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Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization.
Dhume, Ashwini; Lu, Shajia; Horowits, Robert.
Afiliación
  • Dhume A; Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (Department of Health and Human Services), Bethesda, MD 20892, USA.
Cell Motil Cytoskeleton ; 63(8): 493-511, 2006 Aug.
Article en En | MEDLINE | ID: mdl-16767749
N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Interferencia de ARN / Proteínas Musculares / Miofibrillas Límite: Animals Idioma: En Revista: Cell Motil Cytoskeleton Año: 2006 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Interferencia de ARN / Proteínas Musculares / Miofibrillas Límite: Animals Idioma: En Revista: Cell Motil Cytoskeleton Año: 2006 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos