Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors
Abstract Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonica...
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| Format: | Article |
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Nature Portfolio
2023-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-50188-z |
| _version_ | 1797377031146569728 |
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| author | Hayato Ito Kodai Machida Mayuka Hasumi Morio Ueyama Yoshitaka Nagai Hiroaki Imataka Hideki Taguchi |
| author_facet | Hayato Ito Kodai Machida Mayuka Hasumi Morio Ueyama Yoshitaka Nagai Hiroaki Imataka Hideki Taguchi |
| author_sort | Hayato Ito |
| collection | DOAJ |
| description | Abstract Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5′ cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism. |
| first_indexed | 2024-03-08T19:47:09Z |
| format | Article |
| id | doaj.art-33e603ee6d9a4f0fadf353693f19abac |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-08T19:47:09Z |
| publishDate | 2023-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-33e603ee6d9a4f0fadf353693f19abac2023-12-24T12:15:00ZengNature PortfolioScientific Reports2045-23222023-12-0113111310.1038/s41598-023-50188-zReconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factorsHayato Ito0Kodai Machida1Mayuka Hasumi2Morio Ueyama3Yoshitaka Nagai4Hiroaki Imataka5Hideki Taguchi6School of Life Science and Technology, Tokyo Institute of TechnologyGraduate School of Engineering, University of HyogoSchool of Life Science and Technology, Tokyo Institute of TechnologyDepartment of Neurology, Faculty of Medicine, Kindai UniversityDepartment of Neurology, Faculty of Medicine, Kindai UniversityGraduate School of Engineering, University of HyogoSchool of Life Science and Technology, Tokyo Institute of TechnologyAbstract Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5′ cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.https://doi.org/10.1038/s41598-023-50188-z |
| spellingShingle | Hayato Ito Kodai Machida Mayuka Hasumi Morio Ueyama Yoshitaka Nagai Hiroaki Imataka Hideki Taguchi Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors Scientific Reports |
| title | Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors |
| title_full | Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors |
| title_fullStr | Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors |
| title_full_unstemmed | Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors |
| title_short | Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors |
| title_sort | reconstitution of c9orf72 ggggcc repeat associated non aug translation with purified human translation factors |
| url | https://doi.org/10.1038/s41598-023-50188-z |
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