R354X Mutation in PRPF31 Affects Its Capacity in Inammation Response and Splicing

Pre-mRNA processing factor 31(PRPF31) is a key component of RNA splicing and also a disease-causing gene of Retinitis Pigmentosa (RP). Previously, we found that the nonsense mutation R354X in PRPF31induces RP in a Chinese RP family. In order to investigate the underlining molecular mechanisms of RP pathogenesis induced by this mutation, we generated cell lines stably expressing R354X mutant, wild type (WT) of PRPF31 and corresponding empty vector using HEK293T cells ,the resulting cell lines were used for Long non-coding RNA sequencing(LncRNA -sequencing). The results of LncRNA sequencing showed that, comparing to WT, R354X mutation changed the expression and splicing of coding and non-coding transcripts. Interestingly, in HEK293T and APRE-19 cells, inammation-associated genes such as IFI6, OAS3and STAT3, enhanced their expression in response to the overexpression of WT PRPF31; however, in R354X mutation cells, those genes’ expression remained basal levels. Moreover, increased H2AFX expression and attenuated growth capacity were found in cells expressing R354X PRPF31. In contrast with WT, R354X mutant showed varied splicing model for dihydrofolate reductase (DHFR) in HEK293T , reduced CPSF1 and SORBS1 mRNAs binding in ARPE-19 ,and its binding with CTNNBL1 was also interfered in ARPE-19 cells. On the other hand, the R354X mutant also increased the level of transcripts read-through. Taken together, R354X mutation in PRPF31 affected cell survival, changed gene’s expression and splicing. Those ndings indicate that inammation and oxidation might contribute the pathogenesis of RP induced by the R354X mutation.


Introduction
Retinitis pigmentosa(RP) is an inherited ocular disorder, characterized by progressive degeneration of photoreceptors and abnormalities in retinal pigment epitheliump [1]. In RP, the progressive death of Rod photoreceptors can cause early phenotype of RP, night blindness; then, induce the loss of cones photoreceptors ,and neuronal, glial and vascular remodeling, leading to the loss of vision [1][2]. Currently, more than 70 RP disease-causing genes are identi ed including hundreds of mutations [3]. The various disease-causing mutations in RP indicate complex molecular mechanisms during its pathogenesis including in ammation. The pro-in ammatory component is also a hallmark of the pathogenesis of RP [5][6][7]. Accumulated evidences showed that chronic and low-grade in ammatory processes play an important role in the RP progression [5][6][7].An markedly elevated in the levels of pro-in ammatory cytokines detected in vitreous and aqueous humor from RP patients [7].
As a constitutive component of spliceosomes, Pre-mRNAs processing factor 31(PRPF31) involves in the assembly and stabilization of U4/U6/U5 tri-snRNP [8][9][10]. Moreover, loss-of-function analysis showed PRPF31 participates in snRNPs activation and splicing e ciency determination [11]. Insu ciency of PRPF31 can only impair the splicing of a subset of genes using patient-derived lymphocytes or siRNAtreated human organotypic retinal cultures [11]. Knocking PRPF31 out in zebra sh or mouse also severely affected the retinal-speci c gene expression although mild disturbance for global transcriptome is also observed.PRPF31 mutation also causes RP [12][13]. Previously, we found that a nonsense mutation R354X in PRPF31 is a disease-causing mutation for a Chinese RP family [10]. This mutation can lead to an elevated PRPF31 protein expression [10]. In order to further understanding the potential molecular mechanisms of RP pathogenesis in this family, we performed a RNA sequencing analysis. We found that the changed expression for genes involving in in ammation in PRPF31 R354X mutant comparing to WT.

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 modi ed 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 humidi ed 5% CO 2 incubator at 37 °C. Plasmids containing wild type PRPF31 and its R354X mutant were constructed with a GFP (Green uorescence protein) merged at N terminus and a FLAG-tag merged at its C-terminus. These plasmids were used for the stable cell line establishment. Brie y, cells were seeded at a density of 2 × 10 5 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 rst 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. Brie y, 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 Scienti c, 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 puri ed to select desired cDNA fragments of 150-200 bp.The products were puri ed 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 ltered 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 le 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, de ned as FPKM = 10 6 ×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 in uences 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 |log 2 ratio| >1 were identi ed 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 ampli ed 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 ampli cation, 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 quanti cation 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.

