Mammalian cell culture
Abbreviations used in this publication are listed in Supplementary Table S1. HEK293-derived stable cell lines TC4-2A and TL4-2A expressing C2A-2B-3-4 and L2A-2B-3-4, receptively, used here were previously generated from the commercially available T-REx cells44. TC4-2A and TL4-2A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Cat No. 11965-092), supplemented with 10% fetal bovine serum (Gibco, Cat No. 16000-044) and 1× Glutamax (Gibco, Cat No. 35050061). Antibiotics were added following the manufacturers’ instruction and/or experimentally determined values, including 100 µg/mL zeocin, 15 µg/mL blasiticidin, 100 µg/mL hygromycin (Life technologies, Cat No. R250-01, R210-01, R220-05) and 0.1 µg/mL doxycycline (Dox, Sigma-Aldrich, Cat No. D9891-10G). Cells were cultured in CO2 incubator (Thermo Fisher Scientific, NAPCO Series 8000WJ) at 37 ºC and 5% CO2. For induction of expression, TC4-2A, TL4-2A and control T-REx cells were seeded at a density of 2 × 105 cells per 100 mm dish. After incubating for 18 hours (h), the cell culture medium was replaced with fresh medium containing Dox. After 48 h of incubation, the cell culture medium was replaced with fresh medium containing Dox. The cells were collected 48 h after the second addition of Dox (96 h of induction in total).
RNA isolation and RNase R treatment
Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Cat No. 15596018). RNA was digested with RQ1 RNase-free DNase (Promega, Cat No. M6101) to remove contaminating genomic DNA, followed by phenol:chloroform extraction and ethanol precipitation. Then RNA was treated with RNase R (Applied Biological Material Inc., Cat No. E049), a 3¢-to-5¢ exoribonuclease to eliminate linear transcripts. RNase R treatment was performed in 10 µL reaction, in which 0.5 µL (10 U) of RNase R was used to treat 2 µg of RNA. The treatment was performed for 45 min (min) at 37 ºC, followed by inactivation at 65 ºC for 20 min. To estimate the digestion efficiency, mock reactions without enzyme were carried out side by side.
Reverse transcription and PCR (RT-PCR)
Superscript III reverse transcriptase (RT) (Invitrogen, Cat No. 18080044) was used in cDNA synthesis following the manufacturer’s instruction. Per 5 µL RT reaction, 0.5 µg of total RNA was used. Reactions were primed with either a gene-specific or a random primer (Promega, Cat No. C1181) (Supplementary Table S2). For semi-quantitative PCR, 1 µL of cDNA was added per 20 µL reaction. PCR products were separated by electrophoresis on 6% native polyacrylamide gels and visualized by ethidium bromide staining. For qPCR, 3 µL diluted cDNA (1:20 dilution) was used in a 20 µL qPCR reaction containing 1× PowerUp SYBR green master mix (Life Technologies, Cat No. A25742) and the desired primer set. Biological triplicates of qPCR reactions were performed on a QuantStudio 3 thermocycler (Thermo Fisher Scientific). Relative expression was determined using the 2-∆∆Ct method using GAPDH as housekeeping gene. To determine the copy number of circRNA per cell, we used a standard curve defined by serial dilution of a known quantity of linearized plasmid containing the sequence of the expected PCR product44 . All primers were obtained from Integrated DNA Technologies. Primers used in this publication are listed in Supplementary Table S2.
Library Generation and RNA-Seq
To confirm RNA integrity, TRIzol-isolated total RNA was characterized using an Agilent Bioanalyzer on an RNA nano chip (RIN ≥8). 1 µg of total RNA was then subjected to rRNA depletion using the NEBNext rRNA depletion kit v2 (Human/Mouse/Rat). Libraries were generated from rRNA-depleted RNA using the NEBNext Ultra II directional RNA library prep kit for Illumina. Libraries were barcoded for multiplexing using NEBNext Dual Index oligos for Illumina. Size distribution of libraries was determined using an Agilent Bioanalyzer DNA 1000 chip and quantified using a Qubit fluorimeter. Libraries were pooled together and sequenced on an Illumina Novaseq 6000 using an S23 flow cell following a 100-cycle, paired-end protocol. Reads from RNA-Seq were mapped to the human reference genome build GRCh38 using HISAT246. For differential expression, mapped reads were assigned to genes according to the Gencode v33 human transcriptome annotation47 using the featureCounts script from the Subread software package48. Differential expression was estimated using the DESeq2 R package49. RNA-Seq data is available from the NCBI sequence read archive under project number PRJNA1058619.
