ythdf2 knockdown had little effect on ma enrichment in the 5 ′ utr, 3 ′ utr, cds, or the total peak distribution in hacat cells ( fig. 2b, c ). sequence motif analysis of ma peaks showed enrichment for the previously identified ma target site motif ( ggacu ) ( fig. 2d ). overall, the levels of over 3800 genes were changed in cells with knockdown of ythdf2 ( supplementary fig. 7a ). the downstream targets of ythdf2 were selected following 3 criteria in ythdf2 knockdown cells as compared with control cells: ( 1 ) increased rna level, ( 2 ) increased ma enrichment, and ( 3 ) related to inflammation or tumorigenesis. from this analysis, we identified the top 30 upregulated genes that are potential ma targets of ythdf2 ( supplementary fig. s7b ), consistent with ythdf2 ’ s known role in promoting decay of ma - modified mrna. finally, we selected and verified four genes – fos, jun, sox4, and sox9 – as potential ythdf2 targets, based on increased mrna levels, stability, and increased ma enrichment upon ythdf2 knockdown, and as well as ythdf2 binding to the mrnas in hacat cells ( supplementary fig. s7c – f, s8a, b ). these findings indicate that fos, jun, sox4, and sox9 are downstream ma targets for ythdf2. further investigation is needed to elucidate the functional importance of these genes in ythdf2 ’ s function. interestingly, ythdf2 knockdown had little effect on the ma enrichment across the transcripts for tnf - α and il - 1β, while it increased the ma enrichment across the transcripts for il - 6 and cox - 2 in hacat cells ( supplementary fig. s9a – d ). since ythdf2 knockdown had no effect on the mrna stability of cox - 2 or il - 6 ( supplementary fig. s6e ), future investigation is warranted to elucidate the role of ma methylation of cox - 2 and il - 6 in their gene expression, including but not limited to translation. fig. 2ythdf2 binds to ma - methylated self u6 snrna to induce u6 decay. a uhplc – ms / ms for ma enrichment in mrna in hacat cells with or without ythdf2 knockdown ( n = 2 ). no statistical analysis was performed. b distribution of ma peaks across 5 ′ utr, cds and 3 ′ utr. c proportion of ma peaks in the indicated regions across all mrnas. d consensus ma motif identified by homer. e pathway analysis of uvb - induced upregulated genes in hacat cells ( q < 0. 05, deseq2 ). f identification of u5 -, u4 - and u6 - associated rnps among ythdf2 - interacting proteins by mass spectrometry in hacat cells. g – i qpcr of u6 snrna in hacat cells with or without ythdf2 knockdown, with or without ythdf2 overexpression, and with or without uvb irradiation. j qpcr of u6 snrna in mouse skin with or without ythdf2 deletion after sham or chronic uvb for 23 weeks ( n = 3 mice ). k ma - ip qpcr for enrichment of u6 snrna in hacat cells with or without ythdf2 knockdown. l rip of ythdf2 – u6 snrna interaction in hacat cells. m ma - ip qpcr of u6 snrna in hacat cells with or without uvb ( 30 min ). n rip of ythdf2 – u6 snrna interaction in hacat cells with or without uvb ( 30 min ). o qpcr of u6 snrna stability in hacat cells with or without ythdf2 knockdown, treated with actinomycin d ( actd ), with or without uvb ( 4 h time course ). p qpcr of u6 snrna in hacat cells with mettl16 knockdown ( shnc, shm16 - 1, shm16 - 2 ). q ma - ip qpcr of u6 snrna in hacat cells with or without mettl16 knockdown, with or without uvb ( 30 min ). r rip of ythdf2 – u6 snrna interaction in hacat cells with or without mettl16 knockdown, with or without uvb ( 30 min ). 18s rrna served as internal control ( g – j, o – p ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are presented as mean ± se ( n = 4 for g – i, o – p ) or mean ± sd ( n = 2 for a; n = 3 for j – n, q – r ). all experiments were conducted using biologically independent samples. a uhplc – ms / ms for ma enrichment in mrna in hacat cells with or without ythdf2 knockdown ( n = 2 ). no statistical analysis was performed. b distribution of ma peaks across 5 ′ utr, cds and 3 ′ utr. c proportion of ma peaks in the indicated regions across all mrnas. d consensus ma motif identified by homer. e pathway analysis of uvb - induced upregulated genes in hacat cells ( q < 0. 