Figure 5

SRSF1 suppresses binding of U1 snRNP to the intron-distal 5′ SS of DOK7 intron 4. (a) Schematic of 3 x MS2 RNA probes used for isolation of an early spliceosome complex. Control probe contains constitutive 5′ SS of the human β-globin gene encoded in pSP64-HβΔ6-MS2. Probe-1 carries the SRSF1-binding site and the intron-distal (Int-dis) 5′ SS, but lacks the intron-proximal (Int-pro) 5′ SS. Probe-2 carries a disrupted SRSF1-binding site and the intron-distal 5′ SS, but lacks the intron-proximal 5′ SS. Probe-3 carries the SRSF1-binding site, a mutated intron-distal 5′ SS, and the intron-proximal 5′ SS. Probe-4 carries a disrupted SRSF1-binding site, a mutated intron-distal 5′ SS, and the intron-proximal 5′ SS. Sequences of the probes are shown in Supplementary Fig. S3. (b) Immunoblotting of purified spliceosome complexes assembled on control, probe-1, and probe-2 RNA substrates. (c) Immunoblotting of purified spliceosome complexes assembled on control, probe-3, and probe-4 RNA substrates. (d) Schematic of DOK7 minigenes carrying wild-type (Wt and Mut-6) or disrupted (Mut-5 and Mut-7) SRSF1-binding site, with (Mut-6 and Mut-7) or without (Wt and Mut-5) duplicated intron-proximal 5′ SS. Lower panel shows RT-PCR of these minigenes transfected in HeLa cells. Sequences of the Wt and mutant constructs (Mut-5, -6 and -7) are shown in Supplementary Fig. S6. Mut-5 is identical to Mut-5 in Fig. 3d. (e) Distribution of SRSF1-CLIP tags centered around the intron-distal (left panels) and intron-proximal (right panels) 5′ SSs depicted by integrated genome-wide analysis of CLIP-seq of SRSF1 in native HeLa cells and RNA-seq in SRSF1-knocked down HeLa cells. Mean (green lines) and standard error (light green areas) of normalized CLIP-tag densities are shown. In 1445 (upper panels) and 427 (lower panels) genes, SRSF1-konockdown activates intron-distal and intron-proximal 5′ SSs, respectively. A double-headed arrow indicates a peak immediately upstream to the intron-distal 5′ SS in the 1445 genes, indicating the suppressive effect of SRSF1 on the intron-distal 5′ SS, as we observed in DOK7 intron 4.