Protein lysate preparation
Cells were washed in ice-cold phosphate buffered saline (PBS) for three times and collected by scraping in 1 mL PBS followed by centrifugation at × 2500 g for 2 min. PBS supernatants were aspirated, and pelleted cells were lysed using lysis buffer, which contained 500 mM triethylammonium bicarbonate (TeABC, Sigma–Aldrich, Cat No. 18597-100ML), 1% sodium deoxycholate (SDC, Sigma–Aldrich, Cat No. D5670-5G), and 1× Halt™ Protease, Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 100X, Cat No. 78440)50. The lysates were incubated on ice for 45 min, and then sonicated. Samples were centrifuged at 16,000 × g for 30 min at 4 ºC. Clear supernatants were collected and transferred to new tubes. The purified protein was snap-frozen on dry ice and stored at -80 ºC. Protein concentrations were measured by Bio-Rad protein assay (Bio-Rad, Cat No. 5000006).
Label-free proteomic quantification and analysis
About 50 µg crude protein samples were submitted to the Protein Facility of the Iowa State University Office of Biotechnology (https://www.protein.iastate.edu/). The samples were processed by a label-free relative quantification approach, using the Minora Feature Detector to detect and quantify isotopic clusters. Specifically, the samples were digested with trypsin/Lys-C. SDC was removed by acid precipitation with 2% (v/v) trifluoroacetic acid (TFA). Then, PRTC standard (Pierce Biotechnology, Cat No. 88320) was spiked in to serve as an internal control. The fragmented peptides were then separated by liquid chromatography with tandem mass spectrometry (LC-MS/MS), using the Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer system (Thermo Fisher Scientific). The relative abundance of the protein was quantified using peak-area based quantification of the precursor ions of the top three most abundant peptides from the identified protein. Then the raw data was computed using Proteome Discoverer Software (Thermo Fisher Scientific, Version 2.4). The data was searched against Mascot and/or Sequest HT. Common contaminants in LC-MS/MS were excluded for downstream analysis51,52. MetaboAnalyst platform (https://www.metaboanalyst.ca/, Version 5.0) was used for statistical and bioinformatics analysis, and generation of plots 53. Features with more than 50% missing values were removed. Interquartile range (IQR) was used for data filtering. Data was normalized by quantile normalization and log transformation. Enrichment analysis was performed by WEB-based GEne SeT AnaLysis Toolkit (http://www.webgestalt.org/, Version 2019, accessed in 2022 December)54. Analysis included Gene Ontology terms (GO terms) for biological processes, cellular components and molecular functions. Pathways including those associated with core protein complex subunits, diseases, phenotypes and the chromosomal locations were defined by Kyoto Encyclopedia of Genes and Genomes (KEGG), Panther, Reactome and Wikipathways. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with identifier PXD048160.
Western blot
A total amount of 10-50 µg of protein was loaded per lane of a 10% SDS-PAGE gel. Proteins were transferred from gel to a PVDF membrane using a Transblot Turbo fast transfer system (Bio-Rad) after electrophoresis. To block the blots, 5% non-fat milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST) was used. Primary antibody incubation was performed at 4 ºC overnight with gentle agitation. The dilutions of primary antibodies were prepared as follows: mouse anti-α-tubulin 1:4000 (Sigma-Aldrich, Cat No. T6199), mouse anti-CNDP2 1:500 (Proteintech, Cat No. 14925-1-AP), mouse anti-NAMPT/PBEF (Santa Cruz, sc-166946) 1:500, rabbit anti-H1-10 (also known as H1x, Fortis Life Sciences, Cat No. A304-604A-T) 1:2,500. After primary antibody incubation, blots were washed in TBST for 10 min and repeated for a total of three times. Then the blots were incubated with secondary antibodies for 1 h at room temperature, with gentle agitation. The preparation of secondary antibody dilutions was as follows: goat anti-mouse 1:4000 (Jackson ImmunoResearch Laboratories Inc, Cat No. 115-035-003), donkey anti-rabbit 1:2000 (GE Healthcare, Cat No. NA934). After secondary antibody incubation, blots were washed in TBST for 10 min three times and developed using Clarity Western ECL Substrate (Bio-Rad, Cat No. 1705061) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat No. 34094). The UVP Biospectrum AC imaging system was used to visualize the bands. Quantification of band intensity was performed by using ImageJ software.
Statistical analysis
Excel (Microsoft, Version 16.62) was used for all the calculation and generation of plots. Data were expressed as mean ± SEM. The unpaired Student’s t-test was applied in statistical analysis. Unless otherwise mentioned, experiments were performed in triplicate, and p values were two-tailed and the level of statistical significance was set as p < 0.05.