Journal: Oncotarget

Article Title: Landscape of RNAs in human lumbar disc degeneration

doi: 10.18632/oncotarget.11334

Figure Lengend Snippet: ( A – D ) delineate circRNA families binding with corresponding miRNAs. ( E and F ) indicate exceptional cases of multiple binding sites between circRNAs and miRNAs.

Article Snippet: Labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 × 15 K, Arraystar).

Techniques: Binding Assay

Journal: Oncotarget

Article Title: Landscape of RNAs in human lumbar disc degeneration

doi: 10.18632/oncotarget.11334

Figure Lengend Snippet: ( A – B ) indicate miRNA-circRNA interacting machinery. ( C ) is the diagram of RNA transcription and splicing. ( D ) delineates the length scope of each type of RNAs.

Article Snippet: Labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 × 15 K, Arraystar).

Techniques:

Journal: Oncotarget

Article Title: Landscape of RNAs in human lumbar disc degeneration

doi: 10.18632/oncotarget.11334

Figure Lengend Snippet: Extremes of RNA length

Article Snippet: Labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 × 15 K, Arraystar).

Techniques:

Journal: Oncotarget

Article Title: Landscape of RNAs in human lumbar disc degeneration

doi: 10.18632/oncotarget.11334

Figure Lengend Snippet: ( A – B ) Represent the constituent ratio of circRNAs and differentially expressed circRNAs. ( C ) indicates the core interacting miRNA, circRNA, and mRNA in human lumbar discs.

Article Snippet: Labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 × 15 K, Arraystar).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: hsa_circNFXL1_009 modulates apoptosis, proliferation, migration, and potassium channel activation in pulmonary hypertension

doi: 10.1016/j.omtn.2020.09.029

Figure Lengend Snippet: circRNA profile, the genome-wide landscape view, and hierarchical clustering of PAH-COPD patients and healthy controls (A) Volcano plot shows the different expression of circRNAs in two groups of subjects. Red/green dots indicate circRNAs expressed more/less than a 1.5-fold change in PAH-COPD patients. (B) Numbers of dysregulated circRNAs coming from different chromosomes. (C) Pie chart represents the percentage of dysregulated circRNAs coming from different chromosome regions. (D) Heatmap of 158 most significantly dysregulated circRNAs. (E) Heatmap of the top 40 dysregulated circRNAs (20 upregulated and 20 downregulated). Green/red represent a low/high expression level. PAH-COPD, pulmonary arterial hypertension due to chronic obstructive pulmonary disease; circRNA, circular RNA. n = 3 pairs of subjects. Comparisons of data were acquired by a Student’s t test.

Article Snippet: The circRNAs were transcribed into fluorescent cRNA and hybridized onto the Arraystar human circRNA array (8 ×15K, Arraystar) using a random priming method.

Techniques: Genome Wide, Expressing

Journal: Nucleic Acids Research

Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation

doi: 10.1093/nar/gkad763

Figure Lengend Snippet: An endogenous EJC loaded onto mRNA after splicing drives the internal initiation of translation. ( A ) Schematic representation of dicistronic reporter mRNAs either containing or not containing the first intron of the β-globin gene in the intercistronic region. The EJCs deposited after splicing are depicted on the mRNAs. The EJCs that will be displaced by a translating ribosome are semitransparent. ( B ) RNA-IP of endogenous eIF4A3. Extracts of HEK293T cells transiently expressing one of the dicistronic reporter mRNAs were subjected to IP using the α-eIF4A3 antibody or, as a negative control, mouse (m) IgG. The levels of co-IPed reporter mRNAs were normalized to those of intronless HIST2H2AA3 mRNA. The normalized levels of reporter mRNAs obtained in the IPs with mIgG were arbitrarily set to 1.0; n = 4; * P < 0.05. ( C ) Translation efficiency of dicistronic reporter mRNAs. HEK293T cells were transfected with one of the dicistronic reporter plasmids. Next, the activities of proteins RLuc and FLuc and levels of reporter mRNAs were analyzed. FLuc and RLuc activities obtained from Gl1 and Gl3 were arbitrarily set to 1.0, respectively; n = 3; ** P < 0.01. ( D ) The effect of downregulation of an EJC component on dicistronic reporter mRNAs. As performed in ( C ), except that the cells were depleted of either eIF4A3 or Y14. The RLuc activities of Gl2 mRNA were arbitrarily set to 1.0, respectively; n = 3; ** P < 0.01. ( E ) The effect of downregulation of eIF3g, eIF3i, or eIF5 on dicistronic reporter mRNAs. As performed in ( D ), except that the cells were depleted of the indicated protein; n = 3; ** P < 0.01; * P < 0.05.

