Stabilization of the Tau Exon 10 Stem Loop Alters Pre-mRNA Splicing

Authors: Christine P. Donahue, Christina Muratore, Jane Y. Wu, Kenneth S. Kosik, Michael S. Wolfe - Journal of Biological Chemistry 2006 cited by 107

Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5′-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5′-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing. Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5′-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5′-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing. Tauopathies are neurodegenerative disorders characterized by neurofibrillary tangles, intraneuronal masses of paired helical filaments that are composed of highly phosphorylated forms of the microtubule-associated protein tau (1Kosik K.S. Shimura H. Biochim. Biophys. Acta. 2005; 1739: 298-310Crossref PubMed Scopus (115) Google Scholar, 2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2097) Google Scholar). Wild type tau containing neurofibrillary tangles are a common feature in a number of neurodegenerative diseases, including Alzheimer disease, Creutzfeldt-Jakob disease, Pick disease, progressive supranuclear palsy, frontal temporal dementia, and dementia pugilistica (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2097) Google Scholar).Tau is expressed predominantly in neurons where it promotes microtubule assembly, reduces microtubule instability, and plays a role in maintaining neuronal integrity and axonal transport (3Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 74-81Crossref PubMed Scopus (345) Google Scholar). The human gene is found on chromosome 17q21 and consists of 16 exons that are alternatively spliced to generate six tau isoforms (4Neve R. Harris Scopus Google Scholar, K.S. PubMed Scopus Google Scholar, K.S. PubMed Scopus Google Scholar). The tau and is by microtubule by exons N. 1994; PubMed Scopus Google Scholar). splicing of exon 10 tau isoforms with 10 10 as and the human the of to tau is critical role of tau in neurodegenerative with the that mutations in tau cause frontotemporal dementia and to chromosome M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google and mutations to Biochim. Biophys. Acta. 2005; 1739: PubMed Scopus Google Scholar). are silent mutations in exon 10 changes in the that the of the protein and on protein and are found in the exon 10. these mutations the splicing as the of at the of exon 10 a of the of a stem loop M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). of the silent mutations are in this stem loop and in the of and predicted of the stem loop as a of this of the with the in increased splicing of exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). these mutations a on the of to tau splice isoforms and are at the of in the of the stem loop is and M. V.M. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google its with that the in this are to demonstrate that these RNA structure that is a in tau that mutations that increase the splicing of exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar, H. M. M. Biol. PubMed Scopus Google Scholar, M. 2001; PubMed Scopus Google Scholar, M. Goedert M. PubMed Scopus Google Scholar, M. M. H. Goedert M. 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V.M. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). These however, the that the stem loop structure itself is a protein to stability with H. M. M. Biol. PubMed Scopus Google Scholar). this the of the stem show that the of the stem loop is critical to the of the the stem loop we the M. PubMed Scopus Google Scholar, M. Biol. PubMed Scopus Google to the of of the found that the predicted of the substantially the of the stem The predicted of the and the as and substantially the stem these that the The in of to these the predicted of these that the stem loop structure in by demonstrating that the of antisense RNA of exon 10 splicing is on the stability of the stem loop that the of the a of the of antisense and this is PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). antisense RNA to the RNA structure at the exon 10 site as it in the site the antisense RNA is the stem loop antisense to with the structure the the structure is the is the show that as the stability of the stem loop the of the antisense RNA that the RNA stem loop is in regulating the of tau and these results the the of mutations at the exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). These mutations the integrity of a RNA stem loop in the tau to increased of tau The existence of this stem loop that it a that to this structure and stabilize it tau splicing. Tauopathies are neurodegenerative disorders characterized by neurofibrillary tangles, intraneuronal masses of paired helical filaments that are composed of highly phosphorylated forms of the microtubule-associated protein tau (1Kosik K.S. Shimura H. Biochim. Biophys. Acta. 2005; 1739: 298-310Crossref PubMed Scopus (115) Google Scholar, 2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2097) Google Scholar). Wild type tau containing neurofibrillary tangles are a common feature in a number of neurodegenerative diseases, including Alzheimer disease, Creutzfeldt-Jakob disease, Pick disease, progressive supranuclear palsy, frontal temporal dementia, and dementia pugilistica (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2097) Google Scholar). is expressed predominantly in neurons where it promotes microtubule assembly, reduces microtubule instability, and plays a role in maintaining neuronal integrity and axonal transport (3Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 74-81Crossref PubMed Scopus (345) Google Scholar). The human gene is found on chromosome 17q21 and consists of 16 exons that are alternatively spliced to generate six tau isoforms (4Neve R. Harris Scopus Google Scholar, K.S. PubMed Scopus Google Scholar, K.S. PubMed Scopus Google Scholar). The tau and is by microtubule by exons N. 1994; PubMed Scopus Google Scholar). splicing of exon 10 tau isoforms with 10 10 as and the human the of to tau is The critical role of tau in neurodegenerative with the that mutations in tau cause frontotemporal dementia and to chromosome M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google and mutations to Biochim. Biophys. Acta. 2005; 1739: PubMed Scopus Google Scholar). are silent mutations in exon 10 changes in the that the of the protein and on protein and are found in the exon 10. these mutations the splicing as the of pathogenesis. The at the of exon 10 a of the of a stem loop M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). of the silent mutations are in this stem loop and in the of and predicted of the stem loop as a of this of the with the in increased splicing of exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). these mutations a on the of to tau splice isoforms and are at the of in the of the stem loop is and M. V.M. