Extended Data Fig. 9: Characterization of CCAT1-5L and its interaction partner. | Nature

Extended Data Fig. 9: Characterization of CCAT1-5L and its interaction partner.

From: RIC-seq for global in situ profiling of RNA–RNA spatial interactions

Extended Data Fig. 9

a, Genotyping results showing no integration of human papillomavirus at CCAT1-5L locus. b, Northern blot analysis of CCAT1-5L expression across diverse cell lines. c, Western blot showing the purity of nuclear and cytoplasmic fractions. d, e, CCAT1-5L is localized in the nucleus revealed by qPCR and smFISH. CCAT1-E2, CCAT1 exon 2. f, DNA FISH showing CCAT1-5L, MYC and PVT1 loci are colocalized. g, qPCR showing efficient knockdown of hub RNAs DANT2 and PDE3A with LNA ASOs. Non-targeting LNAs, NC-a and NC-b. h, qPCR showing reduced expression of CCAT1-5L, CCAT1-E2, MYC and PVT1 upon CCAT1-5L knockdown with LNA ASOs. i, Western blot showing reduced MYC levels upon CCAT1-5L knockdown. j, Western blot showing reduced MYC levels upon the deletion of the extra extended region of CCAT1-5L by CRISPR–Cas9. k, qPCR showing reduced levels of CCAT1-5L and MYC in mutant cell lines. l, A cartoon depicts the transcriptional blocking of the extra extended region of CCAT1-5L by CRISPRi. m, qPCR showing the expression levels of CCAT1-5L, MYC and CCAT1 exon 1 and 2 (CCAT1 (E1 + E2)) upon CRISPRi. n, Western blot showing reduced MYC levels upon the CRISPRi of CCAT1-5L. o, Motifs enriched in CCAT-5L, MYC and PVT1. p, Enriched motifs in CCAT1-5L RNA. q, HnRNPK monomer and dimer CLIP-seq are highly correlated in HeLa cells (n = 313,762 windows). r, Consensus motifs identified by hnRNPK CLIP-seq. s, 3C–qPCR analysis of the long-distance interactions at the CCAT1-5L, MYC and PVT1 loci upon hnRNPK knockdown with siRNA. t, Co-IP showing hnRNPK (T389A/Q391A) mutant (Flag_K MT) did not form a dimer in HeLa cells. Data in d, g, h, k, m are mean ± s.d.; Data in s are mean ± s.e.m.; n = 3 biological replicates, two-tailed, unpaired t-test. The experiments in b, c, e, f, i, j, n, t were independently repeated three times with similar results.

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