Extended Data Fig. 6: Consequences of ZRSR2 and LZTR1 dysregulation.
From: Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition
a, RNA-seq of LZTR1’s minor intron in normal and ZRSR2-mutant and WT MDS (n = 10 each) marrow. b, Ratio of intron retained (IR) to normal LZTR1 in MDS samples (n = 10 each). Mean ± SD. c, Data from (b) ±SD. P-value; two-sided t-test. d, Qualitative RT-PCR using primers amplifying exons 18-19 (‘e18-e19’) as well as specific to IR isoform. e, Splicing efficiencies of LZTR1 in patient samples (median over n = 4 normal samples; left) or mouse lineage-negative c-Kit+ cells (right). P-values: two-sided Mann-Whitney U. f, LZTR1 expression by level of minor intron retention (‘Low’: <10%; ‘Mid’: 10–20%; ‘High-retention’: >20%). P-values: one-sided Mann-Whitney U. In (e) and (f): middle line, hinges, notches, and whiskers indicate median, 25th/75th percentiles, 95% confidence interval, and most extreme points within 1.5x interquartile range from hinge. g, UPF1 immunoblot in K562 cells with mutation disrupting LZTR1s U12 sequence +/− anti-UPF1 shRNA. h, Expression of U12-retained LZTR1 following actinomycin D +/− anti-UPF1 shRNA. n = 3 biological replicates. Mean +/− SD. i, Immunoblot of K562 cells +/− ZRSR2-targeting sgRNA. j, Expression of LZTR1 (left) or CHD4 (right) isoforms in ZRSR2-null K562 cells with DMSO or NMD inhibitor (PMID 24662918). Mean +/− SD; P values: two-sided t-test. n = 3 biological replicates. k, LZTR1 minigene with mutations generated. l, RT-PCR of LZTR1 minigene and endogenous mRNA from WT or ZRSR2-KO K562 cells. m, RT-PCR of LZTR1 minigene and endogenous using native (‘N’) or mutant minigenes. n, Lztr1 minor intron with ___location of sgRNA, PAM site, 3’ U12 consequence (blue text), and sequence in individual Ba/F3 cell clones (red dash: deleted nucleotides). o, As (n) in K562 cells. p, Immunoblot of Lztr1 in Ba/F3 single-cell clones +/− Lztr1 protein-coding or minor intron sgRNAs. q, Median relative percentage of GFP-labeled K562 cells following Rebastinib. Experiments in (d), (g), (i), and (l) were repeated twice with similar results.
