Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with β-Dystroglycan and α1-Syntrophin

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.

First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene

“…They consist in combinations of single or multiple exon skipping involving exons 71 to 74 and exon 78, with the isoforms lacking exon 71 and/or exon 78 being by far the most represented [ 12 , 13 , 16 , 41 , 42 , 43 ]. In Dp71, the splice isoforms determine the differential subcellular localization and various cellular functions of the short dystrophin isoform in the brain [ 44 , 45 ]. Transcripts spliced out for exon 78 produce a protein with an elongated C-term region by replacing the last 13 amino acids in the protein with an evolutionary conserved sequence of 31 new residues encoding the ancestral alternative amphipathic C-terminal α-helix.…”

Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells

“…Recently, Dp71 has been studied in several types of cancer 12 – 16 but the roles of Dp71 in cancer development are still controversial. Although the splice variants of Dp71 have been implicated in specific functions 9 , 11 , 17 – 19 , the functional characterization of each splice variant has been hampered by the co-expression of isoforms 20 , 21 .…”

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

“…It is expressed at high levels in non‐muscle tissues and is most abundant in adult brain . Dp71 transcripts are alternatively spliced in exons 71, 71–74 and/or 78, generating several isoforms . Although the function of Dp71 has not been fully elucidated, mutations in the DMD gene that disrupt Dp71 expression have been correlated with the severity of the cognitive impairment observed in DMD patients, suggesting that Dp71 might play an important role in the central nervous system .…”