A, associated with episodic and spinocerebellar ataxia10. As modulating IRES activity could have potential therapeutic applications, the study by Wein et al.5 should prompt the search for coding-region IRES activity in other clinically relevant genes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Monaco, A.P., Bertelson, C.J., Liechti-Gallati, S., Moser, H. & Kunkel, L.M. Genomics 2, 90–95 (1988). 2. Arechavala-Gomeza, V., Anthony, K., Morgan, J. & Muntoni, F. Curr. Gene Ther. 12, 152–160 (2012). 3. Gurvich, O.L. et al. Hum. Mutat. 30, 633–640 (2009). 4. Witting, N., Duno, M. & Vissing, J. Neuromuscul. Disord. 23, 25–28 (2013). 5. Wein, N. et al. Nat. Med. 20, 992–1000 (2014). 6. Goyenvalle, A. & Davies, K.E. Skelet. Muscle 1, 8 (2011). 7. Goyenvalle, A. et al. Mol. Ther. 20, 1212–1221 (2012). 8. Jackson, R.J. Cold Spring Harb. Perspect. Biol. 5, a011569 (2013). 9. Heppner Goss, K., Trzepacz, C., Tuohy, T.M. & Groden, J. Proc. Natl. Acad. Sci. USA 99, 8161–8166 (2002). 10. Du, X. et al. Cell 154, 118–133 (2013).
volume 20 | number 9 | september 2014 nature medicine
npg
© 2014 Nature America, Inc. All rights reserved.
Upon assembly, stress granules sequester and inactivate proteins involved in translation of certain mRNAs, promoting a translational shift toward proteins that protect the cell12. Studies have shown that anomalous regulation of the ubiquitin-proteasome system can lead to the formation of RNA stress granules and that these can disturb ubiquitin-proteasome system homeostasis13,14. Notably, anomalous regulation of the ubiquitin-proteasome system and subsequent formation of protein aggregates is found in the hypothalamus of obese mice with diet-induced hypothalamic inflammation15. This process contributes to the long-term deterioration of the neuronal network that maintains whole-body energy homeostasis. Thus, hypothetically, the atypical mechanism
of diet- and aging-induced activation of NF-κB through TGF-β1–induced RNA stress granule formation identified by Yan et al.5 may result in defective regulation of the ubiquitinproteasome system, which could explain the formation of protein aggregates in the hypothalamus in animal models of obesity. To our knowledge, this is the first evidence for an independent hypothalamic mechanism leading to a prodiabetic condition. This places TGF-β1 as a promising potential pharmacological target for the treatment of diabetes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Lam, C.K., Chari, M. & Lam, T.K. Physiology (Bethesda) 24, 159–170 (2009).
2. De Souza, C.T. et al. Endocrinology 146, 4192–4199 (2005). 3. Zhang, X. et al. Cell 135, 61–73 (2008). 4. Zhang, G. et al. Nature 497, 211–216 (2013). 5. Yan, J. et al. Nat. Med. 1001–1008 (2014). 6. Akhurst, R.J. & Hata, A. Nat. Rev. Drug Discov. 11, 790–811 (2012). 7. Wan, Y.Y. & Flavell, R.A. Immunol. Rev. 220, 199–213 (2007). 8. Shull, M.M. et al. Nature 359, 693–699 (1992). 9. Ziyadeh, F.N. et al. Proc. Natl. Acad. Sci. USA 97, 8015–8020 (2000). 10. Yadav, H. et al. Cell Metab. 14, 67–79 (2011). 11. Herder, C. et al. Diabetes Care 32, 1921–1923 (2009). 12. Anderson, P. & Kedersha, N. Trends Biochem. Sci. 33, 141–150 (2008). 13. Takahashi, M. et al. Mol. Cell. Biol. 33, 815–829 (2013). 14. Mazroui, R., Di Marco, S., Kaufman, R.J. & Gallouzi, I.E. Mol. Biol. Cell 18, 2603–2618 (2007). 15. Ignacio-Souza, L.M. et al. Endocrinology 155, 2831–2844 (2014).
