Bes5101

To examine the interaction of SRSF1 protein and the target sequence, RNA immunoprecipitation was conducted and all steps were performed according to the manufacturer’s protocol (BerSinBio# Bes5101). In short, cells were wash with PBS and resuspended with lysis buffer containing protease inhibitors and RNA enzyme inhibitors (lysis buffer: protease inhibitors: RNA enzyme inhibitors = 1700:17:7.5); then, the DNA removal process was carried out. Cell lysate was divided into three groups (input, IP, and IgG groups). Anti-SRSF1 antibodies (Santa Cruz Biotechnology #sc-33652, 5 μg) and IgG antibody (BerSinBio#Bes5101, 5 μg) were incubated with cell lysates of IP and IgG groups, respectively, at 4 °C for 16 h. Next, the RNA-protein complexes were isolated by incubating cell lysates with the protein A/G magnetic beads at 4 °C for 1 h. After proteinase K digestion, protein-bound RNAs were extracted by phenol/chloroform/isoamyl alcohol (125:24:1) (Solarbio). The protein-bound RNAs were detected by qRT-PCR and assessed by %Input (%Input = 2^{−[ΔCtIP-(ΔCtinput-log2 Input Dilution Factor)]}, Input Dilution Factor = (Volume _Input/Volume _Input + Volume _IP + Volume _IgG)⁻¹). The fold enrichment (fold enrichment = 2^{−(ΔCtIP-ΔCtIgG)}) was also calculated to evaluate the alternations of RNA-protein binding caused by HKMT stimulation. The primers used in this assay are listed in Supplementary Table S11.

RIP-qPCR was conducted using an RIP-kit (Bes5101, BersinBio, China). According to the manufacturer’s protocols, we performed RIP assays in the HN6 and FaDu cell lines to evaluate and validate NAT10 and GLMP mRNA. In short, magnetic beads pre-coated with 3ug of antibodies against NAT10 (Santa Cruze, USA) or mouse immunoglobulin G (Abcam, UK) were incubated with pre-frozen cell lysates for more than 16 h at 4 °C. Next, Proteinase K was used to digest the beads containing immunoprecipitated RNA-protein complexes, and RNA extraction was performed using the RNAiso Plus (Code No.9108, TaKara, Japan). The following steps were used to quantify the levels of GLMP mRNA using RT-qPCR.