A novel antiviral lncRNA EDAL shields a T309 O-GlcNAcylation site to promote EZH2 degradation
Summary (6 min read)
- Among infectious diseases of the central nervous system (CNS), those caused by viral pathogens-known as neurotropic viruses-are far more common than bacteria, fungi, and protozoans (2Nd & Mcgavern, 2015 , Ludlow, Kortekaas et al., 2016) .
- Moreover, nearly half of all emerging viruses are neurotropic viruses (Olival & Daszak, 2005) , including the Dengue and Zika viruses (Carod-Artal, 2016 , Meyding-Lamade & Craemer, 2018) .
- It is unclear how this regulation occurs.
Identification of a host lncRNA induced by viral infection
- The authors conducted a time-course RNA-seq analysis of cultured N2a cells that were infected with pathogenic RABV (CVS-B2c strain) or were mock infection treated.
- This identified 1,434 differentially expressed lncRNAs (Fig 1A) .
- QPCR analysis successfully confirmed the significantly up-regulated expression of ten of the most highly up-regulated of these lncRNAs in response to RABV infection (Fig 1B) .
- This long intergenic non-coding RNA had no obvious annotation hits after examining its sequence using tools available with the NONCODEv5 (Fang, Zhang et al., 2017) , lncRNAdb 2.0 (Quek, Thomson et al., 2015) , or LNCipedia 5.0 (Volders, Verheggen et al., 2015) databases.
EDAL inhibits viral replication
- The authors next transfected N2a cells with pcDNA3.1 plasmid expressing either EDAL (pcDNA-EDAL) or an EDAL-specific small interfering RNA and then verified that EDAL was appropriately expressed or specifically silenced in N2a cells (Fig EV3A and 3B) .
- The authors also confirmed that overexpression or silencing of EDAL did not affect cell viability (Fig EV3C and 3D) .
- Next, the authors transfected N2a cells with the EDAL expression plasmid and then infected them with RABV at 12 hours (h) post transfection.
- The authors also analyzed the capacity of the recombinant viruses to spread between infected cells and neighboring cells, the infected N2a cells were covered by low melting agar to inhibit the virus release into the supernatant (Tian et al., 2016) .
EDAL reduces RABV pathogenicity in vivo
- To investigate the role of EDAL in RABV infection in vivo, the authors compared the pathogenicity of rRABV, rRABV-EDAL, and rRABV-revEDAL in the C57BL/6 mouse model.
- Mice were infected intra-nasally (i.n.) with rRABV, rRABV-EDAL, or rRABV-revEDAL (100 FFU).
- The mice infected with rRABV and rRABV-revEDAL exhibited decreased body weights starting from 7 to 9 days post infection (dpi), and these decreases became significant between 9 and 14 dpi.
- In contrast, the body weight of mice infected with rRABV-EDAL only exhibited a slight decrease between 10-14 dpi (Fig 3A Collectively, these results establish that EDAL can dramatically inhibit intranasal-inoculation-induced RABV infection in mice.
EDAL decreases H3K27me3 levels by promoting lysosome-mediated EZH2 degradation
- Having demonstrated that RABV infection induces the accumulation of EDAL and established that EDAL can restrict RABV replication in vitro and in vivo, the authors were interested in potential mechanism(s) through which EDAL may exert its antiviral effects.
- The authors have for some time been interested in the potential contributions of epigenetic regulation on host responses to neurotropic viruses, and they noted that the N2a cells transfected with the pcDNA3.1 plasmid expressing pcDNA-EDAL had significantly decreased levels of histone methylation.
- The authors next used the recombinant viruses that they used for mice infection ( again highlighting an apparently specific contribution of EDAL to the reduced levels of H3K27me3 and its catalyst EZH2.
A 56 nt 5' segment is responsible for EDAL's antiviral activity
- Secondary structures are thus far good candidates for identification of functional elements of lncRNAs (Bonasio & Shiekhattar, 2014 , Johnsson, Lipovich et al., 2014 , Mercer & Mattick, 2013 , Rivas, Clements et al., 2017) .
- Seeking to identify secondary structures of EDAL that affect its specific interaction with EZH2, predictions using the RNAstructure 5.
EDAL reduces EZH2 stability by impeding an O-GlcNAcylation PTM at the T309 site
- RNA tertiary structure prediction revealed a tertiary structure for the 56-nt antiviral RNA segment: the helix-loop tertiary structure folded by the 18-nt terminal hairpin corresponding to 125-142 of EDAL was packed on the second helix folded by the stem base-paired structure, and most of the two structural components were free for contacting other partners (Fig 6C ).
