Fig 2: U1A mRNA levels associate with cancer prognosis and cell growth(A–C) U1A mRNA levels were evaluated in data from TCGA (A) or in paired samples from TCGA (B) or our own cohort of patients (C). (D) Disease-free survival (DFS) and overall survival (OS) were evaluated with the TCGA cohort of LIHC using GEPIA. (E) Top: fold change (FC) increase of U1A mRNA levels in tumor (T) versus peritumor (PT) samples from the TCGA cohort of indicated tumors. Bottom: TCGA patients were divided in two groups of equal size based on the U1A mRNA level in the tumor (high or low) and DFS and OS were calculated for the indicated tumors. (F) Overlap of U1A-bound genes from U1A IP2 iCLIP data and genes whose expression positively correlates with U1A mRNA levels (R > 0.5) in TCGA LIHC data. The result from hypergeometric enrichment analysis is shown. (G) JHH6 cells were transfected with control or two independent U1A-targeting siRNAs and cell number was quantified 3 days later. The percentage of proliferation, shown in the y axis, was calculated by dividing the final cell number by the number of cells seeded prior to transfection. Proliferation percentage at 72 h post transfection is plotted as dots representing each replicate. Error bars show SEM. Two-tailed Student’s t test was employed. * p < 0.05 and **p < 0.01. (H) JHH6 cells were transfected with control or two independent U1A-targeting siRNAs and cell proliferation was assessed through an MTT assay. MTT absorbance is plotted as dots representing each replicate. Error bars show SEM. One-way ANOVA was employed to compare group means: ns, not significant; **p < 0.01; ****p < 0.0001.

Fig 3: CircME1 exerts cis-regulatory effect via interacting with U1 snRNP.A CircME1 was abundantly distributed both in nucleus and cytoplasm as demonstrated by RNA-FISH with CY3-labeled circME1 probe and DAPI labeled nuclei. B, C U1-70K, U1-A, Sm-D2 and Sm-E (protein components of U1 snRNP) were identified as circME1-interacting proteins by RNA pull-down assay. The proteins pulled down by circME1 or CTRL probes were subject to SDS-PAGE and silver staining (B). U1-70K and U1-A were pulled down by circME1 as demonstrated by Western blot analysis (C). D CircME1 was markedly enriched in U1-A-immunoprecipitated RNA as demonstrated by RIP assay with IgG as negative control (left panel). Electrophoresis analysis result of qPCR products of immunoprecipitated RNA is shown (right panel). E U1-A bound to ME1 promoter (around -500 bp–-300 bp) in 786-O cells as demonstrated by ChIP assay. Knockdown of circME1 decreased the enrichment level of this DNA sequence (left panel). Electrophoresis analysis result of the ChIP products is shown (right panel). F The schematic diagram of cis-regulatory effect of circME1 on its parental gene. G, H qPCR and Western blot analyses showing that overexpression of circME1 containing mutant binding site of U1 snRNP failed to increase the expression level of ME1. I CircME1 containing mutant binding site of U1 snRNP failed to be enriched in U1-A-immunoprecipitated RNA as demonstrated by RIP assay (upper panel). Electrophoresis analysis result of the qPCR products of immunoprecipitated RNA is shown (lower panel). J Blockage of U1 snRNA with U1 AMO abrogated the cis-regulatory effect of circME1 at mRNA level of ME1 as demonstrated by qPCR assay.

Fig 4: U1A-binding sequences increase reporter expression(A) Relative expression level, quantified by RT-qPCR, of U1A, AGO1, GMPR2, and UQCR11 mRNAs after IP of crosslinked cell extracts with the antibodies indicated in the x axis. mRNAs from input samples were used as a reference. (B) Quantification of U1A, AGO1, GMPR2, and UQCR11 mRNAs by RT-qPCR in HeLa cells transfected with control or U1A-targeting siRNAs. Cells were collected 2 days after transfection and RPLP0 mRNA levels were used as a reference for normalization. (C) RL activity in HeLa cells transfected with a control (CMVRL) or plasmids expressing an RL transcript with AGO1, GMPR2, and UQCR11 3' UTR sequences (schematized in the upper part of the figure). Cells were also transfected with a Luc plasmid and collected 2 days after transfection to quantify RL and luc activity. The activity of RL was normalized with that of luc to calculate the percentage of reporter activity. (D) 293T cells were co-transfected with a Sleeping Beauty transposase and transposon plasmids expressing an RL transcript with AGO1, SL2WT, or SL2Mut 3' UTR sequences (schematized in the upper part of the figure). Stable clones were isolated and transfected with either control or U1A-targeting siRNAs. Two days later, total RNA and proteins were extracted to measure RL mRNA by RT-qPCR and RL luciferase activity. Each dot represents the mean of three replicates performed on the same clone. The activity of RL was normalized to that of the control for each clone to estimate the relative RL activity. The experiments were performed at least three times in duplicate (B) or triplicate (C and D). Error bars show SEM. Two-tailed Student’s t test was used to compare two independent groups. Results are indicated as ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Fig 5: Inhibition of gene expression by RNAi, U1i, or U1 snRNP proteins(A) HeLa or S2R + Drosophila cells were transfected with a plasmid expressing an RL transcript with a wild-type (WT) (pRL-U1BS) or a mutated (pRL) binding site for endogenous U1 snRNP and/or a plasmid expressing a shRNA (HeLa) or 0.1 ng of dsRNAs (S2R+) against RL. (B) HeLa cells were co-transfected with plasmids expressing RL, plasmids expressing U1WT, U7WT, or U7SmOpt snRNAs, which target RL 3' UTR (BS) and/or plasmids expressing a shRNA against RL. (C) Mammalian cells were co-transfected with a plasmid expressing an RL transcript with 4 MS2-binding sites, plasmids expressing the indicated MCP fused proteins, and/or plasmids expressing a shRNA against RL. (D) As in (C), but the RL 4xMS2 plasmid contains a canonical, a histone, or a MALAT1 3'-end processing signal. Each plasmid was co-transfected with plasmids expressing MCP fused to U1 snRNP-specific proteins. (E) As in (C), but the RL 4xMS2 plasmid was co-transfected with plasmids expressing MCP fused to U1A or U1A-truncated fragments. In all (A–E) cases, a plasmid expressing firefly luciferase (luc) was also co-transfected as a transfection control. Two days after transfection, RL and luc were quantified and the activity of RL was normalized with luc to calculate the percentage of reporter activity. All experiments were performed at least three times in triplicate. Error bars show standard error of the mean (SEM). The synergy index (SI) was calculated as described.15 Either two-tailed Student’s t test or one-way ANOVA was employed to compare two or more independent groups respectively. Results are indicated as ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.