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. to determine the mechanism by which u6 and ythdf2 translocate to endosomes, we assessed the role of dynamin, a key factor in the endocyt osis pathway that mediates biomol ecule trafficking. indeed, the dynamin inhibitor dynasore reduced the endosomal proportion of u6 and the endosomal ythdf2 level in hela cells and reduced u6 stability in a431 cells ( fig. 5g – i, supplementary fig. s14j ), indicating a critical role of endocyt osis in the localization of u6 and ythdf2 to endosomes. to further understand the molecular mechanism by which u6 and ythdf2 are trafficked to endosomes, we assessed the role of sidt2, a rna transporter localized in late endosomes and lyso somes. indeed, knockdown of sidt2 reduced the total and endosomal proportion of u6, endosomal ythdf2 levels, and u6 stability in a431 cells ( fig. 5j – l and supplementary fig. s14k – s14l ). these results mimic the effect of dynasore and indicate that sidt2 is required for the localization of u6 and ythdf2 to endosomes. to examine whether ythdf2 or u6 enters endosomes as a complex or individually, we assessed the effect of knockdown of u6, ythdf2, or mettl16 on endosomal localization. knockdown of u6 reduced the ythdf2 levels in endosomes in a431 cells ( fig. 5m and supplementary fig. s14m ), indicating that u6 is required for ythdf2 localization to endosomes. we found that knockdown of either ythdf2 or mettl16 increased both endosomal u6 levels and total u6 levels in hela cells ( fig. 5n, o ), indicating that neither ythdf2 nor ma methylation is required for u6 entry into endosomes. in addition, we found that the c - terminus of ythdf2 is required for its localization to endosomes, as wild - type ( df2 ( wt ) ) full - length and c - terminal ythdf2 ( df2 ( c ) ), but not n - terminus ythdf2 ( df2 ( n ) ), were detected in endosomes ( fig. 5p, q ). together, our data demonstrate that sidt2 transports either the u6 - ythdf2 complex altogether or u6 alone into endosomes, while ythdf2 entry into endosomes requires u6 snrna. next, to characterize whether ythdf2 or u6 snrna is trafficked intracellularly or extracellularly to endosomes, we assessed whether cells uptake either ythdf2 or u6 snrna secreted by other cells. however, when cultured with conditioned medium from control cells, no ythdf2 or u6 snrna was detected in endosomes of hela cells with ythdf2 deletion or a431 cells with u6 snrna knockdown, respectively ( fig. s14n – s14o ), suggesting that intracellular trafficking pathways deliver ythdf2 and u6 snrna into endosomes. lastly, to determine whether u6 snrna entry into endosomes is required for the increased expression of inflammatory genes, we assessed the effect of dynasore, an inhibitor of u6 endosomal entry ( fig. 5g ), on cytokine expression. we found that dynasore reduced expression of inflammatory genes induced by u6 in hela cells, or by ythdf2 knockdown in a431 cells at baseline condition ( fig. 5r, s ) and in hacat and hela cells treated with uvb irradiation ( fig. s14p, s14q ). in control cells with ythdf2 expression, dynasore also slightly reduced inflammatory gene expression in a431 cells and il - 6 expression in uvb - irradiated hacat cells, but not in uvb - irradiated hela cells ( fig. 5s and fig. s14p, s14q ), suggesting a cell - type - dependent, ythdf2 - independent effect of dynasore. such effect of dynasore may be due to the reduction of endosomal localization of non - ma - modified u6, which can be ythdf2 - independent, as non - ma - modified u6 is bound by tlr3 but not by ythdf2 ( fig. 4k, o ). future investigation is needed to elucidate the specific molecular mechanism for regulating u6 snrna metabolism and function. taken together, these findings demonstrate that the endocyt osis pathway mediates the localization of u6 and ythdf2 to endosomes, thus regulating tlr3 activation and inflammation. since both ythdf2 loss and uvb treatment similarly increase u6 stability ( fig. 2o ), we next asked whether uvb inhibits ythdf2 by modulating ythdf2 post - translational modifications. mass spectrometric analysis showed that at 1 h, uvb induced dephosphorylation of ythdf2 at serine 39 ( s39 ) in hacat cells ( fig. 6a, supplementary fig. s15a ). to confirm this observation, we generated an antibod y specific for ythdf2 phosphorylation at s39 and found that uvb decreases ythdf2 s39 phosphorylation post - irradiation in hacat cells ( fig. 6b ). we next re - examined ythdf2 - interacting proteins and found that uvb inhibits the binding of ythdf2 with several kinases and cnot1, the subunit for the ccr4 – not deadenylase complex that mediates ma - methylated rna decay, but promotes ythdf2 binding to mypt1 ( myosin phosphatase target subunit 1 ) ( supplementary fig. s15b – d ), a regulatory subunit for myosin phosphatase that is a member of protein phosphatase type 1. we hypothesized that uvb might induce ythdf2 dephosphorylation by the mypt1 - phosphatase complex to inhibit the ythdf2 - cnot1 interaction and thus u6 decay. coimmunoprecipitation ( co - ip ) analysis demonstrated that uvb induces the ythdf2 - mypt1 interaction in hela cells ( supplementary fig. s15e ). further co - ip analysis demonstrated that the phosphomimetic s39d ythdf2 mutant shows enhanced binding to mypt1 as compared with wt ythdf2 and the non - phosphorylatable s39a mutant in hela cells with ythdf2 knockout ( fig. 6c ), suggesting a potential role of the mypt1 – phosphatase complex in de phosphorylating ythdf2 at s39. in addition, as compared with wt ythdf2, we found that the phosphomimetic s39d ythdf2 mutant shows increased binding to cnot1, while the s39a mutant shows decreased binding in hela cells with ythdf2 knockout ( fig. 6d ), indicating that s39 phosphorylation of ythdf2 is required for the ythdf2 - cnot1 interaction. consistently, wt ythdf2, but not the s39a mutant, decreased u6 snrna level and u6 - induced - tnf - α expression, while both wt and s39a mutant ythdf2 showed similar binding affinity to u6 in hela cells with ythdf2 knockout ( fig. 6e, supplementary fig. s15f – i ). fig. 6ythdf2 is inhibited by uvb irradiation. a loss of ythdf2 phosphorylation at serine 39 ( s39 ) in hacat cells after uvb irradiation identified by mass spectrometry. b immunoblot of p - ythdf2 ( s39 ) and ythdf2 in hacat cells with or without uvb irradiation ( 1 h ). c co - ip of mypt1 with flag - tagged ythdf2 ( ev, wt, s39d or s39a ) in df2 ko hela cells. d co - ip of cnot1 with flag - tagged ythdf2 ( ev, wt, s39d or s39a ) in df2 ko hela cells. e qpcr of u6 snrna stability in df2 ko hela cells transfected with df2 wt or df2 s39a. f rip of u6 with cnot1 in hela cells. qpcr of u6 snrna stability in hela ( g ) and a431 ( h ) cells with or without cnot1 knockdown. rip of u6 with cnot1 in hela cells with or without ythdf2 knockout ( i ) or mettl16 knockdown ( j ). k immunoblot of flag - tagged ythdf2 in wcl and endosomes from hela cells transfected with ev, df2 wt or df2 s39a. l immunoblot analysis of ythdf2, p62, and lc3 - i / ii in hacat after sham ( c ) or uvb irradiation.