Characterization of FOXP2-∆11, a novel alternatively spliced product of FOXP2 gene

FOXP2 gene encodes a forkhead transcription factor linked to speech and language disorders (SPCH1) and more recently associated with autism spectrum disorders (ASDs). Two major FOXP2 spliced variants were previously described: a full-length (FOXP2-FL) isoform and a shorter transcript truncated at exon 10 (FOXP2-10+), lacking the DNA binding domain sequence. This study aims to investigate FOXP2 variants in cellular models and characterize the ribonucleoproteins involved in transcript generation. In Hek293 cells, FOXP2-FL transcript gives rise to a protein of about 80 kDa, while FOXP2-10+ is present only as untranslated transcript. 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 ribonucleoprotein mainly regulating the exon-skipping process. On this basis, we overexpressed PTBP1 in Hek293 cells and analyzed the pattern of FOXP2 transcripts expression by semi-quantitative RT-PCR. Interestingly, we detected a dose-dependent upregulation of a novel transcript excluding exon 11 (FOXP2-∆11). The lack of exon 11 causes a frameshift change in the FOXP2-∆11 transcript creating a premature stop codon within exon 12, which is predicted to produce a peptide of about 50 kDa, not found in our cells. While in absence of overexpressed PTBP1, FOXP2-∆11 transcripts were barely detectable in Hek293 cells, they appeared highly expressed in H4 glioblastoma cells, suggesting a yet unidentified role for it. To prove that PTBP1 promotes the skipping of exon 11 by direct binding to FOXP2 transcripts, we performed ribonucleoprotein immunoprecipitation (RIP) assay in HeLa cells expressing both FOXP2 and PTBP1 proteins. As a result, FOXP2 transcripts were detected in PTBP1 immunocomplexes, thus confirming its role in regulating FOXP2 transcript maturation. We are now in the process to further define the functional role of FOXP2 alternatively regulated exons.