Cargando…

Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The m...

Descripción completa

Detalles Bibliográficos
Autores principales: Rani, Abdul Qawee Mahyoob, Yamamoto, Tetsushi, Kawaguchi, Tatsuya, Maeta, Kazuhiro, Awano, Hiroyuki, Nishio, Hisahide, Matsuo, Masafumi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278912/
https://www.ncbi.nlm.nih.gov/pubmed/32443516
http://dx.doi.org/10.3390/ijms21103555
_version_ 1783543440680681472
author Rani, Abdul Qawee Mahyoob
Yamamoto, Tetsushi
Kawaguchi, Tatsuya
Maeta, Kazuhiro
Awano, Hiroyuki
Nishio, Hisahide
Matsuo, Masafumi
author_facet Rani, Abdul Qawee Mahyoob
Yamamoto, Tetsushi
Kawaguchi, Tatsuya
Maeta, Kazuhiro
Awano, Hiroyuki
Nishio, Hisahide
Matsuo, Masafumi
author_sort Rani, Abdul Qawee Mahyoob
collection PubMed
description The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.
format Online
Article
Text
id pubmed-7278912
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-72789122020-06-15 Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients Rani, Abdul Qawee Mahyoob Yamamoto, Tetsushi Kawaguchi, Tatsuya Maeta, Kazuhiro Awano, Hiroyuki Nishio, Hisahide Matsuo, Masafumi Int J Mol Sci Article The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin. MDPI 2020-05-18 /pmc/articles/PMC7278912/ /pubmed/32443516 http://dx.doi.org/10.3390/ijms21103555 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rani, Abdul Qawee Mahyoob
Yamamoto, Tetsushi
Kawaguchi, Tatsuya
Maeta, Kazuhiro
Awano, Hiroyuki
Nishio, Hisahide
Matsuo, Masafumi
Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title_full Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title_fullStr Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title_full_unstemmed Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title_short Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients
title_sort intronic alternative polyadenylation in the middle of the dmd gene produces half-size n-terminal dystrophin with a potential implication of ecg abnormalities of dmd patients
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278912/
https://www.ncbi.nlm.nih.gov/pubmed/32443516
http://dx.doi.org/10.3390/ijms21103555
work_keys_str_mv AT raniabdulqaweemahyoob intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT yamamototetsushi intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT kawaguchitatsuya intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT maetakazuhiro intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT awanohiroyuki intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT nishiohisahide intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients
AT matsuomasafumi intronicalternativepolyadenylationinthemiddleofthedmdgeneproduceshalfsizenterminaldystrophinwithapotentialimplicationofecgabnormalitiesofdmdpatients