Fig. 3: Generation of PHF5A-Y36C heterologous expression in rice.
From: Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation
a The structure of SF3B complex binding to U2 and the pre-mRNA intron. The branch point is in contact with SF3B1 and PHF5A. The branch point recognition sequence (BPRS) of U2 snRNA binds to branch point and flanking nucleotides. b GEX1A interrupts these interactions and inhibit this early spliceosome assembly. The human PHF5A protein sequence is aligned with sequences of rice PHF5A homologs. PHF5A-Y36C (highlighted in bold) confers tolerance to splicing modulators (Teng et al., 2017) and is highly conserved among eukaryotes. c The coding sequence of rice OsPHF5A (LOC_Os4g56760) was mutated (Y36C; TAT to TGT) and cloned into a construct under the control of the Ubiquitin promoter with a 3XFLAG-tag. d Confirmation of PHF5A-Y36C heterologous expression in transgenic lines. Total protein was extracted from independent lines of O. sativa cv japonica transformed with UBIp::PHF5A-Y36C. Anti-FLAG antibody was used to detect FLAG-tagged PHF5A-Y36C. The arrow indicates the presence of PHF5A. WT was used as a negative control. e, f Heterologous expression of PHF5A-Y36C confers tolerance to GEX1A. Rice seeds were germinated on 1⁄2 MS media for 3 days, then transferred onto 1⁄2 MS media supplemented with 0.3 μM GEX1A for 3 days. Root tips were marked to observe post-transfer growth. The transgenic lines were termed OGR (Overexpression-PHF5A GEX1A Resistance).
