Fig. 1.

Conditional expression of U2af1(S34F) from the mouse endogenous locus alters splicing similar to that in human cells expressing U2AF1(S34F). (A) Diagrams of the endogenous U2af1 locus, the MGS34F targeting vector, and modified alleles, with sites used for Southern blotting- and PCR-based genotyping. Numbers in boxes indicate exons or exonic sequences in cDNA; lines represent introns; STOP denotes a 3× transcriptional stop signal from SV40; B, BamHI site; E, EcoRI site. The red version of exon 2 encodes the S34F mutation (TCT-to-TTT). A more detailed description of the targeting vector is in SI Appendix, Supplemental Methods and Materials. (B) U2af1(S34F) changes the inclusion levels of indicated exons (or portions of exons; see inserted cartoons) found at six loci of genes (named in boxes) in total bone marrow cells from mice with the indicated genotypes (n = 3). The mice were treated with poly (IC) and were killed 2 wk later for RNA extraction. As noted in the text, alternative splicing of human homologs of these mRNAs was previously reported to be affected by U2AF1(S34F). Asterisks indicate statistically significant changes compared with other genotypes by t test (*P < 0.05). Error bars represent SEM. (C) Representative read coverage of U2af1 cDNA by RNA-seq, which was performed on MPs from mice of the indicated genotypes 4 wk after poly (IC) treatment. Each gray line represents a sequencing read. The reference DNA (noncoding strand) and protein sequences are shown below the sequencing reads, and the numbers of reference (WT) and variant (S34F) alleles are quantified below the graphs. (D) The Venn diagram indicates the numbers of orthologous genes from mouse and human datasets that show at least 10% change in cassette exon inclusion levels in the presence of mutant U2AF1. The mouse dataset is presented in SI Appendix, Table S3. The human dataset was published previously (21), using AML samples from The Cancer Genome Atlas (63). Genes with low levels of expression (median transcripts per million sequenced RNAs <10) or that have no mouse or human ortholog were excluded from the analysis. (E) U2af1(S34F) recognizes similar consensus sequences at 3′ splice sites in the mouse genome as U2AF1(S34F) does in the human genome. mRNA from MPs with or without U2af1(S34F) was sequenced to determine nucleotides at the 3′ splice-acceptor sites of cassette exons and was displayed as sequence logos according to whether inclusion of the exon in mRNA was increased, decreased, or unaffected by U2af1(S34F). The resemblance of these logos to those previously determined with human materials is discussed in the text. Additional molecular characterization of the established mouse lines (including another conditional allele, IES34F) and their cell derivatives is presented in SI Appendix, Figs. S1 and S2.