Alternative splicing regulation of human FOXP2 speech gene

Abstract

FOXP2 gene encodes a forkhead transcription factor linked to speech and language disorders (SPCH1). Three major FOXP2 spliced variants were previously described: a full-length (FOXP2-FL), a transcript starting at exon 4 (FOXP2-III) and an isoform truncated at exon 10 (FOXP2-10+) lacking the DNA binding domain. This study aims to investigate the functional role of FOXP2 variants in cellular models and characterize the RNA-binding proteins involved in transcript maturation. Of the three transcripts, only FOXP2-FL and FOXP2-III were translated into peptides in Hek293 cells. In silico analysis revealed clusters of putative binding sites for Polypyrimidine-Tract Binding protein 1 (PTBP1) in intron sequences flanking exons 3b and 11 of FOXP2. PTBP1 is a hnRNP (heterogeneous nuclear ribonucleoprotein) that mainly regulates exon-skipping. We overexpressed PTBP1 in Hek293 cells and looked at FOXP2 transcripts modulation by RT-PCR and qPCR. No significant change was observed in ratios between FOXP2 variants, but we detected a novel transcript excluding exon 11 (FOXP2-\u39411), which lacks the first of the two nuclear localization signal sequences (NLS1) of FOXP2-FL. Based on this finding we checked for endogenous FOXP2-\u39411 transcript in a panel human cell lines and we found it was expressed in H4 glioblastoma cells. We are in process to verify the translation of FOXP2-\u39411 into peptide: the lack of NLS1 may determine conformation changes influencing FOXP2 dimerization and/or DNA binding and therefore its function in a tissue-specific manner. For further investigations on FOXP2 splicing mediated by PTBP1, we depleted endogenous PTBP1 in Hek293 cells using small interfering RNA (siRNAs). By western blot analysis, we found a significant upregulation of FOXP2-FL, thus supporting the hypothesis that PTBP1 might control FOXP2 expression