an ma immunoprecipitation assay showed that 16. 5 % u6 snrna is ma modified in a431 cells ( fig. 4l ), similar to previous findings in mouse embryonic fibroblasts. additionally, pull - down assays showed that the u6 36 - 60 sequence, where the a43 ma motif is located, preferentially binds with tlr3 in hela cells ( fig. 4m, supplementary fig. s13b, c ). when recombinant tlr3 was incubated with u6 or ma u6 in the presence of recombinant ythdf2, we found that u6 exhibited preferred binding with tlr3 ( fig. 4n ). in comparison, ma u6 preferentially bound with ythdf2, but not tlr3 ( fig. 4n ). in wild - type ( wt ) hela cells, pull - down assays showed that u6 binds with tlr3 but not ythdf2, while ma u6 binds with ythdf2, not tlr3 ( fig. 4o ), confirming the findings from in vitro binding assays ( fig. 4n ). however, in ythdf2 knockout cells, both u6 and ma u6 bound with tlr3 with ma u6 showing more prominent binding with tlr3 than u6 ( fig. 4o ), suggesting that tlr3 prefers to bind with ma u6 in vivo, distinc t from the findings from in vitro binding assays ( fig. 4k ), possibly due to structural differences between endosomal tlr3 in vivo and recombinant tlr3 in vitro. together, these findings demonstrate that ythdf2 specifically inhibits tlr3 binding to ma u6. tlr3 recognizes its ligand, dsrna, through its n - terminal ectodomain, which contains 23 leucine - rich repeats ( lrrs ). mutational analysis has shown that the lrr20 motif of tlr3 binds to poly i: c, a synthetic analog of dsrna and known tlr3 ligand. to determine the specific lrr motif of tlr3 that binds to u6, we generated various lrr deletion mutants of motifs ( δlrr16 - 19, δlrr20, and δlrr21 ) that are in the c - terminus of tlr3 around the poly i: c - binding lrr20 motif to determine which mutants show loss of binding to u6 and activation by u6. a rip assay showed that deletion of lrr21 leads to loss of binding to u6, while deletion of lrr20 had no effect in hela cells with ythdf2 knockout and tlr3 knockdown ( fig. 4p ). intriguingly, deletion of lrr16 - 19 enhanced binding of tlr3 to u6 ( fig. 4p ), suggesting that these motifs suppress the tlr3 - u6 interaction, warranting future investigation to determine the underlying mechanism for such suppression. further analysis showed that deletion of the lrr21 motif, but not the lrr20 motif, resulted in loss of induction of il - 6 by u6 in hela cells with ythdf2 knockout and tlr3 knockdown ( fig. 4q ). in comparison, deletion of the lrr20 motif, but not the lrr21 or lrr16 - 19 motifs, led to loss of induction of il - 6 expression by poly i: c in hela cells with ythdf2 knockout and tlr3 knockdown ( fig. 4r ), consistent with previous studies. furthermore, a pull - down assay confirmed that lrr20 is required for binding to poly i: c in hela cells with ythdf2 knockout and tlr3 knockdown ( fig. 4s ). together, these results demonstrate that the ma motif sequence of u6 snrna interacts with and activates tlr3 via the lrr21 motif. tlr3 is an rna sensor localized in endosomes. to determine the mechanism by which ythdf2 regulates tlr3 activation, we assessed whether u6 and ythdf2 also localize in the cytoplasm, particularly in endosomes, and whether ythdf2, u6, and tlr3 interact. first, we found that both ythdf2 knockdown and uvb irradiation increased the cytoplasmic u6 level in hela, hacat, and a431 cells ( fig. 5a and supplementary fig. s14a – e ). second, uvb irradiation increased both total level and endosomal proportion of u6 snrna in hela cells ( fig. 5b ). third, we performed rna fluorescence in situ hybri diz ation ( fish ) of u6 in combination with immunoflu orescence analysis of the endosome marker rab7 to confirm u6 localization in endosomes. while u6 snrna was mainly detected in the nucleus, we observed that uvb increases u6 snrna levels in the cytoplasm and u6 colocalization with the late endosome marker rab7 in hacat cells, hela cells, and mouse skin ( fig. 5c, supplementary