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 2×10 5 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 su cient cell lysates (more than 2*10 7 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 puri ed by TRIzol methods (ThermoFisher Scienti c) and detected by RT-qPCR with the normalization to input (fold change was calculated for comparison).

Results
1. LncRNA sequencing data was quali ed and large number of useful information was discovered As our previous paper showed, R354X mutation in PRPF31 could induced development of Retinitis pigmentosa(RP). In order to further understand its molecular mechanisms, we generated three lines, which can stably express R354X mutation or WT of PRPF31 or negative control (NC) using HEK293T cells. After that, we used those cell lines for LncRNA sequencing with Illumina Hiseq platform with a read length 150 bp(Ruohe, China). The rRNA but not the mRNAs were removed from the total RNAs samples before cDNA synthesis. Thus, we can analyze both mRNAs and non-coding RNAs in samples. The sequencing data from all samples were quali ed and the effective reads were all close to 99.6%. (Figure.1A).The number of clean reads (about 67.7M), clean bases (more than 10 Gb), Q30 (about 94.5%) and Q20 (about 97.5%) are almost the same (Supplemental Figure.1A). The GC content has no signi cant difference in all three samples (Supplemental Figure.1B).

Differential expression and splicing in transcripts between groups expressing R354X mutant and WT of PRPF31
The number of total reads and total mapped reads were almost the same between these two groups ( Figure.1A). However, we can easily notice the signi cant difference between groups expressing R354X mutant and WT of PPRF31 when we compared the mapped reads number between spliced(18.54%,19.26% for mutant and WT, respectively) and non-spliced (29.23%,27.86% for mutant and WT, respectively)( Figure.1A).In contrast, over-expressing WT or R354X mutant of PRPF31 had higher exon read number than no transfected cells(NC) (exon read for WT 22%,R354X 23%, NC 18.4%); but decreased the number of Intron reads (59.7%(NC),56.4% (WT) and 53.8% R354X mutation, respectively) ( Figure.1B). The detailed information for total signi cantly differential mRNAs and non-coding RNAs were listed in supplemental data1 and supplemental data2.Heatmaps generated from differentially expressed noncoding RNAs and mRNAs showed R354X mutation could affect large numbers of transcripts expression ( Figure.2B, supplemental Figure.1). Gene ontology with KEGG analysis showed that genes involving in infection, immunity and metabolisms are especially signi cantly changed in R354X mutated PRPF31 stable cell lines ( Figure.2A). A similar result was also observed in non-coding RNAs and microRNAs Supplemental Figure.1 .

R354X mutation in PRPF31 disrupts the alternative splicing of RI and A3SS
PRPF31 is a key protein in the regulation of RNA splicing. We thus analyzed the LncRNA sequencing data in order to observe whether alternative splicing events were affected after R354X mutation. Interestingly, R354X mutation leads to the reduction of RNA splicing event, especially in types of RI and A3SS, which were almost removed from R354X mutation PRPF31 cells ( Figure.3A, B) .Other three types of splicing were also affected partially comparing to WT.
3. qPCR and Western blotting were used to con rm the selected differential expression mRNAs We chose two down-regulated (STAT1, IFI6) and one up-regulated (H2AFX) genes for qPCR and western blotting (WB) Analysis in HEK293T or ARPE-18 cells. The results showed that RNA expression for STAT3 ( Figure.  4. R354X mutation in PRPF31 changed the alternative splicing of DHFR. Based on the results of LncRNA sequencing, we found that the MXE type of splicing at the second and forth exon in DHFR was strongly affected in R354X mutant cells ( Figure.5A, B).So we designed two primer sets (FP1/RP1, FP2/RP2) to detect this alternative splicing event using RT-PCR. In contrast to WT group, we observed different sizes of PCR products in R354X mutated PRPF31 via same primer set( Figure.5E). Quantifying the above PCR products with qPCR showed differential expression level of those alternative splicing products between R354X mutant and WT ( Figure.5F), showing a consistent result with the sequencing data.
5. Immunoprecipitation further showed that R354X mutant affected the splicing capacity of PRPF31 CPSF1 and SORBS1 are the substrate of PRPF31 splicing. We thus compared the binding level of WT and R354X mutant to those two RNAs via immunoprecipitation(IP) via anti-Flag antibody. As expected, R354X mutant showed a reduced binding capacity to the above two RNAs, compared to WT (Figure.6C). The IP e ciency was con rmed by checking the pull-downed WT and R354X mutant via WB. Also, we found that CTNNBL1,which was con rmed to be a binding protein during splicing( Figure.6B).

