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Does mRNA translation starting from an alternative initiation site contribute to the pathology of Huntington’s disease?
Medical Hypotheses (2000) 54(5), 689–690 © 2000 Harcourt Publishers Ltd doi: 10.1054/mehy.1998.0815, available online at http://www.idealibrary.com on
Does mRNA translation starting from an alternative initiation site contribute to the pathology of HuntingtonÕs disease? A. D. Santos, E. A. Padlan Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Summary Huntington’s disease is associated with an expanded and unstable trinucleotide repeat (CAG)n. Various possibilities have been suggested to explain the significance of poly-(CAG) length in HD, including changes in the structure of the product (huntingtin) which result in the protein acquiring deleterious properties. We have looked at the nucleotide sequence coding for huntingtin and find that another possibility may exist for the correlation between the occurrence of HD and poly-CAG length. We have noted an alternative reading frame that includes the trinucleotide repeat, now read as (GCA)n. Upon close examination of this alternative gene product, we observe features that suggest it can likewise have deleterious properties. © 2000Harcourt Publishers Ltd
Huntington’s disease (HD) is a neurodegenerative disorder associated with an expanded and unstable trinucleotide repeat, (CAG)n, in the IT15 gene. Patients with HD show repeat lengths of 36-121, while normal individuals show repeat lengths of 9–39 (1). The IT15 gene product, called huntingtin, contains 3144 amino acids (including the 21 glutamines coded by the poly-(CAG)) in the particular gene sequenced [GenBank Entry L12392] (2). Various possibilities have been suggested in an attempt to explain the significance of poly-(CAG) length in HD, including alterations in gene structure and
Received 29 May 1998 Accepted 25 August 1998 Correspondence to: Eduardo A. Padlan, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0560, USA. Phone: +1 301 4021780; Fax: +1 301 4960201 Permanent address for Dr Santos: College of Science, University of the Philippines, Diliman, 1101 Quezon City, Philippines
changes in the structure of huntingtin which result in the protein acquiring new and deleterious properties (see, e.g. (1,3) for reviews). We have examined the nucleotide sequence coding for huntingtin [GenBank Entry L12392] and find that an alternative explanation may exist for the correlation between poly-(CAG) length and the occurrence of HD. The GenBank Entry L12392 is partly reproduced in Figure 1. Huntingtin is presumed to result from a translation starting at the initiation site at position 316 in the above sequence which yields the 3144 amino acids. The poly(CAG) tract starts at position 367. We have underlined a segment of the nucleotide sequence starting from another possible initiation site at position 90 and ending just before the termination codon (in this alternative reading frame) at position 564. The sequence around this alternative initiation site, ‘atc atg ctg’, is very similar to that of human MAP kinase activated protein kinase 2, ‘acc atg ctg’, [GenBank Entry U12779] so that utilization of this alternative initiation site is not implausible. Downstream of the alternative termination codon is a polyadenylation signal, ‘attaaa’ (4), at position 841. 689
690
Santos and Padlan
Fig. 1 Part of GenBank Entry L12392 for the mRNA coding for huntingtin. The sequence coding for the hypothesized polypeptide is underlined.
Translation of the nucleotide segment underlined above would result in the following polypeptide chain which contains 159 amino acids: MLAGVAPPPPARPRLRRRTSGTQGAVGAAGTGPRWTAAQVLLLPAAQSPIHCPGAERRRESARGLRGLPCRAGDRHGDPGKADEGLRVPQVLPAAAAAAAAAAAAAAAAAAAAATAATAAAAAAASSASSAAAAGTAAAASAAAAPAAAPAATRPGCG
where the 21 alanines which result from the poly-(CAG) repeat, now read as poly-(GCA), are underlined. Many other alanines result from this alternative reading frame, most noticeably beyond the polyalanine tract. This reading frame also results in 15 arginines and one lysine (highlighted above using bold and bigger letters), all of which, except for one arginine, are anterior to the (Ala)21 stretch. The polypeptide chain that results from this alternative reading frame is of reasonable size (bigger than many proteins, e.g. myoglobin, lysozyme) and may have a specific function inside the cell. Alternatively, the polypeptide may be innocuous, or nonfunctional, under normal circumstances and is eventually degraded. We hypothesize that, in this alternative gene product, when the polyalanine stretch reaches a sufficient length, it can contribute to the pathology of Huntington’s disease, or in itself can cause the disease. One can envision a number of ways by which this alternative gene product can have deleterious effects. Its sequence suggests that the polypeptide would have an Nterminal part that is positively charged and a C-terminal region that is very hydrophobic. The hydrophobicity, which will increase as the polyalanine tract gets longer, could cause the formation of aggregates which can affect cell viability. Aggregation may also result from the formation of coiled helical structures the probability of which should increase in the presence of more alanines. Further, increased hydrophobicity may protect the polypeptide
Medical Hypotheses (2000) 54(5), 689–690
