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Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1
Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8658563/ https://www.ncbi.nlm.nih.gov/pubmed/34884222 http://dx.doi.org/10.3390/jcm10235520 |
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author | Koehorst, Emma Núñez-Manchón, Judit Ballester-López, Alfonsina Almendrote, Miriam Lucente, Giuseppe Arbex, Andrea Chojnacki, Jakub Vázquez-Manrique, Rafael P. Gómez-Escribano, Ana Pilar Pintos-Morell, Guillem Coll-Cantí, Jaume Ramos-Fransi, Alba Martínez-Piñeiro, Alicia Suelves, Mònica Nogales-Gadea, Gisela |
author_facet | Koehorst, Emma Núñez-Manchón, Judit Ballester-López, Alfonsina Almendrote, Miriam Lucente, Giuseppe Arbex, Andrea Chojnacki, Jakub Vázquez-Manrique, Rafael P. Gómez-Escribano, Ana Pilar Pintos-Morell, Guillem Coll-Cantí, Jaume Ramos-Fransi, Alba Martínez-Piñeiro, Alicia Suelves, Mònica Nogales-Gadea, Gisela |
author_sort | Koehorst, Emma |
collection | PubMed |
description | Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods. |
format | Online Article Text |
id | pubmed-8658563 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-86585632021-12-10 Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 Koehorst, Emma Núñez-Manchón, Judit Ballester-López, Alfonsina Almendrote, Miriam Lucente, Giuseppe Arbex, Andrea Chojnacki, Jakub Vázquez-Manrique, Rafael P. Gómez-Escribano, Ana Pilar Pintos-Morell, Guillem Coll-Cantí, Jaume Ramos-Fransi, Alba Martínez-Piñeiro, Alicia Suelves, Mònica Nogales-Gadea, Gisela J Clin Med Article Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods. MDPI 2021-11-25 /pmc/articles/PMC8658563/ /pubmed/34884222 http://dx.doi.org/10.3390/jcm10235520 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Koehorst, Emma Núñez-Manchón, Judit Ballester-López, Alfonsina Almendrote, Miriam Lucente, Giuseppe Arbex, Andrea Chojnacki, Jakub Vázquez-Manrique, Rafael P. Gómez-Escribano, Ana Pilar Pintos-Morell, Guillem Coll-Cantí, Jaume Ramos-Fransi, Alba Martínez-Piñeiro, Alicia Suelves, Mònica Nogales-Gadea, Gisela Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title | Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title_full | Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title_fullStr | Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title_full_unstemmed | Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title_short | Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 |
title_sort | characterization of ran translation and antisense transcription in primary cell cultures of patients with myotonic dystrophy type 1 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8658563/ https://www.ncbi.nlm.nih.gov/pubmed/34884222 http://dx.doi.org/10.3390/jcm10235520 |
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