Fig. 4

The two-nucleotide single-stranded RNA structure feature at − 1 and − 2 nt positions upstream of 5′ss can regulate splicing. a SHAPE reactivity profiles across 5′ss of the first intron of AT5G56870. High SHAPE reactivities are observed at − 1 and − 2 nt positions (shaded in yellow) upstream of 5′ss under both in vivo (top) and deproteinized (bottom) conditions, which resemble the global SHAPE reactivity profiles for spliced events. b Schematic of experimental design to validate the effect of single-strandedness at the − 1 and − 2 positions of 5′ss on splicing of the first intron of AT5G56870. A short sequence (blue) was inserted immediately upstream of the U1 snRNA binding site (red dashed box) to form a stable hairpin structure with the whole U1 binding site completely base-paired. The exon and intron sequences are colored in black and gray, respectively. A series of mutations were introduced at different positions of the inserted sequence to disrupt the base-pairing status of different nucleotides. Two types of mutations (with/without bracket) were designed for each position to avoid potential effects due to changing the sequence content. c, d Determination of splicing events by transient expression assay in Nicotiana benthamiana. The spliced and unspliced products were distinguished by semiquantitative RT-PCR using the same pair of primers located upstream and downstream of the intron. Spliced and unspliced products are indicated by bands with different sizes. The construct with native sequence was successfully spliced (lane 1). Splicing was completely inhibited in the stem design (lane 2). The mutation “AA” or “GG” disrupted the base-pairing status at − 1 and − 2 positions upstream of 5′ss (Mutation-1) and rescued the splicing (lanes 3 and 4). All other mutations (Mutation-2–10) designed to disrupt other base-pairing sites across the U1 binding site did not rescue the splicing (lanes 5–22). Lane 23, the DNA marker