Activating internal ribosome entry to treat Duchenne muscular dystrophy Shireen R Lamandé & Kathryn N North Mutations in the DMD gene, encoding dystrophin, cause the most common forms of muscular dystrophy. A new study shows that forcing translation of DMD from an internal ribosome entry site can alleviate Duchenne muscular dystrophy symptoms in a mouse model. Duchenne muscular dystrophy (DMD) is characterized by complete or almost complete loss of dystrophin, an intracellular muscle structural protein that is a crucial component of the major connection between the internal cytoskeleton and the extracellular basement membrane. Children with DMD present in early childhood with progressive muscle weakness leading to loss of ambulation and early death due to respiratory or cardiac failure. In most patients, genotype-phenotype relationships follow the ‘reading frame rule’1, where mutations that disrupt the translational reading frame and introduce premature translation termination codons result in little or no protein production and cause DMD. Mutations in DMD that preserve the open reading frame and protein synthesis usually result in Becker muscular dystrophy (BMD), a less severe disorder in which a truncated form of the protein is produced. Restoration of the reading frame is the principle underlying the exon-skipping approach to DMD therapy, currently in clinical trials2, which aims to produce a truncated Shireen R. Lamandé and Kathryn N. North are in the Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne, Victoria, Australia. e-mail: [email protected]
functional dystrophin protein and convert the DMD phenotype to a BMD phenotype. Interestingly, premature termination codons in DMD exons 1 and 2 (such as p.Trp3X (ref. 3), p.Glu5ValfsX3 (ref. 3) and p.Gln17X (ref. 4)) result in a very mild clinical phenotype, with affected individuals retaining the ability to walk well into adulthood. Instead of triggering nonsense-mediated mRNA decay and therefore complete loss of dystrophin protein, the exon 1 mutation p.Trp3X is thought to initiate translation at two AUG codons in exon 6, producing a protein that lacks the amino acids encoded by exons 1–5 (ref. 3). Although this was proposed to be the mechanism behind the mild phenotype of patients with truncating mutations in the first two DMD exons, it was not known how this downstream translation initiation occurred. In this issue of Nature Medicine, Wein et al.5 demonstrate that DMD exon 6 translation initiation is driven by an internal ribosome entry site (IRES) in exon 5, allowing translation of a truncated form of dystrophin. This finding presents a new avenue for therapy in patients with DMD with truncating mutations in early DMD exons, in which translation beginning in exon 6 could be promoted. Wein et al.5 were puzzled as to why a deletion of exon 2, which would result in a translation
nature medicine volume 20 | number 9 | september 2014
frameshift and a premature translation termination codon in exon 3, and hence severe Duchenne muscular dystrophy according to the reading frame rule, had never been reported. They hypothesized that this mutation might have mild symptoms and therefore evade detection, which they confirmed when they identified a DMD exon 2 deletion in an entirely asymptomatic individual. The authors carried out mass spectrometry on muscle biopsy tissue from this individual and detected a form of dystrophin with a smaller molecular weight than normal; this form did not have any peptides encoded by exons 1 through 5, consistent with translation initiation of this protein in exon 6. The second subject, with mild BMD, had an exon 2 frameshift mutation and a similar smaller-molecular-weight dystrophin lacking the normal N terminus. Using muscle RNA from this subject, they were able to carry out ribosome profiling to determine the translation efficiency along the DMD mRNA. They found normal levels of exon 1 translation initiation followed by termination in exon 2 and translation again after the AUG translation start codons in exon 6. The authors then carried out in vitro translation assays using a recombinant version of the DMD mRNA that excluded the 5′ translation 987
news and views DMD mouse model
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Debbie Maizels/Nature Publishing Group
© 2014 Nature America, Inc. All rights reserved.
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Figure 1 Exon 2 skipping in a DMD mouse model with an exon 2 duplication can ameliorate the DMD phenotype. Dmd pre-mRNA and mRNA is shown with coding regions in red and the 5∙ UTR and 3∙ UTR in blue. Duplication of exon 2 in a DMD mouse model (left) results in a premature translation termination codon (PTC) in exon 2 and no translation of Dmd and severe DMD symptoms. Wein et al.5 show that inducing exon 2 skipping in this mouse model using AAV-encoded U7 snRNAs (right) activates a Dmd IRES (red line), produces N-truncated functional dystrophin (green) and restores muscle function. The resulting phenotype is mild.
initiation codon but included sequences from exon 1 to part of exon 6 in frame with a reporter. They showed cap-independent translation in rabbit reticulocyte lysate, which is consistent with IRES activity within DMD exons 1–5. They confirmed this in mouse C2C12 myoblasts and, using deletion constructs, mapped the IRES to 71 nucleotides in exon 5. The IRES was active in two myoblast cell lines but not in HEK293K cells, suggesting the IRES requires muscle-specific factors for activity. Duplication of exon 2 is the most common single-exon duplication in the human DMD gene. It introduces a premature termination codon and results in the severe DMD phenotype. The authors showed in C2C12 myoblasts that the duplication prevents translation from the IRES in exon 5, explaining the severe clinical phenotype associated with this mutation. In the same cell line, they showed that deletion of exon 2 did not disrupt translation from the IRES. This finding suggested the possibility that exon-skipping strategies could be used to delete exon 2 in individuals with exon 2 duplications and activate the IRES to produce functional N-truncated dystrophin to ameliorate the DMD phenotype.
