Fig. 4

SNORA37 directly interacts with nuclear CMTR1 essential for CD44 alternative splicing and aggressiveness of gastric cancer. a Schematic illustration showing the identification of SNORA37-binding proteins. b SDS-PAGE, silver staining (left panel), mass spectrometry (MS) assay, and Venn diagram (middle panel) indicating the differential proteins pulled down by biotin-labeled sense or antisense (AS) SNORA37 from AGS cells, and overlapping analysis with established RBPs from EuRBPDB (http://eurbpdb.gzsys.org.cn) and RBP2GO (https:// rbp2go.dkfz.de) databases. Further over-lapping (right panel) identifying the RBPs essential for both cellular viability and patients’ survival of gastric cancer by using datasets derived from iCSDB (https://www.kobic.re.kr/icsdb/) and KM-Plotter (http://kmplot.com) databases. c Biotin-labeled RNA pull-down and western blot assays showing the proteins pulled down by biotin-labeled SNORA37 from HGC-27 cell lysates, using SNORA37 antisense transcript or beads as controls. d Mass spectrometry assay revealing CMTR1 peptide fragment pulled down by SNORA37. e Dual RNA-FISH and immunofluorescence staining assays indicating the co-localization of SNORA37 (red, arrowheads) and CMTR1 (green, arrowheads) in HGC-27 cells, with nuclei staining with DAPI (blue). f Schematic diagram revealing the domains of CMTR1 truncations. g In vitro binding (upper panel) and RIP (lower panel) assays showing the enrichment of SNORA37 detected by RT-PCR after incubation with GST-tagged recombinant CMTR1 proteins, or lysates of AGS cells transfected with full-length or truncations of HA-tagged CMTR1 constructs validated by western blot. h RT-PCR assay indicating the differential expression levels of CD44v6 and CD44s in gastric cancer AGS and MKN-45 cells stably transfected with empty vetor (mock), CMTR1, scramble shRNA (sh-Scb), sh-CMTR1 #1, or sh-CMTR1 #2. i and j Western blot assay (i) and quantification (j) indicating the differential expression levels of CD44v6 and CD44s in AGS and MKN-45 cells stably transfected with mock, CMTR1, sh-Scb, sh-CMTR1 #1, or sh-CMTR1 #2 (n = 3). Fisher's exact test for over-lapping analysis in b. Student's t-test or one‐way ANOVA analyzed the difference in j. ***P < 0.001. Data are shown as mean ± SEM (error bars) and representative of three independent experiments in c, e and g-j