m6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response

Abstract Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6‐methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele‐specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803‐YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

For YTHDC1 C-term construct the YTH domain together with the C-terminal were cloned in a CMV driven vector using AscI and FseI restriction sites.The primers used for cloning are listed in Table S1.
For LOC339803 overexpression, 200 ng of plasmids per 100.000 cells were used.Cells were seeded and transfection was performed with X-TremeGENE HP DNA transfection reagent (Sigma-Aldrich, #6366546001) for 24 h.
For LOC339803 silencing in biopsies, viral particles were produced in HEK293FT cells transfected with 1 μg pLKO.1 shRNA plasmid, 750 ng psPAX2 packaging plasmid (Addgene, #12260) and 250 ng pMD2.G envelope plasmid (Addgene, #12259) using X-TremeGENE HP DNA transfection reagent (Sigma-Aldrich, #6366244001) in DMEM without antibiotics and cells were incubated o/n at 37ºC.Transfection media was replaced with fresh complete DMEM and viral particle containing media was harvested after 24 h and 48 h.Collected media was centrifuged and concentrated 10 times using Lenti-X concentrator (Takara, #631232).Viral particles were stored in aliquots at -80ºC until used.Human intestinal biopsy samples were infected with sh-LOC339803 or plKO.1 as negative control for 24h.S1.

RNA immunoprecipitation assay (RIP):
For RIP experiments, HCT-15 cells were lysed in RIP buffer (150 mM KCl, 25 mM Tris, 0.5 mM DTT, 0.5 % NP-40, PI), kept on ice for 15 minutes and homogenized using a syringe.Lysates were pre-cleared with protein A-Agarose beads (GE Healthcare, Chicago, USA) for 1 h in a wheel shaker at 4ºC.A-Agarose beads were blocked with 20 % BSA and mixed with pre-cleared lysates and 1 µg of anti-IgG antibody (negative control; Santa Cruz Biotechnologies, #sc-2025) or antibody of interest.After overnight incubation in a wheel shaker at 4ºC, beads were washed 3X with RIP buffer, 3X with low salt buffer (50 mM NaCl, 10 mM Tris-HCl, 0.1 % NP-40) and 3X with high salt buffer (500 mM NaCl, 10 mM Tris-HCl, 0.1 % NP-40).After the washes, 70 % of beads were resuspended in RNA extraction buffer and 30 % was used for WB.

Chromatin immunoprecipitation assay (ChIP):
For ChIP experiments, HCT-15 cells were crosslinked with formaldehyde and collected in PBS with a scratcher.Cell pellet was then resuspended in L1 buffer (50 mM Tris pH8, 2 mM EDTA, 0.1 % NP-40, 10 % glycerol) + PI and incubated in ice for 5 min.Supernatant was discarded and pellet resuspended in 300 uL L2 Buffer (50 mM Tris pH8, 0.1 % SDS, 5 mM EDTA) + PI to disrupt the chromatin using bioruptor sonicator.Centrifuged samples at maximum speed were used for immunoprecipitation.
ChIP dilution buffer (50 mM Tris pH8, 0.5 % NP-40, 0.2 M NaCl, 0.5 mM EDTA) was added up to 1 mL and in order to reduce non-specific background, the samples were pre-incubated with 60 uL of protein A-Agarose beads (GE Healthcare, Chicago, USA) + Salmon Sperm DNA (Invitrogen #15632-011) (1 ug of DNA/20 uL of protA) for 60 mins at 4°C shaking.The supernatant was collected and equal volumes were put into 2 tubes.
Cellular fractionation: For the quantification of RNA amounts in nuclear and cytoplasmic compartments, nuclei were isolated using C1 lysis buffer (1.28 M sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 4 % Triton X-100).The amounts of LOC339803-A/G, MALAT1 (nuclear control) and RPLP0 (cytoplasmic control) were measured by RT-qPCR and compared to the total amount of those RNAs in the whole cell lysate.
For the quantification of RNA amounts in chromatin and nucleoplasm compartments, HCT-15 cells were crosslinked using 16% formaldehyde.After crosslinking, cells were centrifuged and resuspended in NARA buffer (500 mM HEPES pH7.9, 1 M KCl, 500 mM EDTA, 0.05 % NP-40) for cytoplasm separation.Nuclei were resuspended in low-salt buffer (10 mM Tris HCl pH 7.4, 0.2 mM MgCl2, 1 % triton) and after centrifugation nucleoplasm was transferred to fresh tubes.Chromatin was resuspended in HCl 0.2 N centrifuged and neutralized with 1 M Tris-HCl pH8.Obtained lysates were decrosslink prior to RNA extraction.
For the quantification of protein amounts in nuclear and cytoplasmic compartments, cells were resuspended in NARA buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA) with PI and incubated in ice for 10 minutes.After adding NP-40 to final concentration 0.05 %, lysates were incubated 5 minutes in ice and centrifuged at 400 g for 2 minutes.The supernatant was the cytosolic fraction.Pellet was washed 3X with NARA buffer and resuspended in NARC buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA) + PI, shaken at 4ºC for 30 minutes and centrifuged at 16.000 g for 10 minutes.The supernatant was the nuclear extract.
DNase I Hypersensitivity Assay: 1x10 6 cells per condition were transfected with pCMV6 and both LOC339803 overexpression plasmids.Then cells were washed and collected with cold PBS.Cells were resuspended in C1 lysis buffer (1.28 M sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 4 % Triton X-100) and incubated in ice for 15min.Nuclei were pelleted, resuspended in Nuclei wash buffer (10mM Tris pH 7.4, 60mM KCl, 15mM NaCl, 5mM MgCl2, 30mM sucrose) and separated in two tubes.One of the tube was incubated with DNase I at 37ºC for 30min and the other was left untreated as a negative control.DNA was extracted and same amount of DNA was used to quantify by RT-QPCR.Used primers are listed in Table S1.Dotblot: 200 ng of RNA was crosslinked into a nitrocellulose membrane using UV and blocked using 5 % milk in 0.1 % Tween in PBS.Membrane was incubated overnight with a m 6 A antibody (1:200) (Abcam, Cambridge, UK, #ab151230) at 4ºC.After washing in 0.1 % PBST, membranes were incubated with a secondary HRP-conjugated anti-rabbit antibody (1:10000) (Santa Cruz Biotechnology, #sc-2357) and the membrane was developed using Clarity Max ECL Substrate (BioRad, #1705062).

