a, Summary of experiments that we carried out to systematically investigate the effects of acute inhibition of U1 snRNP on lncRNA–chromatin associations, transcription dynamics, and RNA processing and decay. b, Volcano plot of polyA RNA-seq showing the fold change (log 2 ) in the chromatin/whole-cell ratio of lncRNAs upon SNRNP70AID degradation. Red dots and deep grey dots indicate lncRNAs that show a significant decrease or increase, respectively, in their chromatin/cell ratio by comparing SNRNP70AID (4-h auxin treatment) versus control (0 h) (P < 0.05; two-sided t-test with three biological replicates). c, Correlation plot of polyA and total RNA-seq analysis of SNRNP70AID mESCs. The set of chromatin-downregulated lncRNAs shows significantly correlated changes in chromatin localization upon degradation of SNRNP70AID (n = 346). d, SLAM-seq analysis of chromatin and non-chromatin (cytoplasm and nucleoplasm) fractions in SNRNP70AID mESCs. SNRNP70AID mESCs were treated with or without auxin for 2 h and then labelled with 4sU for 3 h. After chemical conversion of the incorporated 4sU nucleotides to cytidine, RNA from various subcellular fractions was isolated for 3′-end polyA-seq library construction. e, Box plots showing the conversion rate detected by SLAM-seq of chromatin fractions in the last exon of genes with detectable new transcript (n = 24,097) before (0 h) and after (4 h) SNRNP70AID degradation. Box plots show 5th, 25th, 50th, 75th and 95th percentiles. f, Pearson correlation analysis of the change in chromatin/non-chromatin ratio for new versus all transcripts of lncRNAs with detectable new transcripts (n = 492) identified by SLAM-seq. g, Volcano plots showing expression changes of mESC-expressed lncRNAs (n = 1,282) after treatment with U1 AMO (i) or degradation of SNRNP70AID (ii). Chromatin-downregulated or non-downregulated lncRNAs were further classified into ‘downregulated (down-)’, ‘upregulated (up-)’ or ‘unchanged’ according to their expression changes in whole-cell samples. LncRNAs with reduced chromatin association upon inhibition of U1 snRNA or SNRNP70AID do not show greater downregulated expression by comparison with all lncRNAs. Only a small proportion of them (84 of 337 U1-regulated and 76 of 346 SNRNP70-regulated) show decreased transcript levels. P-values obtained by two-sided t-test; n = 3 biological replicates. h, Metagene analysis of whole-cell RNA-seq reads for the set of U1-snRNP-regulated, chromatin-downregulated lncRNAs in mESCs. Only lncRNAs that do not overlap with any protein-coding gene on the same strand were analysed (n = 239). Similar read-distribution patterns were observed in control cells and in cells treated with U1, U2 or U1/U2 AMOs (i) or in cells subjected to auxin-induced degradation of SNRNP70AID for 0 h or 4 h (ii). Thus, rapid inhibition of U1 snRNP did not cause global transcription termination, although we did observe decreased downstream RNA signals in a few very long lncRNAs, such as Kcnq1ot1 (83 kb), in agreement with the proposed role of U1 telescripting in protecting the transcription integrity of very large transcripts25. In addition, we conjecture that the slight decreases in total transcript levels are likely to be post-transcriptionally mediated by RNA degradation instead of an effect of U1 inhibition on nascent transcription (see panels i–k below). i, Metagene analysis of TT-seq signals in all mESC-expressed genes (n = 10,675) (i) and chromatin-downregulated (n = 239) and non-downregulated lncRNAs (n = 151) (ii) upon SNRNP70AID degradation. Only lncRNAs that do not overlap with any protein-coding gene on the same strand were analysed. j, k, Metagene analysis of ChIP-seq signals of Pol II S5P (‘paused’ Pol II; i) and S2P (‘elongating’ Pol II; ii) across the gene body and upstream/downstream 5-kb region of all mESC-expressed genes (n = 10,675) and unexpressed genes (n = 7,933) (j) or chromatin-downregulated (n = 155) and non-downregulated (n = 66) lncRNAs upon SNRNP70AID degradation (k). Only lncRNAs that do not overlap with any protein-coding gene on either strand were analysed. For h–k, shadings represent 95% confidence intervals for the average enrichment. l, RT–qPCR analysis of the knockdown efficiency and lncRNA expression level change (i), chromatin/non-chromatin ratio (ii) and relative expression (iii) in SNRNP70AID mESCs depleted of EXOSC3 by RNA inhibition. Knockdown was analysed at 72 h after shRNA viral infection. The observation of increased expression for most lncRNAs analysed is consistent with a role of EXOSC3 in mediating RNA degradation (i). Knockdown of EXOSC3 blocked RNA degradation for most lncRNAs analysed (iii), but failed to rescue their decreased chromatin associations induced by auxin (ii). Thus, the effect of U1 snRNP in promoting lncRNA-chromatin binding is not caused by increased RNA degradation. Means ± s.e.m. are shown; P-values obtained by two-sided t-test for three biological replicates.