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google its with that the in this are to demonstrate that these RNA structure that is a in tau splicing. that mutations that increase the splicing of exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar, H. M. M. Biol. PubMed Scopus Google Scholar, M. 2001; PubMed Scopus Google Scholar, M. Goedert M. PubMed Scopus Google Scholar, M. M. H. Goedert M. PubMed Scopus Google destabilize the stem loop in the stem loop under physiological conditions in as a the stem loop lead to a decrease in exon 10 splicing. stem loop that increase the stability of the stem loop in vitro and lead to a decrease in exon 10 splicing in we the of these stem loop in with antisense RNA the of exon 10 to show that the of antisense on the stability of the tau stem in with and the tau stem loop RNA the the with on the and the and The with and in to the H. N. R. Biol. PubMed Scopus Google Scholar). The the gene mutations in the tau stem loop by the changes are six stem loop in the the wild type wild and These by as Goedert M. PubMed Scopus Google Scholar). RNA at a of in in 10 at The at a with a as PubMed Scopus Google RNA and with the as M. 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Here the of the on splicing is in the as the of the stem loop the of to with the predicted of the stem loop with the and mutations the on exon 10 of on the of the that antisense RNA the splice site of exon 10 exon 10 splicing and to increase in tau Biol. 2001; PubMed Scopus Google Scholar). this antisense RNA to the stability of the stem loop in The antisense RNA site is to to the splice The stem loop as to the stem loop in the antisense RNA to with the RNA structure PubMed Scopus Google Scholar, Biol. 2001; PubMed Google Scholar). The the the the splice site is to by the antisense in antisense in the of antisense on tau we a splicing on the the is to the of exon to as a tau the is in exon 10 is the the minigene it and on in exon as a splicing this and in a to is in the with of the six stem loop and with of a antisense RNA with a to the inclusion of exon 10 in tau Biol. 2001; PubMed Scopus Google Scholar). of the antisense RNA in a decrease in to with a of a to the and the of antisense to that the The to with stability of the tau stem The the stem loop the is the antisense RNA of exon 10 splicing with stem loop RNA the of exon 10 the inclusion of exon 10 in the with tau of the and with antisense decrease in with antisense RNA to with a RNA The to decrease with stem loop this is the of to is on RNA antisense to with a RNA of antisense and RNA are that the increase in in to the of to on RNA the The that the increase in is to a increase in the to of splice isoforms to with the and These results a the changes in the to and stem loop stability of exon 10 inclusion is the stem loop the stem of tau mutations lead to increase in mutations lead to increase in this with exon on that exons and Biol. 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Here the of the on splicing is in the as the of the stem loop the of to with the predicted of the stem loop with the and mutations the on exon 10 splicing. of on the of the that antisense RNA the splice site of exon 10 exon 10 splicing and to increase in tau Biol. 2001; PubMed Scopus Google Scholar). this antisense RNA to the stability of the stem loop in The antisense RNA site is to to the splice The stem loop as to the stem loop in the antisense RNA to with the RNA structure PubMed Scopus Google Scholar, Biol. 2001; PubMed Google Scholar). The the the the splice site is to by the antisense in antisense in splicing. the of antisense on tau we a splicing on the the is to the of exon to as a tau the is in exon 10 is the the minigene it and on in exon as a splicing this and in a to is in the with of the six stem loop and with of a antisense RNA with a to the inclusion of exon 10 in tau Biol. 2001; PubMed Scopus Google Scholar). of the antisense RNA in a decrease in to with a of a to the and the of antisense to that the The to with stability of the tau stem The the stem loop the is the antisense that the increase in in to the of to on RNA the The that the increase in is to a increase in the to of splice isoforms to with the and These results a the changes in the to and stem loop stability of exon 10 inclusion is the stem loop and mutations found in and exon 10 that cause increase the inclusion of exon 10 in tau in the stem loop regulation of tau splicing. that exon 10 splicing is substantially by a stem loop structure that forms at the of exon 10 and the of the The of to in with the of these stem loop in this of the protein this the mutations to the as the of the the base is the splicing where of the stem loop mutations are found M. V.M. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). These however, the that the stem loop structure itself is a protein to stability with H. M. M. Biol. PubMed Scopus Google Scholar). this the of the stem show that the of the stem loop is critical to the of the the stem loop we the M. PubMed Scopus Google Scholar, M. Biol. PubMed Scopus Google to the of of the found that the predicted of the substantially the of the stem The predicted of the and the as and substantially the stem these that the The in of to these the predicted of these that the stem loop structure in by demonstrating that the of antisense RNA of exon 10 splicing is on the stability of the stem loop that the of the a of the of antisense and this is PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). antisense RNA to the RNA structure at the exon 10 site as it in the site the antisense RNA is the stem loop antisense to with the structure the the structure is the is the show that as the stability of the stem loop the of the antisense RNA that the RNA stem loop is in regulating the of tau and these results the the of mutations at the exon 10 M. M. H. M. M. M. Trojanowski H. M. N. PubMed Scopus Google Scholar). These mutations the integrity of a RNA stem loop in the tau to increased of tau The existence of this stem loop that it a that to this structure and stabilize it tau splicing. and mutations found in and exon 10 that cause increase the inclusion of exon 10 in tau in the stem loop regulation of tau splicing. that exon 10 splicing is substantially by a stem loop structure that forms at the of exon 10 and the of the The of to in with the of these stem loop in this of the protein this the mutations to the as the of the the base is the splicing where of the stem loop mutations are found M. V.M. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). These however, the that the stem loop structure itself is a protein to stability with H. M. M. Biol. PubMed Scopus Google Scholar). this the of the stem show that the of the stem loop is critical to the of the the stem loop we the M. PubMed Scopus Google Scholar, M. Biol. 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