Discussion
Mutations in PRPF31 induce the pathogenesis of autosomal dominant retinitis pigmentosa. Recently research showed knockout prpf31 in zebra sh affects the differentiation and viability of retinal progenitor cells [16]. Previously, we found that several mutations in PRPF31 induced the pathogenesis of RP [10]. Of those mutations, R354X mutation showed a more stable protein comparing to the WT although Cterminus is deleted. In order to test its underlining molecular mechanisms, we generated three cell lines stably expressing Empty vector, R354X PRPF31,WT PRPF31 via HEK293T and then did LncRNA sequencing with those cell lines. We found that R354X mutation in PRPF31 affected RNA splicing and expression of certain transcripts. Interestingly, we uncovered that over-expressed PRPF31 in HEK293T strongly activated the expression of in ammation-associated genes such as IFI6. However, in cells stably expressing R354X mutant,IFI6 expression could not be induced. Moreover, the key transcriptional factor for expression of in ammation genes, STAT3 was also activated in WT but not in R354X mutant. Because in ammation, autophagy and oxidative stress are very important factors during the pathogenesis of RP[6- 7,14], mutation at the R354 in PRPF31 might prevent the ability of PRPF31's response to various infections and lead to the damage of infected retinal tissues.
We also noticed that H2AFX expression signi cantly increased in R354X mutant comparing to WT. H2AFX is a marker of DNA damage [15]. Previous ndings showed that knockout prpf31 in zebra sh, retinal progenitor cells (RPCs) displayed signi cant mitotic arrest and DNA damage, which was rescued by the wild type human PRPF31 [12][13]16]. Further analysis showed knockout prpf31caused the skipping of exons with a weak 5' splicing site. The genes involve in the DNA repair and mitotic progression are affected [12][13]16]. Our current nding is in line with this. R354X mutation in PRPF31 affects its splicing capacity, and in turn prevents the biological roles of DNA repair associated genes.
R354X mutant not only alters gene expression, but also undermines its splicing capacity(Figure.5 , Figure. 6B.C). DHFR is an important enzyme required to maintain bacterial growth, and hence inhibitors of DHFR have been proven as effective agents for treating bacterial infections [17].CPSF1 was reported as a regulator of retinal ganglion cell development [18],therefore, un-proper splicing for this gene ,might affect ocular functions.
Taken together, we found R354X mutation in PRPF31 changed its splicing capacity and exposed the retinal tissue to be vulnerable after infection.   Figure 1 Statistical analysis of LncRNA data on mRNAs A, Statistical analysis that reads mapped to reference genome; B, Statistical analysis of transcript structure in three different cell lines; C,Venn diagram illustrating common differentially expressed genes in three groups.NC represent cells stably expressing empty vector; WT represent cells stably expressing wild type of PRPF31; R354X represent cell stably expressing R354X mutated PRPF31. The expression levels of STAT3, IFI6 and H2AFX. Expression levels were normalized to beta-actin. Data are the means + S.D. of three independent experiments. **P<0.01,*p<0.05, one-way analysis of variance followed by Dunnett's multiple comparison test or Student's t-test was performed. ET, represents cells stably expressing empty vector; Figure 5 Con rmation of alternative splicing on DHFR in cells expressing R354X mutant. A, alternative splicing result between WT and R354X on DHFR gene from lncRNA sequencing; B, schematic diagram showed the alternative splicing exons and locations of primers used to con rm the splicing; C,D multi-alignment of three alternative transcripts in DHFR genes showed differential splicing sequence; E,RT-PCR analyzing the alternative splicing exons within cells stably expressing WT or R354X PRPF31;F, qPCR analysis to the alternative splicing exons with the same cell lines as in E. data was expressed as mean relative mRNA expression + S.D. qPCR analysis for the pulldown RNAs with designated primers(C). Using Flag and IgG antibody to IP and Proteins or RNAs pull-downed was used to WB directly or RNA extraction with TRIzol reagent.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementalFigure1.tif