Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation
Fig 5
Point mutation in the third position of the branch sequence converts a Prp4 kinase-independent intron into a kinase-dependent intron.
(A) Proposed base-pairing between the res1 intron branch sequence CUAAC and snRNA U2. Ψ indicates the pseudouridine 39 nucleotides from the 5’ end of snRNA U2, which is suggested to base-pair with the A at position 3 of the branch sequence. (B) res1’-A and res1’-2A: RT-PCR analysis in the absence (-Inh) and presence (+Inh) of inhibitor at the indicated times. (C) res1’-B and res1’-2B. (D) res1’-C and res1’-2C. (E) res1’-D and res1’-2D (F) res1’-E and res1’-2E. H2O, negative control without template. The scheme on the left side of the images show the details of the interactions between exon1/5’ SS and snRNA U1 and between the branch sequence and snRNA U2. Small letters indicate the mutations in exon1/5’ SS and the branch sequence; the corresponding alleles were named as indicated. |, Watson-Crick base-pairing; Ψ, Pseudouridine; ϕ, wobble base-pairing Ψ-A. Asterisks indicate the expected position of fragments if the introns are or are not spliced out. The numbers on the left side of the image represent the sizes of the DNA fragments (bp). M, DNA size marker.