Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with
Jorge Aragón1, Alejandro Martínez-Herrera1, José Romo-Yáñez1,2
1Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN No. 2508, San Pedro Zacatenco, C.P. 07360, México, D. F., Mexico.
Abstract:
Several dystrophin Dp71 messenger RNA (mRNA) alternative splice variants have been described. According to the splicing of exon 78 or intron 77, Dp71 proteins are grouped as Dp71d, Dp71f, and Dp71e, and each group has a specific C-terminal end. In this study, we explored the expression of Dp71 isoforms at the complementary DNA (cDNA) level and the subcellular localization of recombinant Myc-Dp71 proteins in PC12 cells. We determined that PC12 cells express Dp71a, Dp71c, Dp71ab, Dp71e, and Dp71ec mRNA splice variants. In undifferentiated and nerve growth factor-differentiated PC12 Tet-ON cells, Dp71a, Dp71ab, and Dp71e were found to localize and colocalize with β-dystroglycan and α1-syntrophin in the periphery/cytoplasm, while Dp71c and Dp71ec were mainly localized in the cell periphery and showed less colocalization with β-dystroglycan and α1-syntrophin. The levels of Dp71a, Dp71e, and Dp71ec were increased in the nucleus of differentiated PC12 Tet-ON cells compared to undifferentiated cells. Dp71 isoforms were also localized in neurite extensions and growth cones.
Insights
This study reveals distinct subcellular localizations for various dystrophin Dp71 messenger RNA (mRNA) splice variants in PC12 cells. Dp71 isoforms show differential distribution and altered nuclear levels upon differentiation, impacting cellular localization.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroscience
Background:
- Dystrophin Dp71 messenger RNA (mRNA) exhibits alternative splicing, leading to diverse protein isoforms with distinct C-terminal ends.
- These isoforms are implicated in various cellular functions, but their precise localization and behavior in neuronal cells remain incompletely understood.
Purpose of the Study:
- To investigate the expression of Dp71 mRNA splice variants at the complementary DNA (cDNA) level in PC12 cells.
- To determine the subcellular localization of recombinant Myc-Dp71 proteins and their colocalization with known markers in both undifferentiated and differentiated PC12 cells.
Main Methods:
- Analysis of Dp71 mRNA splice variants using complementary DNA (cDNA) sequencing in PC12 cells.
- Subcellular localization studies of recombinant Myc-Dp71 proteins in PC12 Tet-ON cells using immunofluorescence.
- Colocalization assays with β-dystroglycan and α1-syntrophin.
Main Results:
- PC12 cells express Dp71a, Dp71c, Dp71ab, Dp71e, and Dp71ec mRNA splice variants.
- Dp71a, Dp71ab, and Dp71e localized to the periphery/cytoplasm and colocalized with β-dystroglycan and α1-syntrophin.
- Dp71c and Dp71ec showed peripheral localization with less colocalization; differentiated cells exhibited increased nuclear levels of Dp71a, Dp71e, and Dp71ec. Dp71 isoforms were observed in neurites and growth cones.
Conclusions:
- Specific Dp71 mRNA splice variants exhibit distinct subcellular localization patterns in PC12 cells.
- Neuronal differentiation influences the nuclear accumulation of certain Dp71 isoforms.
- Dp71 isoforms are present in neurites and growth cones, suggesting roles in neuronal development and function.
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