from enzymatic degradation, either by the formation of more stable secondary and tertiary structure, or by increasing the likelihood that the polypeptide will be imbedded in a lipid membrane. The presence in the cell, especially in the nucleus, of a polypeptide rich in positively charged amino acids can have a deleterious effect, since it can bind indiscriminately to nucleic acids and interfere with normal cellular activities. The 15 arginines and lysines among the 93 amino acids (16.1%) anterior to the poly-alanine stretch in the polypeptide above is less than, but nonetheless approaches, the 19.4–24.5% occurrence of arginines and lysines in the human core histones (H2A, H2B, H3 and H4) which bind tightly to double-stranded DNA. Can the polypeptide also bind tightly to DNA? It is relevant to mention at this point that anti-DNA and anti-RNA antibodies are also found to have an overwhelming number of arginine residues in their antigen-binding sites (5). There is already evidence in support of this hypothesis. It has been found that a gene segment containing only exon 1 of the HD gene, but which includes the code for the alternative gene product we propose above, is sufficient to cause symptoms akin to HD in transgenic mice (6). It should also be mentioned that an expanded polyalanine tract has been implicated in the genetic disorder called synpolydactyly (7). We wonder if translations employing alternative initiation sites, or mutations which result in, or coupled with, alternative initiation sites, may be the cause of other diseases also.
REFERENCES 1. 2.
3.
4.
5.
6.
7.
Albin R. L., Tagle D. A. Genetics and molecular biology of Huntington’s disease. Trends Neurosci 1995; 18: 11–14. The Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993; 72: 971–983. Perutz M. F. Glutamine repeats and inherited neurodegenerative diseases: molecular aspects. Curr Opin Struct Biol 1996; 6: 848–858. Sheets M. D., Ogg S. C., Wickens M. P. Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic Acid Res 1990; 18: 5799–5805. Eilat D., Anderson W. F. Structure-function correlates of autoantibodies to nucleic acids. Lessons from immunochemical, genetic and structural studies. Mol Immunol 1994; 31: 1377–1390. Mangiarini L., Sathasivam K., Seller M. et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 1996; 87: 493–506. Muragaki Y., Mundlos S., Upton J., Olsen B. R. Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. Science 1996; 272: 548–550.
© 2000 Harcourt Publishers Ltd
Does mRNA translation starting from an alternative initiation site contribute to the pathology of HuntingtonÕs disease? A. D. Santos, E. A. Padlan Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Summary Huntington’s disease is associated with an expanded and unstable trinucleotide repeat (CAG)n. Various possibilities have been suggested to explain the significance of poly-(CAG) length in HD, including changes in the structure of the product (huntingtin) which result in the protein acquiring deleterious properties. We have looked at the nucleotide sequence coding for huntingtin and find that another possibility may exist for the correlation between the occurrence of HD and poly-CAG length. We have noted an alternative reading frame that includes the trinucleotide repeat, now read as (GCA)n. Upon close examination of this alternative gene product, we observe features that suggest it can likewise have deleterious properties. © 2000Harcourt Publishers Ltd
Huntington’s disease (HD) is a neurodegenerative disorder associated with an expanded and unstable trinucleotide repeat, (CAG)n, in the IT15 gene. Patients with HD show repeat lengths of 36-121, while normal individuals show repeat lengths of 9–39 (1). The IT15 gene product, called huntingtin, contains 3144 amino acids (including the 21 glutamines coded by the poly-(CAG)) in the particular gene sequenced [GenBank Entry L12392] (2). Various possibilities have been suggested in an attempt to explain the significance of poly-(CAG) length in HD, including alterations in gene structure and
Received 29 May 1998 Accepted 25 August 1998 Correspondence to: Eduardo A. Padlan, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0560, USA. Phone: +1 301 4021780; Fax: +1 301 4960201 Permanent address for Dr Santos: College of Science, University of the Philippines, Diliman, 1101 Quezon City, Philippines
changes in the structure of huntingtin which result in the protein acquiring new and deleterious properties (see, e.g. (1,3) for reviews). We have examined the nucleotide sequence coding for huntingtin [GenBank Entry L12392] and find that an alternative explanation may exist for the correlation between poly-(CAG) length and the occurrence of HD. The GenBank Entry L12392 is partly reproduced in Figure 1. Huntingtin is presumed to result from a translation starting at the initiation site at position 316 in the above sequence which yields the 3144 amino acids. The poly(CAG) tract starts at position 367. We have underlined a segment of the nucleotide sequence starting from another possible initiation site at position 90 and ending just before the termination codon (in this alternative reading frame) at position 564. The sequence around this alternative initiation site, ‘atc atg ctg’, is very similar to that of human MAP kinase activated protein kinase 2, ‘acc atg ctg’, [GenBank Entry U12779] so that utilization of this alternative initiation site is not implausible. Downstream of the alternative termination codon is a polyadenylation signal, ‘attaaa’ (4), at position 841. 689
690
Santos and Padlan
Fig. 1 Part of GenBank Entry L12392 for the mRNA coding for huntingtin. The sequence coding for the hypothesized polypeptide is underlined.
Translation of the nucleotide segment underlined above would result in the following polypeptide chain which contains 159 amino acids: MLAGVAPPPPARPRLRRRTSGTQGAVGAAGTGPRWTAAQVLLLPAAQSPIHCPGAERRRESARGLRGLPCRAGDRHGDPGKADEGLRVPQVLPAAAAAAAAAAAAAAAAAAAAATAATAAAAAAASSASSAAAAGTAAAASAAAAPAAAPAATRPGCG
where the 21 alanines which result from the poly-(CAG) repeat, now read as poly-(GCA), are underlined. Many other alanines result from this alternative reading frame, most noticeably beyond the polyalanine tract. This reading frame also results in 15 arginines and one lysine (highlighted above using bold and bigger letters), all of which, except for one arginine, are anterior to the (Ala)21 stretch. The polypeptide chain that results from this alternative reading frame is of reasonable size (bigger than many proteins, e.g. myoglobin, lysozyme) and may have a specific function inside the cell. Alternatively, the polypeptide may be innocuous, or nonfunctional, under normal circumstances and is eventually degraded. We hypothesize that, in this alternative gene product, when the polyalanine stretch reaches a sufficient length, it can contribute to the pathology of Huntington’s disease, or in itself can cause the disease. One can envision a number of ways by which this alternative gene product can have deleterious effects. Its sequence suggests that the polypeptide would have an Nterminal part that is positively charged and a C-terminal region that is very hydrophobic. The hydrophobicity, which will increase as the polyalanine tract gets longer, could cause the formation of aggregates which can affect cell viability. Aggregation may also result from the formation of coiled helical structures the probability of which should increase in the presence of more alanines. Further, increased hydrophobicity may protect the polypeptide
Medical Hypotheses (2000) 54(5), 689–690
from enzymatic degradation, either by the formation of more stable secondary and tertiary structure, or by increasing the likelihood that the polypeptide will be imbedded in a lipid membrane. The presence in the cell, especially in the nucleus, of a polypeptide rich in positively charged amino acids can have a deleterious effect, since it can bind indiscriminately to nucleic acids and interfere with normal cellular activities. The 15 arginines and lysines among the 93 amino acids (16.1%) anterior to the poly-alanine stretch in the polypeptide above is less than, but nonetheless approaches, the 19.4–24.5% occurrence of arginines and lysines in the human core histones (H2A, H2B, H3 and H4) which bind tightly to double-stranded DNA. Can the polypeptide also bind tightly to DNA? It is relevant to mention at this point that anti-DNA and anti-RNA antibodies are also found to have an overwhelming number of arginine residues in their antigen-binding sites (5). There is already evidence in support of this hypothesis. It has been found that a gene segment containing only exon 1 of the HD gene, but which includes the code for the alternative gene product we propose above, is sufficient to cause symptoms akin to HD in transgenic mice (6). It should also be mentioned that an expanded polyalanine tract has been implicated in the genetic disorder called synpolydactyly (7). We wonder if translations employing alternative initiation sites, or mutations which result in, or coupled with, alternative initiation sites, may be the cause of other diseases also.
REFERENCES 1. 2.
3.
4.
5.
6.
7.
Albin R. L., Tagle D. A. Genetics and molecular biology of Huntington’s disease. Trends Neurosci 1995; 18: 11–14. The Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993; 72: 971–983. Perutz M. F. Glutamine repeats and inherited neurodegenerative diseases: molecular aspects. Curr Opin Struct Biol 1996; 6: 848–858. Sheets M. D., Ogg S. C., Wickens M. P. Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic Acid Res 1990; 18: 5799–5805. Eilat D., Anderson W. F. Structure-function correlates of autoantibodies to nucleic acids. Lessons from immunochemical, genetic and structural studies. Mol Immunol 1994; 31: 1377–1390. Mangiarini L., Sathasivam K., Seller M. et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 1996; 87: 493–506. Muragaki Y., Mundlos S., Upton J., Olsen B. R. Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. Science 1996; 272: 548–550.
© 2000 Harcourt Publishers Ltd