12/30/2023 0 Comments Dp116 sparkle and co![]() RNA was extracted from these cells using High Pure RNA isolation kits (Roche Diagnostics). Louis, MO, USA) and collected using the lysis/binding buffer from High Pure RNA isolation kits (Roche Diagnostics, Basel, Switzerland). Identification of Dp116 in U-251 cells facilitates studies on the roles of Dp116.Ĭultured cells were rinsed twice with phosphate buffered saline (PBS Sigma-Aldrich Co., St. Splicing patterns of DMD exons were compared in Dp116 and Dp71. Furthermore, five splice variants and two novel splicing patterns of the DMD gene were identified. ![]() Dp116 mRNA and protein were shown to be expressed in glioblastoma cells. This study was designed to characterize Dp116 and the alternative splicing of DMD exons in glioblastoma cells. We previously showed that glioblastoma cells express Dp71, with this Dp71 composed of six splice variants, suggesting that glioblastoma provides a specific environment for regulating the splicing of DMD exons.ĭuring the study on Dp71, we have obtained a signature that indicates the expression of Dp116 in U-251 cells. The DMD gene is regarded as a tumor suppressor gene, with Dp71 shown to have tumor suppressive activity. One method of treatment may be the manipulation of RNA processing of tumor drivers. Molecular characterization of glioblastoma may help in designing effective therapies. Glioblastoma is an aggressive brain tumor highly resistant to treatment. Analysis of Dp71 transcripts has identified multiple exon skipping patterns in the region from exon 71 to exon 78. Though most skipping of DMD exons occurs in-frame exons, skipping of exon 78 shifts the reading frame to produce a large dystrophin with an elongated C-terminal amino acid sequence. The most frequent type of alternative splicing of the DMD gene consists of the skipping of exons. Īlternative splicing is a mechanism that enables cells to generate various diverse proteins from a limited number of genes, as well as having important physiological functions in different developmental processes in humans. It is necessary to understand physiological roles of Dp116 well, since cardiomyopathy is a leading cause of early death in DMD. However, exact mechanism of Dp116 to enhance cardiomyopathy remains unknown. We recently reported that Dp116 plays a role in the development of cardiac dysfunction in DMD patients, suggesting that Dp116 expression is not limited to Schwann cells. Due to its limited expression and large size, the pathophysiological roles of Dp116 are largely unknown. The Dp116 promoter is characterized by its very specific activation in Schwann cells. Dp116 transcript is 5.2 kb long and consists of the Dp116-specific exon S1 joined to DMD exons 56–79. Thus, DMD exons 63–79 are incorporated into the mRNA of not only Dp71 but also all other isoforms.ĭp116, the second shortest isoform of the DMD gene, is transcribed from the Dp116 promoter in intron 55. ![]() Dp71, the shortest isoform, is transcribed from a promoter in intron 62 of the DMD gene, with the Dp71 transcript consisting of Dp71-specific exon G1 and DMD exons 63–79. Four alternative promoter-first exon regions are embedded in downstream introns and produce the Dp260, Dp140, Dp116 and Dp71 isoforms. Dp427 m is a full-length muscle-specific isoform, a lack of which causes Duchenne muscular dystrophy (DMD) (OMIM310200), a fatal progressive muscle wasting disease. The gene exhibits a highly complex arrangement, with eight alternative promoters scattered in its introns driving the expression of four full-length and four short dystrophin isoforms in a tissue- or development-specific manner. The DMD gene is one of the largest genes in the human genome, encoding a 14-kb long transcript consisting of 79 exons spread over more than 2.4 Mb on the X chromosome. However, the splicing patterns in glioblastoma cells of DMD exons in Dp116 and Dp71 showed no significant differences. Two novel splicing patterns were also observed, one with a deletion of exons 68 and 69 (Dp116bΔ68-69) and the other with a 100 bp deletion in the 5’ terminal end of exon 75 (75s), which was produced by the activation of a cryptic splice acceptor site (Dp116b75s). A third transcript lacking exons 71–74 and 78 was also identified (Dp116bc). The most abundant transcript lacked only exon 78 (Dp116b), whereas the second most abundant transcript lacked both exons 71 and 78 (Dp116ab). Sequencing of the amplified product revealed five splice variants, all skipping exon 78. Western blotting of U-251 cell lysates revealed a signal at a position corresponding to vector-expressed Dp116 protein, indicating that Dp116 is expressed in glioblastoma cells. ![]() The full-length Dp116 transcript was amplified as nearly 3 kb in size. Full-length Dp116 cDNA, extending from exon S1 to exon 79, was PCR amplified to avoid confusion with other DMD isoforms.
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