“Language” gene FOXP2 major trascripts are FOXP2 full-length (FOXP2-FL), comprising 17 exons plus 2 alternatively spliced exons (3b and 4a) and a shorter variant ending with an elongated exon 10 (FOXP2-10+). Since little is known on the regulation of FOXP2 alternative splicing, we undertook a study on the ribonucleoproteins involved. In silico analysis showed putative binding sites for the Polypyrimidine-Tract Binding protein 1 (PTBP1) in FOXP2 introns flanking exon 3b but also exon 11. We overexpressed PTBP1 in Hek293 cells and performed a semi-quantitative RT-PCR (RT-SqPCR) on FOXP2 transcripts. Untransfected cells showed three major transcripts: FOXP2-10+, FOXP2-FL and a novel low abundant transcript 200 bp shorter than FOXP2-FL, missing exon 11 (FOXP2-Δ11). Interestingly, overexpression of PTBP1 caused a diminution of FOXP2-FL and an increase of FOXP2-Δ11 transcripts. Sequencing of FOXP2-Δ11 transcript revealed a premature stop codon within exon 12, questioning its function. To confirm PTBP1 effect on FOXP2-Δ11 generation, we: 1) created a FOXP2 minigene by cloning the 7.5 kb genomic sequence between exons 9 and 13 in pcDNA3.0; 2) co-trasfected FOXP2 minigene together with PTBP1; 3) performed RT-SqPCRs using T7 and SP6 primers amplifying only FOXP2 minigene transcripts. As a result, we detected almost exclusively the transcript missing exon 11, confirming exon-skipping actions by PTBP1 on FOXP2. Of note, protein analysis showed a significant decrease of FOXP2-FL while the predicted protein for translated FOXP2-Δ11 transcript was not detected. The latter observation together with FOXP2-Δ11 sequence analysis lead us to the hypothesis of a controlled degradation of FOXP2-Δ11 transcripts via Non Sense mediated Decay (NMD) that we are now testing. Interestingly, we detected FOXP2-Δ11 transcripts also in H4, T47D and MDA cells. We propose a regulatory role for the newly identified FOXP2-Δ11 variant in controlling the levels of FOXP2-FL expression, mediated by PTBP1.
PTBP1 promotes FOXP2 alternative splicing in Hek293 cells
F. Ferrarini
;F. Martinetto
;R. Galavotti
;D. De Pietri Tonelli
;M. G. Romanelli
;P. M. Lievens
2018-01-01
Abstract
“Language” gene FOXP2 major trascripts are FOXP2 full-length (FOXP2-FL), comprising 17 exons plus 2 alternatively spliced exons (3b and 4a) and a shorter variant ending with an elongated exon 10 (FOXP2-10+). Since little is known on the regulation of FOXP2 alternative splicing, we undertook a study on the ribonucleoproteins involved. In silico analysis showed putative binding sites for the Polypyrimidine-Tract Binding protein 1 (PTBP1) in FOXP2 introns flanking exon 3b but also exon 11. We overexpressed PTBP1 in Hek293 cells and performed a semi-quantitative RT-PCR (RT-SqPCR) on FOXP2 transcripts. Untransfected cells showed three major transcripts: FOXP2-10+, FOXP2-FL and a novel low abundant transcript 200 bp shorter than FOXP2-FL, missing exon 11 (FOXP2-Δ11). Interestingly, overexpression of PTBP1 caused a diminution of FOXP2-FL and an increase of FOXP2-Δ11 transcripts. Sequencing of FOXP2-Δ11 transcript revealed a premature stop codon within exon 12, questioning its function. To confirm PTBP1 effect on FOXP2-Δ11 generation, we: 1) created a FOXP2 minigene by cloning the 7.5 kb genomic sequence between exons 9 and 13 in pcDNA3.0; 2) co-trasfected FOXP2 minigene together with PTBP1; 3) performed RT-SqPCRs using T7 and SP6 primers amplifying only FOXP2 minigene transcripts. As a result, we detected almost exclusively the transcript missing exon 11, confirming exon-skipping actions by PTBP1 on FOXP2. Of note, protein analysis showed a significant decrease of FOXP2-FL while the predicted protein for translated FOXP2-Δ11 transcript was not detected. The latter observation together with FOXP2-Δ11 sequence analysis lead us to the hypothesis of a controlled degradation of FOXP2-Δ11 transcripts via Non Sense mediated Decay (NMD) that we are now testing. Interestingly, we detected FOXP2-Δ11 transcripts also in H4, T47D and MDA cells. We propose a regulatory role for the newly identified FOXP2-Δ11 variant in controlling the levels of FOXP2-FL expression, mediated by PTBP1.
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