Fig. 3

Knocking down UPF1 expression as a tool for assessing NMD-induced effects on FMR1 mRNA. A Schematic diagrams for FMR1 gene and transcripts. Full-length Fmr1 coding exons 1 to 17 (numbered boxes) and FMRP encoded domains, nuclear localization (NLS) and export (NES) signal motifs. FMR1 transcripts with or without exon 14 and selection of splice acceptor sites 15A, 15B or 15C are partially illustrated (exons 13 to 17). Location of translation termination codons is indicated by asterisks. B, C Western blot analysis of lysates of HEK293T transfected with UPF1 or control dsRNA, as indicated, treated with antibodies for UPF1 or α-tubulin, and plot of densitometry intensities. D RTqPCR data normalized by PPIA for HEK293T cells transfected with UPF1 or negative control dsRNAs, for SMG6 (positive control), FMR1 exons 10 and 11, exons 13 and 14, and amplicons 13–15A, 13–15B and 13–15C. Student´s t test: P < 0.05 (*), P < 0.01 (**), and P < 0.001(***). E Illustration of Fmr1 exon 15 sequence RNA (length legend in nucleotide, nt) and location of splice sites A, B and C, as well as the segment able to form a G-quartet structure stabilized by a counterion (K⁺). A suggested location of a UPF1 binding site is indicated between sites 15A and 15B