Fig. 6.

SF3B3 regulates mTOR exon 8 skipping in CRC cells. A Schematic diagram of alternative splicing types, and alternative splicing events regulated by SF3B3 from RNA-seq data. B The mTORβ transcript levels were examined by 1% agarose gel electrophoresis of PCR products. The 2.1 kb product (full-length mTORβ transcript) was amplified with the primers (F, R2), whereas the 100 bp product (partial mTORβ transcript) was amplified with the primers (F, R1). C Representative western blots of full-length mTOR (mTORα) and its short-length isoform (mTORβ) in CRC cells using the mTOR antibody (sc-517464). LoVo and HT29 cells were treated with siRNAs for 72 h. D Schematic diagram of the protein-coding mTOR transcript variants and isoforms. mTOR variant 1 and variant 2 include exon 8, whereas mTOR variant 3 exclude exon 8 in NCBI dataset. Exon 8 is also skipped in mTORβ transcript. E Exon 8 skipping of mTOR was examined by 3% agarose gel electrophoresis of PCR products in CRC cells. LoVo and HT29 cells were treated with siRNAs for 48 h. Data are shown as mean ± SD. F Exon 8 skipping was determined by 3% agarose gel electrophoresis of PCR products in CRC cells after 48 h transfection with a minigene contains exons 7–9 as well as two introns of mTOR. G The immunoprecipitated RNAs by anti-SF3B3 or IgG were purified and analyzed by PCR using primers specific for mTOR and EZH2. EZH2 used as positive control. H 3% agarose gel electrophoresis of PCR products of mTOR exon 8 skipping and SF3B3 mRNA in control and SF3B3-knockdown LoVo xenografts (shNC vs shSF3B3#1). I Representative agarose gel electrophoresis of PCR products of mTOR exon 8 skipping and SF3B3 mRNA in paired CRC tissues and adjacent normal tissues