Article Snippet: For the transcriptome-wide identification of circRNAs that associate with polysomes in an eIF4A3-dependent manner, circRNA microarray analysis was performed using either polysomal or subpolysomal fractions from HeLa cells depleted or not depleted of eIF4A3, following Arraystar's standard protocols for the Human Circular RNA Array (8 × 15 K; AS-S-CR-H-V2.0, Arraystar).

Techniques: Expressing, Negative Control, Transfection

Journal: Nucleic Acids Research

Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation

doi: 10.1093/nar/gkad763

Figure Lengend Snippet: An eIF4A3 drives internal translation in the in vitro system. ( A ) The effect of an SL on in vitro translation. In vitro -synthesized BoxB-R or SL-BoxB-R RNA (either capped or uncapped) and in vitro -synthesized capped FLuc RNA were mixed with the cytoplasmic extracts of HeLa cells. RLuc activities were normalized to FLuc activities. The normalized levels of RLuc activity obtained from BoxB-R RNA were arbitrarily set to 1; n = 3; ** P < 0.01. ( B ) The in vitro translation assay using in vitro -synthesized linear reporter RNAs. In vitro -synthesized reporter RNA (SL-BoxB-R mRNA, either capped or uncapped) and in vitro -synthesized capped FLuc RNA were mixed with the cytoplasmic extracts of HeLa cells expressing the indicated effector protein. RLuc activity was normalized to FLuc activity. The normalized levels of RLuc activity in the extracts of the cells expressing λN-HA-GFP were arbitrarily set to 1; n = 3; * P < 0.05; ** P < 0.01. ( C ) A schematic diagram of in vitro transcription followed by in vitro circularization. ( D ) Validation of in vitro -synthesized circRNAs by denaturing agarose gel electrophoresis. To maximize the yield of circRNAs, we carried out the circularization reaction twice. The details are described in the Methods section. ( E ) The in vitro translation assay using in vitro -synthesized circRNAs. As performed in panels ( A ) and ( B ), except that in vitro -synthesized circRNAs were mixed with the cytoplasmic extracts of HeLa cells expressing the indicated effector protein; n = 3; ** P < 0.01. ( F ) The effect of the tethered EJC on the translation of in vitro -synthesized circRNAs. As performed in ( E ), except that in vitro -synthesized Circ-BoxB-R RNAs were mixed with the cytoplasmic extracts of HeLa cells expressing the indicated effector protein; n = 6; ** P < 0.01.

Article Snippet: For the transcriptome-wide identification of circRNAs that associate with polysomes in an eIF4A3-dependent manner, circRNA microarray analysis was performed using either polysomal or subpolysomal fractions from HeLa cells depleted or not depleted of eIF4A3, following Arraystar's standard protocols for the Human Circular RNA Array (8 × 15 K; AS-S-CR-H-V2.0, Arraystar).

