however, mettl16 knockdown, u6 knockdown, or the combined knockdown had no effect on dsrna levels in a431 cells ( fig. s11f - s11g ), suggesting that mettl16 regulates inflammatory response via a distinc t mechanism from mettl3 and that the effect of u6 depletion on inflammatory gene expression is not mediated by secondary dsrna fragment accumulation. together, these results demonstrate that ythdf2 suppresses inflammatory gene expression, at least in part, by controlling the metabolism of ma methylated u6. our rna - seq data analysis showed that genes in the tlr3 cascade are upregulated by ythdf2 depletion ( fig. 1a ), suggesting that tlr3 signaling may contribute to the inflammatory response induced by ythdf2 loss. tlr3 is a pattern recognition receptor best known for detecting dsrna and structured rnas resembling dsrna to activate immune responses. indeed, a small - scale sirna screening for tlr3, tlr7, tlr8, mda5, and rig - i, which are all known sensors for rna, showed that knockdown of tlr3 at least partially reverses the effect of ythdf2 knockdown in hacat cells ( fig. 4a, b ). in addition, tlr7 / 8 knockdown also partially reversed the effect of ythdf2 knockdown in hacat cells ( fig. 4a, b ), suggesting an important role of these receptors in ythdf2 ’ s function and warrants further investigation. moreover, inhibition of tlr3 at least partially reversed the effect of either ythdf2 knockdown or mettl16 knockdown both under uvb stress in hacat cells and under baseline condition in a431 cells ( fig. 4c – g and supplementary fig. s12a – j ). together, these data demonstrate that the effect of ythdf2 on inflammation is mediated, at least in part, through tlr3. fig. 4ythdf2 interacts with ma u6 and thus inhibits ma u6 binding to tlr3. a, b qpcr of tnf - α and cox - 2 mrnas in shnc and shdf2 hacat cells transfected with sinc, sitlr3, sitlr7, sitlr8, simda5 or sirig - i ( sirnas targeting tlr3, tlr7, tlr8, mda5 and rig - i, respectively ). c, d qpcr of tnf - α and il - 6 mrnas in shnc and shdf2 hacat cells transfected with sinc or sitlr3, 6 h after sham or uvb irradiation. e - g. qpcr of il - 8, il - 6 and il - 1β mrnas in shnc and shdf2 a431 cells. h – j qpcr of il - 6, u6 snrna and tlr3 in a431 cells with or without u6 and / or tlr3 knockdown. k pull - down showing interaction between biotin - labelled u6 or ma - u6 and recombinant tlr3. l qpcr of u6 snrna in ma - ip or flow - through fractions in a431 cells, showing the proportion of ma - modified u6. m pull - down of biotin - labelled trna, u6 or truncated u6 oligos ( 1 – 26, 36 – 60, 54 – 91, 63 – 85 ) with tlr3 in lysates of hela cells overexpressing tlr3 – flag. n pull - down of biotin - labelled trna, u6 or ma - u6 with recombinant ythdf2 and tlr3. o. pull - down of biotin - labelled trna, u6 or ma - u6 with ythdf2 or tlr3 in lysates from wt and df2 ko hela cells overexpressing tlr3 – flag. p rip of u6 interaction with tlr3 ( wt and mutants ) in df2 ko hela cells. q – r qpcr of il - 6 mrna in df2 ko hela cells with tlr3 knockdown and overexpression of wt or mutant tlr3, following treatment with synthetic u6 or poly i: c ( polyinosinic – polycytidylic acid, synthetic dsrna analog ). s pull - down of biotin - labelled poly i: c with wt or mutant tlr3 in df2 ko hela cells with tlr3 knockdown and overexpression of wt or mutant tlr3 – flag. housekeeping genes: hprt1 ( a – g, q – r ), gapdh ( h, j ) and 18s rrna ( i ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are shown as mean ± se ( n = 4 for a – g ) or mean ± sd ( n = 3 for h – j, l, p – r ). all experiments used biologically independent samples. a, b qpcr of tnf - α and cox - 2 mrnas in shnc and shdf2 hacat cells transfected with sinc, sitlr3, sitlr7, sitlr8, simda5 or sirig - i ( sirnas targeting tlr3, tlr7, tlr8, mda5 and rig - i, respectively ). c, d qpcr of tnf - α and il - 6 mrnas in shnc and shdf2 hacat cells