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. 2023 Jul 24;14:4447. doi: 10.1038/s41467-023-40105-3

Fig. 1. ORC1 binds in vivo to RNAs produced from active replication origins.

Fig. 1

a Schematic representation of human ORC1 protein domains4,24,103. b Cross-correlation between endogenous ORC1, and unlabeled (control) or EU-labeled RNA (short or long pulse), comparing STORM experimental (EXP) and randomized (RND) analysis in the chromatin fraction of U2OS cells synchronized in G1. Data were presented as mean values (n > 50 cells). Indicated p values (ns denoting p value >0.05) derive from unpaired two-sample t-test. c Schematic of RIP-seq and iCLIP experimental approaches, where endogenous or Flag-tagged ORC1 is immunoprecipitated from native or UV-crosslinked nuclear extracts, followed by recovery of full-length or digested bound RNAs. Below, the number of genes identified by both methods, with different iCLIP stringencies, and hypergeometric test p values of the experimental overlap (RIP-iCLIP) on top of the bars; red for selected high confidence (HC) ORC1-RNAs. d Genomic distributions of ORC1 iCLIP crosslinks, and (below) their density around TSSs (−/+ 10 kb) of ORC1-bound genes. e Gene length and expression level of high confidence (HC) ORC1-RNAs (iCLIP-RIP overlap) and ORC1-RNAs (iCLIP-RIP union), relative to sample size-matched control genes with different fold changes (FC) in ORC1 RIP-seq. n = number of genes in each category (Supplementary Data 2; from iCLIP data [>5 crosslink sites and <0.05 FDR] and RIP-seq data [log2 fold change >1 and p value <0.05]). Box plots show the median distribution between Q1 and Q3. *** denotes p value <0.001, derived from unpaired two-tailed Student’s t-test. f Gene biotypes of ORC1-RNAs. g Percentage of ORC1-RNA and high confidence ORC1-RNA (HC) genes with mutual interactions according to Hi-C analysis, compared to controls shown in Fig. 1e. Bars represent mean values. **** denotes p value <0.0001, derived from a two-proportion z-test. h, i Density plots of h ORC1 ChIP-seq and i SNS-seq normalized reads across six quantiles (Q) of ORC1-RNA genes, defined by ORC1 iCLIP, centered around their TSSs (−/+ 5 kb). j Browser snapshot of representative high confidence ORC1-RNA genes, showing data of ORC1 RNA-binding (ORC1 RIP-seq or iCLIP crosslinks), and replication origins (SNS-seq) at their TSSs, in HCT116 cells. Green arrows indicate positions of GAA repeats.