Alternative BP usage reveals previously unknown nonsense-mediated mRNA decay splice isoform. (A) Distance from 5′SS to BP for first and second BPs in introns that use two BPs. (Red line) x = y. Two BPs in BBP (MSL5) were also reported by Qin et al. (2016) (B) Three genes from A where novel BP (red) is located close to the 5′SS and far from the annotated BP (blue). Intronic transcript position shown below each intron; direction indicated with white arrows. (C) Motif of upstream BP (top) and downstream BP (bottom) for 11 introns that use two BPs. (D) Branch-seq reads support a novel 5′SS-BP shifted pair in the MCR1 intron with an “atypical” 5′SS motif. (E) Branch-seq read coverage from the top, middle, and bottom sections of the 2D gel arc (Supplemental Fig. S1A) correspond to usage of the canonical LSM2 BP (blue dotted line and circle) and a “new” BP (red dotted line and circle). Potential alternative 3′SS usage would insert a premature termination codon (octagon stop sign). (F) RT-PCR and subsequent sequencing confirmed the novel LSM2 PTC isoform. (G) qPCR verification that LSM2 PTC isoform is up-regulated in upf1 null yeast.
