human ythdf3  (Genecopoeia)

Journal: Cancer letters

Article Title: m 6 A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition

doi: 10.1016/j.canlet.2023.216178

Figure Lengend Snippet: YTHDF3 specifically interacts with DRACH RNA motifs in cyclin D1 and c-myc IRESs to regulate IRES activity and mTOR inhibitor sensitivity. (A) Screening of YTH domain family proteins binding to the c-myc DRACH RNA motifs (motifs 1–3, see fig. 2A) in U373MG(Uppsala) or HK296 GBM cells in the absence or presence of PP242 as indicated (100 nM, 18 h). The indicated proteins were immunoprecipitated from lysates and bound RNAs were amplified using primers specific for the hairpin sequences. + S.D., n = 4. (B) as in (A) except binding to the two CCND1 DRACH RNA motifs (motifs 1–2, see fig. 2A) were assessed. (C) Induction of YTHDF3 protein expression following PP242 exposure (100 nM, 18 h) in the indicated lines. (D) siRNA mediated knockdown of YTHDF3 sensitizes PP242 resistant GBM lines to PP242 (100 nM, 48 h). ATP-release assays were performed (Promega CellTiter-Glo® assay) and cell proliferation determined as a percentage of control vehicle treated cells +S.D.; n = 3. (E) siRNA mediated knockdown of YTHDF3 inhibits PP242 induced IRES activity. U373MG(Uppsala) or HK296 cells were transfected with the indicated non-targeting control (scrambled sequence; shscr) or two individual (siYTHDF3–1 or −2) YTHDF3 targeting siRNAs and IRES activity determined from CCND1 or c-MYC dicistronic mRNA reporters in the absence or presence of PP242 as indicated. Luciferase activity was normalized to the luciferase mRNA level. * P-values are shown. Mean + S.D., n = 3.

Article Snippet: Site-specific m 6 A mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) with the appropriate mutagenic primers to generate c-myc and cyclin D1 IRES luciferase reporter constructs. shRNA constructs (psi-LVRH1P) targeting human YTHDF3 and a scrambled sequence control were obtained from GeneCopoeia (#HSH069323).

Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Amplification, Expressing, Knockdown, Glo Assay, Control, Transfection, Sequencing, Luciferase

Journal: Cancer letters

Article Title: m 6 A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition

doi: 10.1016/j.canlet.2023.216178

Figure Lengend Snippet: hnRNP A1 and YTHDF3 interact and hnRNP A1-induced nucleic acid annealing activity is stimulated by YTHDF3. (A) Extracts from U373MG(Uppsala) cells were immunoprecipitated with nonspecific IgG (control) or antibody against YTHDF3 (left panel) or hnRNP A1 (right panel) and immunoprecipitates immunoblotted for the indicated proteins. Input lysates were probed for the indicated proteins as shown. (B) Recombinant YTHDF3 or hnRNP A1 was purified and analyzed for reannealing activity. 1 pmol of each protein was used in the annealing reactions and the migration positions of the indicated species are displayed.

Article Snippet: Site-specific m 6 A mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) with the appropriate mutagenic primers to generate c-myc and cyclin D1 IRES luciferase reporter constructs. shRNA constructs (psi-LVRH1P) targeting human YTHDF3 and a scrambled sequence control were obtained from GeneCopoeia (#HSH069323).

Techniques: Activity Assay, Immunoprecipitation, Control, Recombinant, Purification, Migration

Journal: Molecular Cancer

Article Title: A positive feedback circuit driven by m 6 A-modified circular RNA facilitates colorectal cancer liver metastasis

doi: 10.1186/s12943-023-01848-1

Figure Lengend Snippet: YTHDF3 recruits eIF4G2 complex to driver circ-YAP translation. A . Western blot testing the levels of the indicated proteins in circ-YAP-overexpressing 293T cells after knockdown of YTHDF1/2/3. B , C . Western blot testing the effect of YTHDF3 knockout on YAP-220 level. D . The indicated vectors were transfected into LoVo and SW620 cells, followed by western blot analysis of YAP-220 level. E . RIP assay using anti-YTHDF3 antibody, followed by qRT-PCR analysis of circ-YAP enrichment. F , G . The in vivo and in vitro pull-down assays using biotin-labeled circ-YAP probes, followed by western blot analysis of YTHDF3 level. H . Co-IP assay testing the interaction between YTHDF3 and eIF4G2 proteins. I . Western blot testing the effect of eIF4G2 silencing on YAP-220aa expression. J , K . RNA pull-down assays using biotin-labeled circ-YAP probes in YTHDF3 −/− SW620 and LoVo cells, followed by western blot analysis of the indicated protein levels. L , M . Transwell assay testing cell invasion in circ-YAP-overexpressing SW480 and HT29 cells after YTHDF3 knockout or eIF4G2 silencing. * P < 0.05, *** P < 0.001. Data (E, L, M) are the mean ± SD of three independent experiments carried out in triplicate. The uncropped western blot data are provided as a Original Blot Image file

Article Snippet: The above synthesized circ-YAP was incubated with recombinant human YTHDF3 protein (#ab166020, Abcam) in binding buffer (20mM Tris, 150mM NaCl, 1% Triton X-100, 2mM DTT, 1mM EDTA) at 4 °C for 1 h, followed by incubation with the Streptavidin Magnetic Beads (Invitrogen) and western blot analysis.

Techniques: Western Blot, Knock-Out, Transfection, Quantitative RT-PCR, In Vivo, In Vitro, Labeling, Co-Immunoprecipitation Assay, Expressing, Transwell Assay