epidermis is minced and suspended in 6 ml of growth medium ( 10 % fbs / dmem, hacat medium ) in a 15 ml tube, following by pipetting up and down to release keratinocytes from the epidermis. the suspension was then filtered through a sterile, 70 μm nylon filter ( thermo fisher, # 22363548 ) into a fresh 50 ml tube to remove cornified sheets. the samples were then rinsed with 5 ml fresh growth medium, filtered through the same filter into the same 50 ml tube, and centrifuged at 160 g for 5 min at room temperature. cells were resuspended in kgm® gold keratinocyte growth medium ( lonza, 00192060 ), counted, and seeded at 0. 5 – 1 × 10 cells in 35 mm dishes. 48 – 72 h after plating, medium was removed and replaced with fresh culture medium for uvb irradiation experiments. wt and df2 chet cells were irradiated with uvb ( 30 mj / cm ) and then samples were collected at 6 h post - sham or - uvb irradiation for ma - ip qpcr analysis. plko. 1 plasmids of shnc, shythdf2, shmettl16, shatg5, shatg7, shp62, and shtlr3 _ gfp were obtained from sigma. plenti plasmids of overexpression of ythdf2 were purchased from applied biological materials inc. ( abm ). lentivirus was produced by co - transfection into hek - 293t ( human embryonic kidney ) cells with lentiviral constructs together with the pcmvdelta8. 2 packaging plasmid and pvsv - g envelope plasmid using genjet™ plus dna in vitro transfection reagent ( signagen, ijamsville, md ). virus - containing supernatants were collected 24 – 48 h after transfection and used to infect recipients. target cells were infected in the presence of polybrene ( 8 μg / ml ) ( sigma - aldrich, st. louis, mo ) and selected with puromycin ( santa cruz biotechnology, santa cruz, ca ) at 1 μg / ml for 6 days. ythdf2 ( wt, n, and c ) plasmids were kindly provided by dr. chuan he. the pcmv3 - c - flag - tlr3 construct was obtained from sino biological ( catalog # hg10190 - cf ). primers for this study were designed individually based on the specific deletions. the ythdf2 mutants ( s39a and s39d ) and tlr3 deletion mutants were generated using a quikchange site - directed mutagenesis kit ( catalog # 200518 ), according to the manufacturer ’ s instructions. sequences of the primers for cloning of tlr3 or ythdf2 mutants are shown in supplementary table s2. newly generated plasmids are available upon request from the corresponding authors. cells were transfected with sirna targeting negative control ( sinc ), an individual gene, or the combination of sidt2, cnot1, tlr3, tlr7, tlr8, mda5, rig - 1, ythdf2, u6, mettl16, tlr9, or mypt1 ( dharmacon, lafayette, co ). siu6, u6 snrna, and ma - modified u6 snrna ( ma43 ) were purchased from integrated dna technologies ( idt, coralville, ia ). trna ( roche, trnabre - ro sku10109517001 ) was used as the control for structured rna. rnas were transfected using pepmute™ sirna transfection reagent ( signagen, ijamsville, md ), according to the manufacturer ’ s instructions. quantitative real - time pcr assays ( qpcr ) were performed using a cfx connect real - time system ( bio - rad, hercules, ca ) with bio - rad iq sybr green supermix ( bio - rad, hercules, ca ). cdnas were prepared by using iscript reverse transcription supermix for rt - qpcr ( bio - rad, hercules, ca ) for mrnas, or mirna all - in - one cdna synthesis kit ( applied biological materials inc., bc, canada ) for non - coding rnas. the threshold cycle number ( cq ) for each sample was determined in triplic ate or quadruplicate. the cq for values for ythdf2, tnf - α, il - 6, cox - 2, il - 1β, gm - csf, il - 8, mmp - 9, vegf, il - 16, il - 1α, fos, jun, sox4, sox9, cd74, u6 snrna, atg5, atg7, p62, sidt2, cnot1, mettl16, and tlr3 were normalized against gapdh, actb, 18s rrna, or hprt1, which are specifically indicated in figure legends. sequences of the primers for qpcr are shown in supplementary table s3. rna stability was assessed using the transcriptional inhibitor actinomycin