Techniques: In Vitro, Synthesized, Activity Assay, Expressing, Biomarker Discovery, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation

doi: 10.1093/nar/gkad763

Figure Lengend Snippet: Polysomal association of endogenous circRNAs depends on the eIF4A3 and is resistant to serum depletion. (A, B) The effect of Puro treatment on relative polysomal distributions of endogenous circRNAs. Subpolysomal fractions (containing subunits the 40S, 60S and 80S) and polysome fractions of HeLa cells either treated or not treated with Puro for 2 h were pooled. Next, in vitro -synthesized FLuc RNA, as a spike-in control, was added to the pooled fractions. ( A ) Polysome profiles of cytoplasmic extracts of HeLa cells either treated or not treated with Puro. ( B ) Quantitative representation of endogenous circRNAs in subpolysomal and polysomal fractions. The endogenous circRNAs in the subpolysomal and polysomal fractions were quantitated by RT-qPCRs using circRNA-specific divergent oligonucleotides; n = 3; ** P < 0.01; * P < 0.05; #, not significant. ( C–E ) The change in relative polysomal distributions of endogenous circRNAs after downregulation of eIF4A3 and complementation with siRNA-resistant FLAG-eIF4A3. ( C ) Western blotting validating specific downregulation of endogenous eIF4A3 and induction of FLAG-eIF4A3 up to a level comparable to that of endogenous eIF4A3. ( D ) Polysome profiles (upper) and relative distributions of endogenous proteins (lower). ( E ) Relative polysomal distributions of endogenous circRNAs. After polysome fractionation, in vitro -synthesized FLuc RNA, as a spike-in control, was added to the subpolysomal and polysomal fractions. The endogenous circRNAs in the subpolysomal and polysomal fractions were quantitated by RT-qPCRs using divergent oligonucleotides. Next, the levels were normalized to those of FLuc RNA. Relative distributions of endogenous circRNAs in the subpolysomal and polysomal fractions are presented as percentages; n = 3; * P < 0.05; ** P < 0.01; #, not significant.

Article Snippet: For the transcriptome-wide identification of circRNAs that associate with polysomes in an eIF4A3-dependent manner, circRNA microarray analysis was performed using either polysomal or subpolysomal fractions from HeLa cells depleted or not depleted of eIF4A3, following Arraystar's standard protocols for the Human Circular RNA Array (8 × 15 K; AS-S-CR-H-V2.0, Arraystar).

Techniques: Serum Depletion, In Vitro, Synthesized, Control, Western Blot, Fractionation

Journal: Nucleic Acids Research

Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation

doi: 10.1093/nar/gkad763

Figure Lengend Snippet: Polysomal enrichment of endogenous circRNAs is dependent on eIF4A3 at the transcriptome level. ( A ) The experimental scheme of polysome fractionation procedures followed by circRNA microarray. Cytoplasmic extracts of HeLa cells, either depleted or not depleted of eIF4A3, were subjected to polysome fractionation. Next, RNA samples purified from either the pooled subpolysomal or polysomal fractions were subjected to the circRNA microarray analysis; n = 2. ( B ) Scatter plots of the relative change in polysomal association of circRNAs after eIF4A3 downregulation. The x - and y -axes represent the relative percentage distributions of circRNAs in the polysomal fraction in the undepleted cells and eIF4A3-depleted cells, respectively. The dots corresponding to cGSE1, cCTNNB1 and cZNF609 are depicted in cyan. Three types of cCTNNB1 were observed in our analysis. Statistical analysis was performed using the Wilcoxon signed-rank test. ( C ) Scatter plots showing a correlation between the polysomal association of circRNAs and eIF4A3 dependency. The x -axis represents the relative percentage distributions of circRNAs in the polysomal fraction in the undepleted cells. The y -axis denotes the relative ratio (on the log 2 scale) of polysomal to subpolysomal level of circRNAs (PS ratio) in eIF4A3-depleted cells to PS ratio in the undepleted cells. (D, E) Scatter plots showing the relative change in polysomal association of circRNAs ( D ) or the effect of eIF4A3 downregulation on the PS ratios of circRNAs ( E ) according to its length. (F, G) Violin plots for the relative difference in the polysomal association of circRNAs ( F ) or effect of eIF4A3 downregulation on the PS ratios of circRNAs ( G ) according to their types. Statistical analysis was performed using one-way ANOVA with post hoc assessment via Tukey's honestly significant difference test. ** P < 0.01, *** P < 0.001. (H, I) Violin plots for the relative difference in the polysomal association of circRNAs ( H ) or an effect of eIF4A3 downregulation on the PS ratios of circRNAs ( I ) according to the number of EJCs in circRNAs. *** P < 0.001.