transfected with sinc or sitlr3, 6 h after sham or uvb irradiation. e - g. qpcr of il - 8, il - 6 and il - 1β mrnas in shnc and shdf2 a431 cells. h – j qpcr of il - 6, u6 snrna and tlr3 in a431 cells with or without u6 and / or tlr3 knockdown. k pull - down showing interaction between biotin - labelled u6 or ma - u6 and recombinant tlr3. l qpcr of u6 snrna in ma - ip or flow - through fractions in a431 cells, showing the proportion of ma - modified u6. m pull - down of biotin - labelled trna, u6 or truncated u6 oligos ( 1 – 26, 36 – 60, 54 – 91, 63 – 85 ) with tlr3 in lysates of hela cells overexpressing tlr3 – flag. n pull - down of biotin - labelled trna, u6 or ma - u6 with recombinant ythdf2 and tlr3. o. pull - down of biotin - labelled trna, u6 or ma - u6 with ythdf2 or tlr3 in lysates from wt and df2 ko hela cells overexpressing tlr3 – flag. p rip of u6 interaction with tlr3 ( wt and mutants ) in df2 ko hela cells. q – r qpcr of il - 6 mrna in df2 ko hela cells with tlr3 knockdown and overexpression of wt or mutant tlr3, following treatment with synthetic u6 or poly i: c ( polyinosinic – polycytidylic acid, synthetic dsrna analog ). s pull - down of biotin - labelled poly i: c with wt or mutant tlr3 in df2 ko hela cells with tlr3 knockdown and overexpression of wt or mutant tlr3 – flag. housekeeping genes: hprt1 ( a – g, q – r ), gapdh ( h, j ) and 18s rrna ( i ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are shown as mean ± se ( n = 4 for a – g ) or mean ± sd ( n = 3 for h – j, l, p – r ). all experiments used biologically independent samples. to further determine whether the effect of ythdf2 inhibition on the inflammatory response is mediated through u6 / tlr3, we assessed the effect of u6 knockdown, tlr3 knockdown, and their combined depletion. we found that either u6 knockdown or tlr3 knockdown decreased il - 6 expression, and that combined depletion of tlr3 and u6 had a similar effect to single knockdown of either u6 ( rnu6 - 1 ) or tlr3 in a431 cells ( fig. 4h – j ), suggesting that the effect of u6 on inflammation is mediated through tlr3 signaling. in addition, u6 knockdown had no effect on the dsrna level in a431 cells ( fig. s11f ), suggesting that u6 depletion does not affect secondary dsrna fragment accumulation in regulating inflammatory response. given the well - established role of u6 snrna in regulation of splicing, we aimed to assess whether splicing inhibition affects the inflammatory response driven by ythdf2 loss through u6 upregulation. we found that the splicing inhibitor pla dien olide b ( plab ) decreased the expression of the proinflammatory cytokines in both control and ythdf2 - knockdown cells ( supplementary fig. s12k ), mimicking the effect of u6 knockdown ( fig. 3 ). in parallel, plab also decreased the u6 snrna level, which correlates with the decrease in cytokine expression caused by plab ( supplementary fig. s12k ). these findings raise the possibility that splicing inhibition by plab reduces global transcription or alters rna metabolism, leading to decreased expression of inflammatory genes and u6 snrna. to determine whether uvb - induced dna damage also contributes to the induction of inflammation, we assessed the effect of tlr9 knockdown or cgas inhibition. we found that either tlr9 knockdown or inhibition of cgas had no effect on uvb - induced expression of inflammatory genes ( fig. s12l – s12m ). these data suggest that uvb - induced inflammation is not mediated by uvb - induced dna damage. to determine the specific mechanism by which ythdf2 regulates u6 and tlr3 signaling, we performed several in vitro binding and pull - down assays. consistent with observations in mrna, we found that recombinant ythdf2 preferentially binds to ma u6 more than u6 in vitro ( supplementary fig. s13a ). in contrast, recombinant tlr3 bound to both u6 and ma u6 with a similar affinity in vitro ( fig. 4k ).