fig. s14f – s14g ), supporting u6 localization in endosomes. ythdf2 inhibition in hacat cells, mouse skin, or a431 cells increased u6 snrna levels in the nucleus and cytoplasm and increased u6 colocalization with rab7 ( fig. 5c, supplementary fig. s14f – s14i ), while u6 knockdown decreased them in a431 and hacat cells ( supplementary fig. s14h – s14i ). these orthogonal validations further suggest a role for endosomal u6 snrna in the inflammatory response. next, we demonstrated ythdf2 colocalization with endosomes using multiple approaches, including endosome fractionation in a431 cells and endo - ip in 293 t cells, which were selected for their high plasmid transfection and protein expression ( fig. 5d, e ). endo - ip enabled rapid isolation of early / sorting endosomes through affinity capture of the early endosome - associated protein eea1. further, confocal imaging analysis in hela cells confirmed that ythdf2 localization in endosomes, as shown by the colocalization of ythdf2 with the late endosome marker rab7 ( fig. 5f ). fig. 5both u6 and ythdf2 are localized in endosomes. a qpcr of nuclear ( nuc ) and cytoplasmic ( cyto ) fractions in hela cells with or without uvb irradiation ( 30 mj / cm², 6 h ). b. qpcr in hela cells with or without uvb irradiation ( 30 mj / cm², 6 h ). c confocal images of hacat cells with or without ythdf2 knockdown, 6 h after sham or uvb irradiation ( 20 mj / cm² ). d immunoblot in a431 cells. e endo - ip of endosomal fractions in 293 t cells. f confocal images of hela cells; arrows indicate colocalization of ythdf2 and rab7. g qpcr in endosomes of hela cells with or without dynasore. h immunoblot in a431 cells with or without dynasore. i qpcr of mrna stability in a431 cells treated with or without dynasore. j qpcr in endosomes in a431 cells with or without sidt2 knockdown. k immunoblot of wcl and endosomes in a431 cells with or without sidt2 knockdown. l qpcr of mrna stability in a431 cells with or without sidt2 knockdown. m immunoblot of wcl and endosomes in a431 cells with or without u6 knockdown. n qpcr in endosomes of hela cells with or without mettl16 knockdown. o qpcr in endosomes of a431 cells with or without ythdf2 knockdown. p schematic of human ythdf2 and truncations ( n - or c - terminal ). q immunoblot of wcl and endosomes in hela cells transfected with ev, df2 ( wt ), df2 ( n ) or df2 ( c ). unmarked bands > 25 kda are nonspecific. qpcr of mrnas in hela cells transfected with trna or u6 and treated with vehicle or dynasore ( r ), and in a431 cells with or without ythdf2 knockdown treated with vehicle or dynasore ( s ). housekeeping genes: 18s rrna ( b, i, l, n, o ) and gapdh ( r – s ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are shown as mean ± sd ( n = 3 for a – b, g, i, j, l, n, o, r; n = 6 for s ). all experiments used biologically independent samples. a qpcr of nuclear ( nuc ) and cytoplasmic ( cyto ) fractions in hela cells with or without uvb irradiation ( 30 mj / cm², 6 h ). b. qpcr in hela cells with or without uvb irradiation ( 30 mj / cm², 6 h ). c confocal images of hacat cells with or without ythdf2 knockdown, 6 h after sham or uvb irradiation ( 20 mj / cm² ). d immunoblot in a431 cells. e endo - ip of endosomal fractions in 293 t cells. f confocal images of hela cells; arrows indicate colocalization of ythdf2 and rab7. g qpcr in endosomes of hela cells with or without dynasore. h immunoblot in a431 cells with or without dynasore. i qpcr of mrna stability in a431 cells treated with or without dynasore. j qpcr in endosomes in a431 cells with or without sidt2 knockdown. k immunoblot of wcl and endosomes in a431 cells with or without sidt2 knockdown. l qpcr of mrna stability in a431 cells with or without sidt2 knockdown. m immunoblot of wcl and endosomes in a431 cells with or without u6 knockdown. n qpcr in endosomes of hela cells with or without mettl16 knockdown. o qpcr in endosomes of a431 cells with or without ythdf2 knockdown.