Extended Data Fig. 1: HEM1 is the causal gene for the dst5 mutant phenotype.

a, Schematic of the uORFs-LUC reporter and representative pictures of the basal LUC activity. TBF1 exon1 with uORFs 1/2 and sequence of the N-terminal 73 amino acids was in-frame fused with firefly luciferase (LUC) driven by constitutive CaMV 35 S promoter. NOS ter, NOS terminator. Translational reporter uORFs-LUC/Col-0 was used for the EMS mutagenesis screen. dst5, a mutant with increased LUC activity from M3 generation. C1 and C2, two independent genetic complementation lines of dst5 with a genomic region of the HEM1 expression cassette. T-DNA, a SALK_135634 insertion line with a homozygous uORFs-LUC reporter. Scale bars, 1 cm. b, Whole-genome resequencing identified three closely-linked homozygous mutations on chromosome 2, Chr. 2: C11572722T (AT2G27100), Chr. 2: C14798110T (AT2G35110/HEM1), and Chr. 2: C15262299T (AT2G36380) in the dst5 mutant. Arrowheads indicate the gene orientation on the chromosome. Gene models, two gene models annotated on the TAIR website; red lines, introns; brown boxes, 5' leader and 3' UTR regions; green boxes, CDS regions with dashed boxes signifying the different CDSes in the two gene models. RNA-seq read coverage from TAIR JBrowser was further confirmed in our cDNA amplification assay and RNA-seq/Ribo-seq data. All evidence supported AT2G35110.2 as the dominant transcript model. The hem1 mutant is caused by a mutation on one splicing acceptor site, which leads to the retention of an intron and the gaining of an early stop codon in this retained intron. c, HEM1-related mutant information. d, HEM1’s acronyms and role in WAVE complex-mediated actin nucleation regulation. e, Profiling HEM1 expression pattern among different tissues by RT-qPCR. The top and bottom lines of the box plot represent the 25th and 75th percentiles, the center line is the median, and the whiskers are the full data range (n = 6). f, RNA-seq and Ribo-seq to show early translational termination on the hem1 mutant. No Ribo-seq read was found after the mutation site because of the premature stop in the retained intron indicated by the blue dashed rectangle.