Journal: Journal of Translational Medicine
Article Title: SRSF9 promotes colorectal cancer progression via stabilizing DSN1 mRNA in an m6A-related manner
doi: 10.1186/s12967-022-03399-3
Figure Lengend Snippet: Selective binding of SRSF9 to m6A-modified RNAs stabilizes DSN1 mRNA in an m6A-related manner in colorectal cancer. A RNA affinity assay using single-stranded RNA probes containing methylated (red) or unmethylated (green) adenosine. The consensus sequence is shown in bold type. Silver staining (left) and immunoblotting (right) showed selective pull-down of 26 kDa SRSF9 proteins from LOVO cell extracts. IB: Immunoblotting. B Gene-specific m6A qRT-PCR was used to detect enrichment of m6A modifications in DSN1 transcripts (***p < 0.001, Student’s t test). C Bioinformatic analysis of RNA modifications within the SRSF9 protein-binding sites of DSN1 mRNA. D Schematic diagram of the wild-type (WT) and mutant (MUT) firefly luciferase reporters. The A-T substitutions (red) were made within the m6A consensus sequence (green). Only the portion of the SRSF9-DSN1-binding sequence that contains the mutation sites is shown. E Relative luciferase activity of WT and MUT reporters in 293T cells ectopically expressing SRSF9 (****p < 0.0001, Student’s t test). F Relative luciferase activity of the WT reporter in cells cotransfected with the indicated amounts of the SRSF9 expression vectors (***p < 0.001; ****p < 0.0001, Student’s t test). G , H The expression of SRSF9 and METTL3 ( G ), DSN1 and METTL3 ( H ) was positively correlated in colon adenocarcinoma tumors in the GEPIA database. I , J Western blot assay showing the protein level of DSN1 in METTL3-deficient CRC cells upon overexpression of SRSF9 in CRC cells. K – P The indicated stable cells were subjected to Western blotting ( K , M and O ). qRT-PCR assays were used to analyze the half-life of DSN1 mRNA in SRSF9 knockdown ( L ), SRSF9-overexpressing ( N ) and SRSF9-overexpressing with METTL3 interfered CRC cells after treatment with actinomycin D (normalized to 0 h). Each experiment was performed in triplicate independently
Article Snippet: Single-stranded RNA oligonucleotide probes containing the m6A-binding consensus sequence GGACU with methylated (ss-m6A, 5′-biotin-CGUCUCGG(m6A) CUCGG(m6A)CUGCU-3′) or unmethylated (ss-A, 5′-biotin-CGUCUCGGACUC GGACUGCU-3′) adenosine were synthesized by RIBOBIO Company (Guangzhou, China) and validated by mass spectrometry.
Techniques: Binding Assay, Modification, Methylation, Sequencing, Silver Staining, Western Blot, Quantitative RT-PCR, Protein Binding, Mutagenesis, Luciferase, Activity Assay, Expressing, Over Expression