05, deseq2 ). f identification of u5 -, u4 - and u6 - associated rnps among ythdf2 - interacting proteins by mass spectrometry in hacat cells. g – i qpcr of u6 snrna in hacat cells with or without ythdf2 knockdown, with or without ythdf2 overexpression, and with or without uvb irradiation. j qpcr of u6 snrna in mouse skin with or without ythdf2 deletion after sham or chronic uvb for 23 weeks ( n = 3 mice ). k ma - ip qpcr for enrichment of u6 snrna in hacat cells with or without ythdf2 knockdown. l rip of ythdf2 – u6 snrna interaction in hacat cells. m ma - ip qpcr of u6 snrna in hacat cells with or without uvb ( 30 min ). n rip of ythdf2 – u6 snrna interaction in hacat cells with or without uvb ( 30 min ). o qpcr of u6 snrna stability in hacat cells with or without ythdf2 knockdown, treated with actinomycin d ( actd ), with or without uvb ( 4 h time course ). p qpcr of u6 snrna in hacat cells with mettl16 knockdown ( shnc, shm16 - 1, shm16 - 2 ). q ma - ip qpcr of u6 snrna in hacat cells with or without mettl16 knockdown, with or without uvb ( 30 min ). r rip of ythdf2 – u6 snrna interaction in hacat cells with or without mettl16 knockdown, with or without uvb ( 30 min ). 18s rrna served as internal control ( g – j, o – p ). statistical analyses were performed using a two - tailed unpaired student ’ s t - test. data are presented as mean ± se ( n = 4 for g – i, o – p ) or mean ± sd ( n = 2 for a; n = 3 for j – n, q – r ). all experiments were conducted using biologically independent samples. as we did not observe canonical inflammatory genes in the candidate target list for ythdf2 by ma - seq, we elected to re - examine the pathways for uvb - induced genes from our previous rna - seq data ( gse145924 ). indeed, uvb irradiation induced the expression of genes related to ( non - coding rna ) ncrna metabolic processes and ncrna processing in hacat cells ( fig. 2e ), suggesting a role for ncrna in ythdf2 regulation of the uvb stress response. in addition, our mass spectrometric analysis of ythdf2 - interacting proteins showed that ythdf2 binds to several u6 snrna - associated small nuclear ribonucleoproteins ( u6 snrnps ) such as u6 snrna - associated sm - like protein lsm4, lsm6, u4 / u6 small nuclear ribonucleoprotein prp4 ( prpf4 ), u4 / u6 small nuclear ribonucleoprotein prp3 ( prpf3 ), and u4 / u6. u5 tri - snrnp - associated protein 1 / 2 ( snut1 / 2 ), as well as several u5 - associated ribonucleoproteins in hacat cells ( fig. 2f, supplementary fig. s10a ). these findings led us to hypothesize that ythdf2 acts as a reader for ma - methylated self u6 snrna. indeed, we found that ythdf2 knockdown increased u6 snrna levels in hacat cells ( fig. 2g ). similarly, uvb irradiation also increased u6 levels in hacat cells by qpcr and fluorescence in - situ hybri diz ation ( fish ) analysis ( fig. 2h - i, supplementary fig. s10b ). both baseline and uvb - induced u6 levels were decreased by ythdf2 overexpression, while they were further increased by ythdf2 knockdown in hacat cells ( fig. 2h - i ). skin - specific ythdf2 deletion in mice increased u6 levels in the skin, which were further increased by uvb irradiation ( fig. 2j ). moreover, ythdf2 knockdown increased ma enrichment in u6 snrna in hacat cells and primary mouse keratinocytes ( fig. 2k, supplementary fig. s10c ). furthermore, using rna immunoprecipitation ( rip ) analysis, we found that ythdf2 binds to u6 snrna in hacat cells ( fig. 2l ). at 30 min in hacat cells, uvb irradiation increased ma enrichment in u6 snrna ( fig. 2m ) and enhanced the interaction of u6 with ythdf2 ( fig. 2n ). we then hypothesized that ythdf2 regulates u6 stability. indeed, both ythdf2 knockdown and uvb stress increased u6 snrna stability in hacat cells ( fig. 2o ).