- The authors co-transfected N2a cells with plasmids expressing wild type EZH2 and EZH2 mutant variants together with the pcDNA3.1, pcDNA-EDAL or pcDNA-revEDAL plasmids.
- Then the authors confirmed that the specific EDAL interaction sites on EZH2 are in its N-terminal region (1-337 aa) using an RNA pull-down analysis (Fig These results support that EDAL specifically contacts T309, shielding T309 from O-GlcNAcylation.
The EZH2 inhibitor gsk126 protects neuronal cells from viral infection
- If EDAL's antiviral effects are indeed mediated by its reduced EZH2 methyltransferase activity, then the authors could anticipate that chemical inhibition of EZH2 should cause antiviral effects.
- The authors findings indicated that EDAL binds to EZH2 at a site different from that of lncRNA-HOTAIR binding of human EZH2 via residues in 342-368 region (Kaneko et al., 2010) .
- Alteration of the host epigenetic dynamics by virus-elicited host lncRNAs might not be limited to EZH2 and H3K27me3 mark.
- As a result, lnc-DC indirectly promotes STAT3 phosphorylation on tyrosine-705 and controls human dendritic cell differentiation (Wang, Xue et al., 2014) .
- PCP4L1 display a distinct expression pattern which is dominantly expressed in the CNS, and mostly expressed in circumventricular organs and modulate the production of the cerebrospinal fluid in the adult brain (Bulfone et al., 2004) .
Cell lines, viruses, and mice
- BV2 (murine microglia, BNCC337749) were obtained from BeNa Culture Collection.
- Cells grown in a 37°C humidified 5% CO 2 atmosphere, growth media was DMEM or RPMI1640 supplemented with 10% (vol/vol) FBS and 1% antibiotics (penicillin and streptomycin) .
- The recombinant rRABVs were cloned from RABV strain challenge virus standard-B2c (CVS-B2c) and constructed as described previously (Tian et al., 2016) HZAUMO-2016-009).
RNA-seq library construction, sequencing and lncRNA prediction pipeline
- Total RNA from RABV infected N2a cells or mock-infected cells were isolated by using Trizol ® reagent following the manufacturer's instructions, and then treated with RQ1 DNase to remove DNA.
- Each RNA-seq library was prepared using 5 μg of total RNA.
- Purified RNAs were iron fragmented at 95°C, followed by end repair and 5' adaptor ligation.
- Reverse transcription was performed using RT primers harboring a 3' adaptor sequence and randomized hexamer.
- The cDNAs were purified, amplified by PCR, and products 200-500 bp in length were isolated, quantified, and used for sequencing.
RNA-seq data processing and alignment
- Raw reads containing more than two unknown (N) bases were discarded.
- The mus musculus genome sequence (GRCm38) and annotation file (gencode.vM6 basic annotation) were obtained from the GENCODE database (Mudge & Harrow, 2015) .
- Clean reads were aligned end-to-end to the mouse genome by TopHat2 (Kim, Pertea et al., 2013) , allowing 2 mismatches.
- Reads that aligned to more than one genomic location were discarded, and uniquely localized reads were used to calculate the number of reads and RPKM values (RPKM represents reads per kilobase and per million) for each gene.
- For each gene, the fold changes, p-values, and adjusted p-values (FDR) were also determined by the edgeR package.
LncRNA prediction pipeline
- The lncRNA prediction pipeline was implemented following the methods described by Liu et al.
- The detailed descriptions of the prediction pipeline and filtering thresholds are as follows: (1) First, using the aligned RNA-seq data (see above), transcripts were assembled by Cufflinks V2.2.1 (Trapnell et al., 2012) using default parameters.
- After the initial assembly, transcripts with FPKM greater than or equal to 0.1 were subjected to a series of filters.
- (3) To remove potential protein-coding transcripts, coding potential score (CPS) was evaluated using the Coding Potential Calculator (CPC) (Kong, Zhang et al., 2007) .
- A total of 1662 novel lncRNA transcripts were identified, originating from 1377 lncRNA loci.
Quantitative real-time PCR (qPCR)
- Total RNA was isolated from cells and tissues by using Trizol ® reagent .
- The genomic DNA was eliminated with TURBO DNA-free TM Kit (Ambion, AM1907) as the manufacturer's instructions.
