Alternative splicing of BAF45d contributes to the pathogenesis of GBM
Guillermo Aldave-Orzaiz, Marisol Gonzalez-Huarriz, Enric Xipell, Fernando Pastor, Roeland Verhaak, Angel Ayuso, Cristobal Belda, Juan Valcárcel, Ricardo Diez-Valle, Sonia Tejada and Marta M. Alonso
Changes in splicing have been implicated in numerous pathologies including cancer. Previously, our group identified Baf45d as aberrantly spliced in GBM using paired normal looking/GBM samples. Baf45d is part of the SWI/SNF complex and plays a key role in the development of the CNS. The objective of this work was to elucidate the function of Baf45d tumoral-spliced isoform (Baf45d-T) and to understand the mechanism underlying its aberrant regulation.
Overall Baf45d mRNA levels (all isoforms) are unchanged when we compare normal brain with low grade astrocytomas and GBM. However, Baf45d-T is overexpressed meanwhile Baf45d normal isoform (Baf45d-N) is significantly downregulated in GBM. Baf45d-T loses exon 7, leading to the formation of a zinc finger domain which increases the DNA binding. TCGA data support our results demonstrating the loss of exon 7 in 166 GBM. The inhibition of Baf45d-T in GBM cells resulted in a reduction in proliferation and in morphology and expression changes towards a more differentiated phenotype. Interestingly, Baf45d-T was the predominant transcript in early postnatal murine neural precursors and its expression disappeared as these cells were instructed towards neurons. In vivo studies, using nude mice bearing intracranial U87-MG cells overexpressing Baf45d-N resulted in a significant increase in overall survival. Analyses of Baf45d sequence revealed several binding sites for HnRNPA1 (an RNA protein that participates in splicing and is overexpress in GBM) flanking exons 6, 6a, 7 and 8. Inhibition of HnRNPA1 in U87 MG cells lead to an increase of Baf45d-N and a reduction of Baf45d-T. Moreover, RIP assays demonstrated the binding of HnRNPA1 to Baf45d mRNA.
Our results suggest that the aberrant splicing of Baf45d in GBM could participate in the malignant phenotype of these tumors and define a pathway that regulates an alternative splicing event required for the maintenance of an undifferentiated state in tumor cells.