Ethical statement
The study was approved by the Ethics Committee of the Joint Shantou International Eye Center (JSIEC), Shantou University & the Chinese University of Hong Kong, Shantou, Guangdong province, China,and conducted in accordance to the Declaration of Helsinki.
Cell culture and stable cell line establishment
HEK293T and human retinal pigment epithelial cell line (ARPE-19) were grown in Iscove’s modified Dulbecco’s medium (Invitrogen, U.S.A) with 10% fetal bovine serum and 1% penicillin and streptomycin (Sigma-Aldrich, U.S.A.), and maintained in a humidified 5% CO2 incubator at 37 °C. Plasmids containing wild type PRPF31 and its R354X mutant were constructed with a GFP (Green fluorescence protein) merged at N terminus and a FLAG-tag merged at its C-terminus. These plasmids were used for the stable cell line establishment. Briefly, cells were seeded at a density of 2 × 105 cells per well in 6-well plates and cultured for 24 hours. Thereafter, each plasmid was transfected into cells using the Lipofectamine3000 Transfection Reagent (Invitrogen, U.S.A.). The cells were incubated for 48 hours and then subjected to stable cell line screening by adding the puromycin to the medium. Cells stably expressing the wild type PRPF31 or PRPF31 R354X mutant were used for RNA extraction or other experiments.
RNA extraction and Reverse transcription
Total RNAs were extracted from cells using TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s instruction. RNA integrity and concentration were assessed using RNA Nanodrop ND-2000 Spectrophotometer (Thermo, U.S.A.). Thereafter, the first stand of DNA synthesis was performed using the reverse transcription kit(Takara,Dalian) based on the manual instruct. The cDNA was used for qPCR or RT-PCR with the designated primer sets (Table.1).
LncRNA sequencing
The LncRNA sequencing services were provided by HUADA(BGI) Shenzhen, China. Cells stably expressing wild type PRPF31 and PRPF31 R354X were sent to BGI. Briefly, total RNA were extracted from the above cells with TRIzol reagent and further digested with DNase I. The resultant total RNAs were then treated with RiboMinus Eukaryote Kit (Qiagen, USA) to remove the rRNA and fragmented using Ambion Fragmentation Solution (Thermo Fisher Scientific, USA).The fragmented RNAs were used for the cDNA synthesis. Following adenylation of DNA 3′-ends and hairpin loop structure ligation, the library fragments were purified to select desired cDNA fragments of 150-200 bp.The products were purified with the AMPure XP system and the library quality was assessed (Agilent Bioanalyzer 2100 system). The generated libraries were sequenced by BGI on an Illumina Hiseq 2500 platform, and 150-bp paired-end reads were produced.
Raw RNA sequence data analysis
The raw data of sequencing was filtered out step-by-step. We obtained the clean data through removing the adapter-polluted reads, low-quality reads and reads with more than 5% N bases accounting in the law data. Then, the resultant reads were mapped to the reference genome using HISAT2. (http://ccb.jhu.edu/software/hisat2/index.shtml). The reference genomes and the annotation file were downloaded from the ENSEMBL database (http://www.ensembl.org/index.html).
HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) was used for read count of each gene in each sample, and FPKM (Fragments Per Kilobase Million Mapped Reads) was then calculated to represent the expression level of genes in each sample, defined as FPKM = 106×F/ (NL×10-3), where F is the number of fragments assigned to a certain gene in a certain sample, N is the total number of mapped reads in the certain sample, and L is the length of the certain gene. Thus, FPKM eliminates the influences of different transcript lengths and sequencing discrepancies on the calculation of expression. DESeq (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html) were used for differential expression analysis of two samples without replicates. A P-value could be assigned to each gene and adjusted by BH. Genes with q <0.05 and |log2 ratio| >1 were identified as differentially expressed genes.
Quantitative real-time polymerase chain reaction
qRT-PCR was performed using the TB Green Premix Ex Taq (Tli RNaseH Plus) (TAKARA,Dalian,China) according to the manufacturer’s instructions. Each reaction contained 5 μl TBGreen Premix Ex Taq qPCR Master Mix (Promega), 5 μl cDNA sample, nuclease-free water and 0.5 μl primers (10 μM). All amplified products ranged from 100 to 200 bp in size. The plates were run on an Applied Biosystems 7500 fast Real Time PCR machine. The cycling program consisted of a hot-start activation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing/extension at 60 °C for 30 s and denaturation 95 °C for 1 min. Following amplification, a melt-curve analysis was performed from 65 to 95 °C with 0.5 °C increments every 10 s. Each sample was run in triplicate, and the average quantification cycle (Cq) value was determined. Control reactions were run with water instead of template for each primer pair to check for primer-dimmers and reagent contamination. Normalised gene expression values (against GAPDH or beta-actin) were obtained using the ΔΔCT method. All primer details are shown in table.1.
Western Blotting
Cells transfected with designated plasmids were lysated in 1×NP40 Lysis buffer containing the complete protease inhibitor (Roche,USA). Then the lysates were centrifuged at 4oC, 15000g for 15 min. The supernatants were used for western blotting analysis. Proteins (ranging from 20 to 50 µg) from each sample were electrophoresed on a 10% Bis-Tris Criterion XT Precast gel (Bio-Rad, U.S.A), then blotted on 0.2 nitrocellulose membrane using a transfer system (Bio-Rad). The protein blot was blocked by exposure to 5% non-fat milk TBST solution at room temperature for 1 hour. Nitrocellulose filters were then incubated overnight at 4°C with the primary antibodies: anti-STAT3 (Ab226942,abcam), anti-IFI6 [G1P3 Antibody (H-84)] (IC162417; X-Y biotechnology, China), anti-Flag (SAB4200071,Sigma), Anti-H2AFX (biovision),anti-CTNNBL1(ab95170) and secondary antibodies. The GAPDH (ab8245,abcam) was used as loading control.
Examination of cell viability
Cell viability was determined using an MTT assay (Sigma-Aldrich, USA). Cells stably expressing PRPF31 WT or R354X were cultured in 24-well plates at a concentration of 2×105 and allowed to grow to 24,48 and 72 hours. Following growth for different time intervals, 100 µl MTT (0.5 mg/ml) was added into the cells and the mixture was incubated for 4 h at 37° C. Subsequently, the supernatant was removed and dimethyl sulfoxide (Sigma-Aldrich,USA) was used to dissolve the resultant formazan crystals. The absorbance was read at 570 nm (EL309 Automated Microplate Reader; Bio-Tek Instruments). Six wells were measured for each group and the experiment was repeated three times.
RNA Immunoprecipitation(RIP)
RIP assay was carried out in ARPE-19 cells using Magna RIP Kit (Millipore, USA) following the manufacturer’s instructions. In brief, magnetic beads pre-coated with 5 mg normal antibodies against Flag (Sigma,USA) or mouse IgG (Millipore) were incubated with sufficient cell lysates (more than 2*107 cells per sample) at 4 °C overnight. And the beads containing immunoprecipitated RNA-protein complex were either used for WB analysis to test the CTNNBL1 binding or treated with proteinase K to remove proteins. Then interested RNAs were purified by TRIzol methods (ThermoFisher Scientific) and detected by RT-qPCR with the normalization to input (fold change was calculated for comparison).