m 6 A
KO cell generation using CRISPR Cas9: For m 6 A KO cell line generation, two sgRNAs flanking the m 6 A motif were designed and cloned in px458 GFP and px330 mCherry vectors.HCT-15 cells were transfected with 250 ng of each plasmid.HCT-15 cells were sorted by cell sorter BD FACSJazz (2B/4YG) 48 hours post-transfection for the generation of clonal cell lines.The sequences for the sgRNAs are shown in Table Bioinformatic packages: UCSC Multiz Alignments of 30 Vertebrates track was used in UCSC genome browser (http://genome.ucsc.edu)1 to study the evolutionary conservation of LOC339803 locus in Rhesus, mouse, dog, elephant, chicken, X_tropicalis and zebrafish species.WashU Epigenome Browser 2 provides visualization, integration and analysis tools for epigenomic datasets.WashU Epigenome Browser v46.2 was used to visualize RNAseq counts from Epigenome Roadmap project 3 of LOC339803 gene in different cells types.MeT-DB V2.0 m 6 A database 4 was used for assessing the existence of m 6 A peaks in LOC3399803.MeT-DB V2.0 records predicted transcriptome-wide m 6 A peaks and single-base m 6 A sites from a significantly expanded collection of Methylated RNA Immunoprecipitation Sequencing (MeRIP-Seq) samples.It provides a genome browser to help visualize the m 6 A sites from different studies.SRAMP (sequence-based N 6 -methyladenosine (m 6 A) modification site predictor) 5 was used to predict m 6 A modification sites on the allele specific RNA sequences of LOC339803 and for secondary structure prediction.GO MOLECULAR FUNCTION analysis 6-8 was used with LOC339803 bound nuclear proteins in HCT-15 intestinal cells from our RIP-MS and p-value results were illustrated using GraphPad Prism 8 (GraphPad Software).GENT2 (Gene Expression database of Normal and Tumor tissues 2) 9 is an updated version of GENT, a user friendly platform gathering gene expression from diverse normal and tumor tissues from public data sets.It was used to analyze the expression profile of LOC339803 and the proinflammatory cytokines in different intestinal tumors.GEPIA (Gene Expression Profiling Interactive Analysis) 10 is an interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and the GTEx projects.It was used to study the overall survival and expression profiles in GI cancers using survival analysis and boxplots functions.

Figure S1 .
Figure S1.The genotype of the intestinal inflammation associated SNP rs11498 affects m 6 A methylation levels and stability of LOC339803 lncRNA

Figure S2 .
Figure S2.YTHDC1 m 6 A reader interacts with LOC339803 influencing its cellular localization

Figure S4 .
Figure S4.LOC339803 expression is increased in inflammatory intestinal disorders and

Figure S5 .
Figure S5.Schematic representation of LOC339803 mechanism of action