988
The authors then infected myoblasts derived from a subject with an exon 2 duplication with adeno-associated virus (AAV) vectors encoding optimized U7 small nuclear RNAs (snRNAs) designed to achieve exon 2 skipping. In this manner exon 2 was skipped and N-truncated dystrophin was synthesized by the myoblasts. To test the potential for a similar therapeutic approach in vivo, they injected the tibialis anterior muscle of a DMD mouse model carrying a Dmd exon 2 duplication with their AAV exon 2–skipping vector. Exon 2 was efficiently skipped and N-truncated dystrophin was expressed in the muscle (Fig. 1). Crucially, exon 2 skipping improved muscle integrity and function and restored the dystrophin-associated glycoprotein complex. This research now opens the way for a new therapy for the up to 6% of patients with DMD that carry the exon duplication; however, some important questions remain. Although patients with exon 2 duplications are obvious candidates for exon 2–skipping therapy, we do not currently know which premature termination mutations trigger nonsense-mediated mRNA decay causing DMD and which ones activate the IRES and ameliorate the phenotype. Some
clues can be found in the Leiden Open Variation Database (www.lovd.nl), which contains 13 different nucleotide substitutions that introduce premature termination codons in exons 1–5. Although the phenotype details for these mutations are incomplete, premature terminations before and including codon 45 (in exon 3) result in BMD, whereas those from codon 60 to 119 produce DMD in most people, suggesting that these termination codons are too far from the initiation codon to escape nonsense-mediated mRNA decay. Thus, in principle, patients with DMD with mutations that introduce premature termination codons in exons 3, 4 and 5 that trigger mRNA decay could also benefit from exon 2–skipping therapies. AAV delivery of U7 snRNAs to induce DMD exon skipping, restore the dystrophin reading frame and produce functional dystrophin with an internal deletion has potential advantages over the antisense oligonucleotide exonskipping approaches currently in human clinical trials. In contrast to antisense oligonucleotide therapies6, AAV vectors produce stable in vivo expression, reducing the need for repeated administration, and can efficiently infect most muscles including cardiac muscle7. Although the existence and activity of vertebrate cellular mRNA IRESs is somewhat controversial8, the evidence presented for an IRES in exon 5 of the DMD gene that is active in vivo is compelling. There are currently only a few known examples of IRES elements in the coding region of genes (rather than in the 5′ untranslated region (UTR)), notably one in the APC gene, associated with colorectal cancer9, and one in CACNA1A, associated with episodic and spinocerebellar ataxia10. As modulating IRES activity could have potential therapeutic applications, the study by Wein et al.5 should prompt the search for coding-region IRES activity in other clinically relevant genes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Monaco, A.P., Bertelson, C.J., Liechti-Gallati, S., Moser, H. & Kunkel, L.M. Genomics 2, 90–95 (1988). 2. Arechavala-Gomeza, V., Anthony, K., Morgan, J. & Muntoni, F. Curr. Gene Ther. 12, 152–160 (2012). 3. Gurvich, O.L. et al. Hum. Mutat. 30, 633–640 (2009). 4. Witting, N., Duno, M. & Vissing, J. Neuromuscul. Disord. 23, 25–28 (2013). 5. Wein, N. et al. Nat. Med. 20, 992–1000 (2014). 6. Goyenvalle, A. & Davies, K.E. Skelet. Muscle 1, 8 (2011). 7. Goyenvalle, A. et al. Mol. Ther. 20, 1212–1221 (2012). 8. Jackson, R.J. Cold Spring Harb. Perspect. Biol. 5, a011569 (2013). 9. Heppner Goss, K., Trzepacz, C., Tuohy, T.M. & Groden, J. Proc. Natl. Acad. Sci. USA 99, 8161–8166 (2002). 10. Du, X. et al. Cell 154, 118–133 (2013).
volume 20 | number 9 | september 2014 nature medicine
