notably, the combination of uvb irradiation and ythdf2 knockdown had a similar effect on u6 stability as either uvb irradiation or ythdf2 knockdown alone ( fig. 2o ), suggesting that uvb irradiation increases u6 stability through inhibiting ythdf2. notably, the half - life of u6 in control cells is approximately 4 h, substantially shorter than the ~ 24 h half - life reported previously. it is possible that actinomycin d ( actd ) is not specific for rna pol iii, as it inhibits all three eukaryotic polymerases ( i, ii, and iii ) by binding to dna. therefore, the shorter half - life observed in our data suggests that the effect of actd on other rna polymerases might influence u6 levels and stability, as other rna polymerases may modulate upstream regulators involved in u6 transcription, maturation, and / or stability. to determine whether binding of ythdf2 to u6 snrna is dependent on ma methylation, we assessed the effect of knocking down the known ma methyltransferase mettl16 on u6 snrna levels. mettl16 knockdown increased u6 levels in hacat cells ( fig. 2p, supplementary fig. s10d ), while it decreased both baseline and uvb - induced ma enrichment of u6 in hacat cells ( fig. 2q ). furthermore, mettl16 knockdown reduced ythdf2 binding to u6 under baseline condition and uvb stress in hacat cells ( fig. 2r ). these results demonstrate that ythdf2 binds to ma - modified u6 snrna to mediate u6 decay. to determine the role of u6 in ythdf2 ’ s function, we assessed whether u6 knockdown rescues the effect of ythdf2 knockdown on inflammatory gene expression. indeed, u6 ( rnu6 - 1 ) knockdown drastically inhibited the effect of ythdf2 knockdown on the expression of cytokine genes in hacat cells ( fig. 3a - c and supplementary fig. s11a ). prior literature has shown that uvb exposure directly damages the snrna u1 to promote inflammation. to determine whether the effect of u6 on cytokine expression occurs through direct uvb damage to u6 snrna, we assessed the effect of uvb - irradiated synthetic u6 snrna. trna was used as a negative control rna. however, we found that in vitro uvb - irradiated u6 snrna had no effect on il - 6 expression when transfected into hacat cells ( fig. 3d, e ). next, we assessed whether the effect of ythdf2 inhibition is mediated by the direct damage on u6 snrna by uvb irradiation. we examined the difference in inflammatory response in hela cells, a cell line with high transfection efficiency, with or without ythdf2 inhibition transfected with sham - or uvb - irradiated synthetic non - ma - modified ( u6 ) - and ma - modified u6 ( ma u6 ) in vitro. consistently, in vitro uvb irradiation of u6 or ma u6 molecules had no effect on il - 6 expression compared to sham ( fig. 3f ). in wt hela cells, while both u6 and ma u6 induced il - 6 expression, an inflammatory gene robustly upregulated by uvb irradiation and / or ythdf2 inhibition ( figs. 2i and 3b ), u6 lead to a higher induction than ma u6 ( fig. 3f ). we also observed that ythdf2 deletion in hela cells increased il - 6 expression and abolished the difference between u6 and ma u6 ( fig. 3f ). fig. 3ythdf2 controls inflammatory gene expression through u6 ma methylation. a – c qpcr of tnf - α, il - 6 and il - 1β mrnas in hacat cells with or without ythdf2 or u6 knockdown, with or without uvb irradiation. siu6, small interfering rna targeting u6 snrna. qpcr of u6 snrna ( d ) and il - 6 mrna ( e ) in hacat cells 30 h after transfection with sham - or uvb - irradiated purified trna or synthetic u6 snrna. f qpcr of il - 6 mrna in wild - type ( wt ) or ythdf2 knockout ( ko ) hela cells 30 h after transfection with sham - or uvb - irradiated purified trna, synthetic u6 snrna, or synthetic ma - modified u6. g, h qpcr of tnf - α and il - 6 mrnas in a431 cells transduced with shnc ( short hairpin rna negative control ), shmettl16 - 1 or shmettl16 - 2. i – l qpcr of tnf - α, il - 6, mettl16 and u6 snrna in a431 cells with