Fig. 2. Global alteration of mRNA splicing in mtl16Δ cells.
a Five classifications of RNA-seq reads mapped to each intron with its neighboring exons annotated in the S. pombe genome: 5′ exon–intron junction (EIJR), intron-3′ exon junction (IEJR), exon–exon canonical splicing junction (CSR), 5′ alternative splice site selection (A5R), and 3′ alternative splice site selection (A3R). b Scatter-plot of IRS in mtl16Δ versus WT. Each plot represents each annotated intron with sufficient number of reads. The plots are classified based on quartiles of the Z-score of each intron. The red and blue arrows represent plots of SPAC18B11.09c intron #1 and sec71 intron #1, respectively. The black line represents an equal value of IRS. c Semi-quantitative RT-PCR analyses of two introns with large IRS difference. The upper and lower bands on the gel represent retained and spliced introns, respectively. Intron retention in the mtl16Δ strain was rescued by ectopic expression of plasmid-encoded WT mtl16 (p-mtl16WT), but not by its active-site mutant (P169A/P170A) (p-mtl16mut). This result was repeated once with a similar result. Source data are provided as a Source Data file. d Sequence logos of 5′ and 3′ splice sites with Z-scores over the top quartile (upper panels) and under the 3rd quartile (lower panels). The adenine base at the fourth position of the intron is highlighted and is associated a higher probability of intron retention in the mtl16Δ strain. e DiffLogo analysis was used to compare sequence enrichment of the 5′SS between introns with high Z-scores versus low Z-scores. The 5′SS is recognized by a part of the loop I sequence of U5 snRNA and the portion of U6 snRNA that contains m6A. Enriched and depleted nucleotides at the 5′SS are shown above and below the axis, respectively. The enrichment of A at position 4 against other nucleotides was significant by a p-value under 1.0 × 10−98 by two-sided Fisher’s exact test. The depletion of A, A, and G at positions −3, −2, and −1 against other nucleotides was significant by p-values under 0.01, 5.0 × 10−14, and 5.0 × 10−30 by two-sided Fisher’s exact test, respectively. The loop I sequence of U5 snRNA and a portion of U6 snRNA pair with the 5′ exon and intron at the 5′SS, respectively. m6A pairs with the fourth nucleotide of introns.