d ( 2 μm ). cells were harvested at different time points after treatment with actinomycin d. total rna was isolated using an rneasy plus mini kit ( qiagen, hilden, germany ) or trizol, following the manufacturer ’ s protocol. the hprt1 housekeeping gene was used as a loading control, as hprt1 mrna does not contain ma modifications, is not bound by ythdsf2, and is rarely affected by actinomycin d treatment. immunoflu orescence analysis was performed as described previously in refs.. for immunoflu orescence analysis of cells, cells were fixed with 4 % paraformaldehyde / pbs for 30 min and permeabilized with 0. 5 % trit on x - 100 ( sigma - aldrich, t8787 ) / pbs for 20 min. cells were then washed with pbs with 0. 05 % trit on x - 100. the samples were then incubated with a blocking solution of 3 % albumin from chicken egg white ( sigma - aldrich, a5503 ) in pbs for 1 h. for immunoflu orescence analysis of tissues, formalin - fixed, paraffin - embedded tissue sections were pre - treated by antigen retrieval and incubated with a blocking solution of 3 % albumin from chicken egg white ( sigma - aldrich, a5503 ) in pbs for 1 h. after removal of the blocking solution, samples were incubated with primary antibod ies at 4 °c overnight, and then the samples were washed three times with 0. 1 % trit on x - 100 / pbs for 10 min at room temperature ( rt ). samples were then incubated with fluorochrome - conjugated secondary antibod ies for 1 h at rt and washed three times with 0. 1 % trit on x - 100 / pbs for 10 min at room temperature. cells were then fixed in prolong gold antifade with dapi ( invitrogen, p36931 ), fluoromount ™ aqueous mounting medium ( sigma - aldrich, f4680 ), or anti - fade fluorescence mounting medium ( abcam, ab104135 ), and were then observed under a fluorescence microscope ( olympus ix71, olympus life science, japan ). for confocal microscopy, cells were imaged with the sora marianas spinning disk confocal microscope ( intelligent imaging innovations, 3i ). hematoxylin and eosin ( he ) staining of tissues and immunoh istochemical analysis of ythdf2 ( proteintech, # 247441 - 1 - ap ) were performed by the human tissue resource center ( htrc ) core facility at the university of chicago. epidermal thickness was measured on he stained sections, as described previously. two investigators independently scored the staining intensity blindly, as 3 ( strong ), 2 ( medium ), 1 ( weak ), and 0 ( negative ). imagej ( nih ) was also used for analyzing cells and tissue. information for the antibod ies for the if staining are shown in supplementary table s4. cells were fixed with 4 % paraformaldehyde ( pfa ) for 30 minutes at room temperature ( rt ). permeabilization was performed using 0. 5 % trit on x - 100 in pbs supplemented with rnase inhibitor ( 40 u / ml ) for 30 min at 4 °c. samples were then washed with buffer a ( 20 % buffer a ( cat: smf - wa1 - 60, biosearch ) + 10 % formamide ) for 5 min at rt. hybri diz ation was carried out overnight at 37 °c using a 150 nm dig - labeled u6 probe ( u6 probe: 5 ’ - cacgaatttgcgtgtcatcctt - 3 ’, idt ) in hybri diz ation solution ( cat: smf - hb1 - 10, biosearch ) containing 10 % formamide and the rnase inhibitor ( 40 u / ml ). after hybri diz ation, samples were thoroughly washed with pbs to prepare for immunoflu orescence staining. blocking was performed using goat serum for 30 minutes at rt. the anti - rab7 ( ab137029 ) primary antibod y was diluted in blocking buffer and incubated with the samples for 2 h at rt. after washing with pbs, dig was detected using the anti - dig alexa fluor 488 antibod y ( 1: 50 ), and rab7 was visualized using an alexa fluor 594 - conjugated secondary antibod y by incubation for 1 h at rt. finally, samples were washed with pbs, counterstained with dapi, and then mounted for imaging. for mouse tissue, formalin - fixed, paraffin - embedded ( ffpe ) tissue sections were first subjected to antigen retrieval.