- RNA quality was assessed by using NanoDrop 2000 (Thermo Scientific).
- The cDNAs were synthesized by ReverTra Ace qPCR RT Master Mix (Toyobo, FSQ-201) or First-Strand cDNA Synthesis Kit (Toyobo, FSK-101).
- QPCR was performed using SYBR Green Supermix (Bio-Rad).
Rapid amplification of cloned cDNA ends (RACE)
- Total RNA from N2a cells was isolated by using Trizol ® reagent and 5'-or 3'-RACE was performed with SMARTer ® RACE 5'/3' Kit (Takara, 634858) following the manufacturer's instructions.
- Primers used for 5'-or 3'-RACE was designed based on the known sequence information.
Fluorescent in situ hybridization
- The red fluorescence labeled probe (Ribo-lncRNA FISH Probe Mix) against EDAL lncRNA was designed by Ribobio Co (Guangzhou, China) and was detected by Fluorescent In Situ Hybridization Kit (Ribobio, R11060.1) according to the manufacturer's instructions.
- Briefly, N2a cells grown on cover slips in 24-well plates were fixed with 4％ (v/v) paraformaldehyde for 10 minutes (min) at room temperature then washed three times with cold PBS.
- And the cells were permeabilized in PBS containing 0.5％ Triton X-100 for 5 min in 4°C, then blocked in pre-hybridization buffer for 30 min at 37°C.
- After hybridization, cells were washed in the dark with washing buffer (4×SSC/2×SSC/1×SSC) then stained with DAPI for 10 min.
- Cells were again washed three times with PBS, and then imaged with an Olympus FV10 laser-scanning confocal microscope.
EDAL specific siRNA
- EDAL specific siRNA was designed and synthesized by Ribobio Co.
- The target sequence was 5'-GGTAGACACCCAGTGACAA-3', and siEDAL sequence was 5'-GGUAGACACCCAGUGACAA -3'.
Cell viability assay
- N2a cells were transfected with plasmids, siRNAs or treated with EZH2 specific inhibitor gsk126 (Apexbio, A3446) for indicated time.
- The viability of N2a cells was evaluated by Cell Titer 96 AQueous One Solution cell proliferation assay kits (Promega, G3582) according to the manufacturer's instruction.
Construction of the recombinant RABVs (rRABV)
- Mouse lncRNAs, reverse EDAL were amplified from the total RNA extracted from RABV-infected N2a cells using the ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201) with Phanta Max Super-Fidelity DNA polymerase (Vazyme, P505-d1).
- The primer sets used were designed by Primer 6 (PREMIER Biosoft Biolabs) .
- PCR products were digested with BsiWI and NheI (New England Biolabs) then ligated into the genome of recombinant RABV strain B2c (rB2c) digest used the same enzymes as previously described (Tian et al., 2016) .
Rescue of rRABVs
- Recombinant RABVs were rescued as reported previously (Tian et al., 2016) .
- Four days post transfection, supernatants was harvested and examined for the presence of rescued viruses using FITC-conjugated anti-RABV N antibodies (Fujirebio Diagnostics, Malvern, PA).
- To determine rRABV and VSV titers, BSR cells were infected with serial dilutions of the viruses.
- After 1 h incubation in 37°C, the cell supernatant was discarded and washed once with PBS, and then overlaid with DMEM containing 1% low melting point agarose (VWR, 2787C340).
- Then the fluorescent foci were counted under a fluorescence microscope.
- For SFV and HSV-1 titration, Vero cells were seeded in 12-well plates and infected with serial dilutions of the viruses.
- After staining for 4 h, the plates were washed with water, and the plaques were counted.
- Eight-week-old female C57BL/6 mice were randomly divided into indicated groups and infected intranasally with rRABV, rRABV-EDAL, rRABV-revEDAL (100 FFU) or mock infected with DMEM in a volume of 20 µl.
- When moribund, the mice were euthanized with CO 2 , and then the brains were collected for qPCR or immunohistochemistry analysis.
- Groups of female C57BL/6 mice were infected intranasally with rRABV or rRABV-EDAL.
- At indicated times post infection (pi), mouse brains were harvested and fixed in 4% paraformaldehyde for 2 days at 4°C.
- For immunohistochemistry (IHC), the sections were deparaffinized and rehydrated in xylene and ethanol.
- After washing, sections were incubated with diaminobenzidine (ServiceBio, G1211) for color development then photographed and analyzed using an XSP-C204 microscope (CIC).
