6a283b78d99936d135e591f3dbc08aef pmc_34884222.pdf 6808690a1220a82eece1bfc9266b98392ea2b717 pmc_34884222.pdf 29c01b82a613705d8ac257f518c4f3ca33d8d2b06d56c24ce7259b92d517ec98 pmc_34884222.pdf Title: Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 Subject: 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. Keywords: RAN translation; antisense transcription; myotonic dystrophies; primary cell cultures; phenotypic modulators Author: Emma Koehorst, Judit Núñez-Manchón, Alfonsina Ballester-López, Miriam Almendrote, Giuseppe Lucente, Andrea Arbex, Jakub Chojnacki, Rafael P. Vázquez-Manrique, Ana Pilar Gómez-Escribano, Guillem Pintos-Morell, Jaume Coll-Cantí, Alba Ramos-Fransi, Alicia Martínez-Piñeiro, Mònica Suelves and Gisela Nogales-Gadea Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Nov 26 04:13:54 2021 CET ModDate: Fri Nov 26 04:23:44 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4501460 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CMRYXI+VnURWPalladioL Type 1 Custom yes yes no 10 0 MYAWPB+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 MLSUMG+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 ZXHCPS+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 LSAWBX+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 XPUSBJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 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edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Small Pool PCR for Sizing the CTG Repeat Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: cDNA Construct Huntingtin and HEK293 Transfection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: RNA Isolation and Subcellular Fractionation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: RT-PCR Analysis for DM1-AS Transcript Detection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Immunoblotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Fluorescence In Situ Hybridization (FISH) and Immunofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Image and Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Children: Item: Title: The DM1 Clinical Phenotype of the Studied Cohort Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Lower Expression of DM1-AS Transcripts Found in DM1 Patients Compared to Controls Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: DM1-AS Transcripts Are Present in the Cytoplasm of DM1 Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: RAN Protein Was Undetectable in All Three Primary Cell Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: The Polyglutamine Containing Protein Found Resides in the Golgi Apparatus Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Info: Title: Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1 Author: Emma Koehorst, Judit Núñez-Manchón, Alfonsina Ballester-López, Miriam Almendrote, Giuseppe Lucente, Andrea Arbex, Jakub Chojnacki, Rafael P. Vázquez-Manrique, Ana Pilar Gómez-Escribano, Guillem Pintos-Morell, Jaume Coll-Cantí, Alba Ramos-Fransi, Alicia Martínez-Piñeiro, Mònica Suelves and Gisela Nogales-Gadea Subject: 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. 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