or without simettl16 or siu6. m immunoblot of cox - 2 in hacat cells with or without mettl16 knockdown, with or without uvb irradiation. n – q qpcr of tnf - α, cox - 2, il - 1β and il - 6 mrnas in hacat cells with or without ythdf2 or mettl16 knockdown, with or without uvb irradiation. r – w qpcr of tnf - α, il - 1β and il - 6 mrnas in wt and ythdf2 knockout ( df2 ko ) hela cells with or without mettl16 knockdown, with or without uvb irradiation. sim16, small interfering rna targeting mettl16. housekeeping genes used were hprt1 ( a – c, n – w ), 18s rrna ( d, k ) and gapdh ( e – j, l ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are presented as mean ± se ( n = 4 for a – c, n – w ) or mean ± sd ( n = 3 for d – l ). all experiments were conducted using biologically independent samples. a – c qpcr of tnf - α, il - 6 and il - 1β mrnas in hacat cells with or without ythdf2 or u6 knockdown, with or without uvb irradiation. siu6, small interfering rna targeting u6 snrna. qpcr of u6 snrna ( d ) and il - 6 mrna ( e ) in hacat cells 30 h after transfection with sham - or uvb - irradiated purified trna or synthetic u6 snrna. f qpcr of il - 6 mrna in wild - type ( wt ) or ythdf2 knockout ( ko ) hela cells 30 h after transfection with sham - or uvb - irradiated purified trna, synthetic u6 snrna, or synthetic ma - modified u6. g, h qpcr of tnf - α and il - 6 mrnas in a431 cells transduced with shnc ( short hairpin rna negative control ), shmettl16 - 1 or shmettl16 - 2. i – l qpcr of tnf - α, il - 6, mettl16 and u6 snrna in a431 cells with or without simettl16 or siu6. m immunoblot of cox - 2 in hacat cells with or without mettl16 knockdown, with or without uvb irradiation. n – q qpcr of tnf - α, cox - 2, il - 1β and il - 6 mrnas in hacat cells with or without ythdf2 or mettl16 knockdown, with or without uvb irradiation. r – w qpcr of tnf - α, il - 1β and il - 6 mrnas in wt and ythdf2 knockout ( df2 ko ) hela cells with or without mettl16 knockdown, with or without uvb irradiation. sim16, small interfering rna targeting mettl16. housekeeping genes used were hprt1 ( a – c, n – w ), 18s rrna ( d, k ) and gapdh ( e – j, l ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are presented as mean ± se ( n = 4 for a – c, n – w ) or mean ± sd ( n = 3 for d – l ). all experiments were conducted using biologically independent samples. to explore the role of ma methylation of u6 in ythdf2 ’ s function in skin cancer, we next assessed the consequence of mettl16 inhibition on the expression of cytokines in the human a431 skin carcinoma cells. mettl16 knockdown enhanced the expression of il - 6 and tnf - α, two inflammatory genes robustly upregulated by uvb irradiation and / or ythdf2 inhibition ( figs. 2i and 3b ), under baseline conditions in a431 cells ( fig. 3g – h, supplementary fig. s11b ). the effect of mettl16 knockdown was reduced by u6 knockdown in a431 cells ( fig. 3i - l ), indicating that mettl16 suppresses cytokine expression through regulating u6. it is noteworthy that as compared with hacat and hela cells, a431 cells showed reduced basal mettl16 expression, which may render a431 cells sensitive to mettl16 knockdown - induced cytokine expression ( supplementary fig. s11c ). mettl16 knockdown increased uvb - induced cox - 2 expression in hacat cells ( fig. 3m ). however, dual knockdown of both mettl16 and ythdf2 mimicked the effect of ythdf2 knockdown alone in hacat cells ( fig. 3n – q ). similarly, ythdf2 knockout abolished the effect of mettl16 knockdown in hela cells ( fig. 3r – w, supplementary fig. s11d, e ). finally, to determine whether mettl16 inhibition affects dsrna levels, as previously observed following mettl3 inhibition, and to assess whether u6 snrna contributes significantly to the dsrna pool, we compared dsrna levels in a431 cells under control, mettl16 knockdown, and / or u6 knockdown conditions using immunoflu orescence analysis with the anti - j2 antibod y.