Fig. 2: Sequence at the exon-intron boundary of HEH1 determines alternative 5’SS selection.
From: Mechanism of alternative splicing of yeast HEH1 through competing 5’ splice sites
a The 16-nucleotide segment at the exon/intron (1913–1928) boundary of HEH1 was investigated for alternative splicing by mutagenesis. The sequence was selected based on the pairing of U1 snRNA, U5 snRNA loop 1, and U6 snRNA ACAGA box. HEH1S (orange) and HEH1L (red) sequences represent HEH1 pre-mRNA isoforms. b The HEH1 gene was mutated by site-directed mutagenesis, sequentially from left to right (except for the essential ‘G’ of the splicing donors), and alternative selection was monitored by western blot. Heh1 isoforms in wt yeast are used as the reference for alternative splicing. Data shown as mean ± s.d. (n = 3 independent repeats). c Analysis of pre-mRNA variants pairing with U1 snRNA (brown), U5 snRNA (blue) and U6 snRNA (green) for both HEH1S and HEH1L is tabulated. The mutants were analyzed for presumed pairings with U1, U5, and U6 snRNAs, adapted from73,74,75. Nucleotide changes are underlined. Western blot lanes are taken from (b). The number in the left column shows the ID of the HEH1 variant. The changes for HEH1S and HEH1L are shown in curly brackets. Filled dots show non-Watson-Crick pairing, standing lines represent Watson-Crick pairing, upward arrows indicate the increase in U5 and U6 pairing compared to WT HEH1S or HEH1L, downward arrows show the decrease in U5 and U6 pairing compared to WT, and double upward arrows represent changes resulting in canonical 5’SS for yeast (GUAUGU or GUAAGU). The underlined nucleotides show mutation sites. The variants leading to premature stop codons, indicated by an asterisk, could be analysed only by cDNA sequencing. The values on the right of the peaks indicate the relative abundance of respective mRNA isoforms.