Article Snippet: For the transcriptome-wide identification of circRNAs that associate with polysomes in an eIF4A3-dependent manner, circRNA microarray analysis was performed using either polysomal or subpolysomal fractions from HeLa cells depleted or not depleted of eIF4A3, following Arraystar's standard protocols for the Human Circular RNA Array (8 × 15 K; AS-S-CR-H-V2.0, Arraystar).

Techniques: Fractionation, Microarray, Purification

Journal: Nucleic Acids Research

Article Title: An interaction between eIF4A3 and eIF3g drives the internal initiation of translation

doi: 10.1093/nar/gkad763

Figure Lengend Snippet: A full-length intact protein can be generated from circRNAs in an eIF4A3-dependent manner. ( A ) The schematic diagram of three isoforms of cCTNNB1 circRNA generated from the β-catenin gene. ( B ) Validation of endogenous cCTNNB1 isoforms. The obtained RT-PCR products shown in were subjected to Sanger sequencing. The circRNA-specific exon–exon junctions generated by back-splicing are indicated by vertical dashed lines. ( C ) A schematic diagram of monitoring Wnt/β-catenin signaling using TOPFlash or FOPFlash reporters. An increased amount of β-catenin caused by Wnt3a treatment activates FLuc expression from TOPFlash but not FOPFlash. (D, E) The effect of endogenous cCTNNB1c circRNA downregulation on Wnt/β-catenin signaling. HEK293T cells were treated with siRNA specific to cCTNNB1c (#1 or #2) or linear β-catenin mRNA (#1 or #2) that annealed to the 3′UTR of β-catenin mRNA. Two days later, the cells were retransfected with a FLuc reporter [either TOPFlash (TOP) or FOPFlash (FOP)] and a reference plasmid expressing RLuc. The cells were either treated or not treated with 300 ng/ml Wnt3a for 24 h before harvesting. ( D ) Validation of specific downregulation of endogenous cCTNNB1c RNA and linear β-catenin mRNA; n = 3. ( E ) The FLuc activities were normalized to RLuc activities. The normalized FLuc levels obtained in the Wnt3a-treated cells transfected with FOPFlash were arbitrarily set to 1.0; n = 3; ** P < 0.01. ( F ) Complementation experiments with pFLAG-cCTNNB1c expressing a circular form of RNA encoding FLAG-cCTNNB1c. As performed in panels D , E , except that the cells were transiently transfected with pFLAG-cCTNNB1c; n = 3; #, not significant; ** P < 0.01. ( G ) Relative distributions of endogenous cCTNNB1c RNA and its corresponding linear β-catenin mRNA in subpolysomal and polysomal fractions before or after eIF4A3 downregulation; n = 2. ** P < 0.01. ( H ) Relative distributions of endogenous cCTNNB1c RNA and linear β-catenin mRNA in subpolysomal and polysomal fractions before or after eIF3g downregulation; n = 3. ** P < 0.01.

Article Snippet: For the transcriptome-wide identification of circRNAs that associate with polysomes in an eIF4A3-dependent manner, circRNA microarray analysis was performed using either polysomal or subpolysomal fractions from HeLa cells depleted or not depleted of eIF4A3, following Arraystar's standard protocols for the Human Circular RNA Array (8 × 15 K; AS-S-CR-H-V2.0, Arraystar).

Techniques: Generated, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Sequencing, Expressing, Plasmid Preparation, Transfection