- N2a cells were lysed in RIPA buffer (Beyotime, P0013B) supplemented with 1x protease inhibitor cocktail .
- Total cell lysates were separated on 8-12% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad).
- Membranes were blocked with TBST with 5% (w/v) non-fat dry milk for 4 h, and probed with primary antibodies which were diluted with TBST and 5% (w/v) non-fat dry milk overnight in 4°C.
- Images were captured with an Amersham Imager 600 (GE Healthcare) imaging system.
EDAL-EZH2 interaction 3D structure modeling
- Murine EZH2 3D structure was predicted with SWISS-MODEL (https://swissmodel.expasy.org/interactive) based on human EZH2 3D structure (PDB code: 5HYN).
- Then amino acid sequence comparison was conducted between human EZH2 and Murine EZH2, and 98.24% similarity was calculated by Clustal2.1 (a multiple sequence alignment software, https://www.ebi.ac.uk/Tools/msa/muscle/).
- EDAL-FD 3D structure model was predicted with RNAComposer (A automated RNA structure 3D modeling server, http://rnacomposer.ibch.poznan.pl/).
- In order to predict the interaction between EDAL functional domain (98-153 nt) and Murine EZH2, the template-based docking method PRIME (Zheng, Kundrotas et al., 2016 ) (If a template can be found, it is often more accurate than the free docking method) was used to dock the EDAL and EZH2 monomer structures at first.
- Two atoms between EZH2 and EDAL with distance less than 5 angstroms in the predicted complex structure are considered to have interactions.
RNA pull-down assay
- The synthesized RNA was treated with Rnase-free DNase I (Thermo, EN0521) and then purified with MicroElute RNA Clean-Up Kit (OMEGA, R6247-01).
- The RNA was heated to 95°C for 2 min, put on ice for 5 min and then put it at room temperature for 20 min to form secondary structure.
- The RNA was then added to the lysed cell containing overexpressed EZH2-1-337-flag and incubated for 2 h at 4°C.
- After being washed with wash buffer for three times, the samples were then analyzed by Western blotting.
O-GlcNAcylation labeling and detection
- The plasmid pCAGGS-EZH2-S73/S75/S725A-flag was co-tranfected with Chromatin was eluted from the beads by two washes with 100 µl elution buffer (100 mM NaHCO 3 , 1% SDS), the Na + concentration was adjusted to 300 mM with 5 M NaCl and the crosslinks were reversed by overnight incubation in a 65°C water-bath.
- DNA was purified by phenol extraction and ethanol precipitation.
- For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions (ThruPLEX DNA-seq 48S Kit, R400427) and analyzed using an Illumina NextSeq-500 system for 150 nt pair-end sequencing (ABlife Inc., Wuhan, China).
ChIP-seq data analysis
- Adaptors and low quality bases were trimmed from raw sequencing reads using Cutadapt (Martin, 2011) .
- Reads were aligned to the mouse-GRCm38 genome using Bowtie2 (Langmead & Salzberg, 2012) .
- To evaluate the quality of ChIP-seq data, the authors performed a cross-correlation analysis, as well as FRiP and IDR analyses for the ChIP-seq data, according to the ChIP-seq guidelines provided by the ENCODE and modENCODE consortia (Kheradpour & Kellis, 2012) .
- All peaks from each sample were clustered by BEDTools (Quinlan & Hall, 2010) .
- To associate peaks with genes, the authors set 10000 bp as the upstream limit for the distance from the peak maximum to the TSS (transcript start site), and 3000 bp as the downstream limit for distance from the peak maximum to the TSS.
- Formaldehyde crosslinking of N2a cells, chromatin sonication and immunoprecipitation were performed following the same procedures as the ChIP-seq section described above.
- Real-time PCR was then performed using a QuantStudio 6 Flex System (ABI) according to the manufacturer's standard protocol.
- Input was used to normalize the amount of each sample as an internal control.
- Assays were repeated at least three times and expressed as Ct values.
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"A novel antiviral lncRNA EDAL shiel..." refers methods in this paper
...Reads were aligned to the mouse-GRCm38 genome using Bowtie2 (Langmead & Salzberg, 2012)....
"A novel antiviral lncRNA EDAL shiel..." refers methods in this paper
...ChIP-seq data analysis Adaptors and low quality bases were trimmed from raw sequencing reads using Cutadapt (Martin, 2011)....
...Adaptors and low quality bases were trimmed from raw sequencing reads 936 using Cutadapt (Martin, 2011) ....
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