Fig 2: Knockdown of the expression of direct targets of miR-193a-5p and miR-193a-3p suppressed growth and motility of breast cancer cells.(a) The expression levels of target genes were examined after transfection of MDA-MB-231 cells with siRNA. (b) The colony formation assay was performed after transfection of MDA-MB-231 cells with si-NLN, si-CCND1, si-PLAU, and si-SEPN1 for 2 wk, and a graph shows quantified values (c,d) The expression levels of target genes in MCF-7 cells were examined after siRNA transfection. (e) The colony formation assay was performed after transfection of MCF-7 cells with si-NLN, si-CCND1, and si-SEPN1 for 2 wk, and a graph shows quantified values (f–j) The migration and invasion abilities were assessed using the Transwell assay after transfection of MDA-MB-231 cells with siRNA. The cell images of a representative experiment are shown, and the graph shows values quantified using Ascent software. Data was reported as the number of colonies relative to the control (means ± SD). ***P < 0.001 vs control.

Fig 3: Stage-specific expression profiles and dynamic alteration of Alu exonization of SELENON during skeletal myogenesis of Hu5/KD3 cells.a Experimental schedule for differentiation of Hu5/KD3 cells. b Phase-contrast images showing Hu5/KD3 cell differentiation from Day 0 to 5. This experiment was repeated more than three times independently with similar results. c Expression analyses of the differentiation markers (MYOG, MYH1), SELENON, hnRNP C, ADAR1, and GAPDH (control) by western blotting on the indicated days during differentiation of Hu5/KD3 cells. SELENON (full-length and short isoforms), hnRNP C2 and hnRNP C1, and p150 and p110 isoforms of ADAR1, are indicated by arrowheads. The samples derived from the same experiment and the gels/blots were processed in parallel. This experiment was repeated more than three times independently with similar results. d Distribution of Log2(fold-change) of expressed genes in RNA-seq data in Hu5/KD3 cells on each day of differentiation relative to Day 0, shown as violin and box plots. For the box plot, center lines indicate median, box limits indicate upper and lower quartiles, whiskers indicate 1.5× interquartile range and points indicate outliers; n = 58,278 genes. e Heat map of RNA-seq transcriptome analysis of differentiation markers, SELENON, hnRNP C, ADAR1, UGA/Sec recoding factors, and ER stress markers on the indicated days in differentiating Hu5/KD3 cells. Colors correspond to the per-gene z-score calculated from log10 fragments per million (FPM) reads mapped. f RT-qPCR to estimate the Alu exonization level of SELENON mRNAs during myoblast differentiation. Transcripts of each isoform were quantified using Hu5/KD3 total RNA from each day of differentiation. The graph represents the ratio between the inclusion and skipping isoforms. Data are presented as mean values ±S.D.; n = 3 biologically independent samples. Source data and unprocessed gel images are provided in the Source Data file.

Fig 4: Mechanisms underlying post-transcriptional regulation of SELENON expression during myoblast differentiation.At the early stage, hnRNP C binds to AS-Alu1 of SELENON pre-mRNA and inhibits U2AF65 recognition, thereby suppressing Alu exonization. At the middle stage, the level of hnRNP C decreases, thereby releasing AS-Alu1, which forms a long double strand with S-Alu to be recognized by ADAR1. ADAR1 edits AS-Alu1, including the 5'SS of the Alu exon, thereby inhibiting U1 snRNA recognition to suppress Alu exonization. Sequential regulation of Alu exonization by the two consecutive mechanisms maintains SELENON expression from the early-to-middle stage of differentiation. At the late stage, the level of ADAR1 also decreases, thereby promoting Alu exonization and generating aberrant mRNA with PTC, followed by degradation by NMD. Moreover, at the middle-to-late stage, the levels of SEPSECS and other UGA/Sec recoding factors decrease to downregulate Sec-tRNASec formation, thereby suppressing UGA/Sec recoding. As a result, SELENON mRNA is subjected to degradation (probably by NMD) or translation to yield the truncated isoform of SELENON.

Fig 5: Dynamic regulation of UGA/Sec recoding during myoblast differentiation.a Constructs of the FLAG-SELENON expression vector and its mutants ?SECIS and GGA. TM (transmembrane domain), EF-hand domain, and Sec residue in the catalytic domain are indicated. b Expression of FLAG-SELENON and its mutants in HEK293T cells, detected by western blotting with an anti-FLAG antibody. This experiment was done once. c Northern blotting of cytoplasmic tRNASec in total RNA obtained on each day of differentiation. Relative steady-state level of tRNASec on each day of differentiation compared with the level on Day 0 is shown below. Results were normalized relative to the band intensity of 5.8 S rRNA in the same sample. Data are presented as mean values ±S.D. Statistical significance was determined by unpaired two-tailed t test; n = 3 biologically independent samples. d Volcano plot of RNA-seq transcriptome data showing altered gene expression [log2(fold-change)], with p values, on Day 5 versus Day 0 of Hu5/KD3 cell differentiation. UGA/Sec recoding factors are indicated, and significantly differentially expressed genes (two-sided p value <0.05 determined by likelihood ratio test) are highlighted in red. e Expression analyses of GAPDH (control) and SEPSECS in differentiating Hu5/KD3 cells by western blotting on the indicated days. The expression level of SEPSECS was normalized to that of GAPDH, making the relative expression level on Day 0 = 1.00 (shown below). The samples derived from the same experiment and the gels/blots were processed in parallel. This experiment was repeated more than three times independently with similar results. f Subcellular localization of SEPSECS on the indicated days in differentiating Hu5/KD3 cells immunostained with an anti-SEPSECS antibody (green). Nuclei were stained with DAPI (blue) and myotubes were stained with an anti-myosin antibody (red). Magnification: ×100. Exposure time: 150 ms (Day 0) or 300 ms (Day 3 and 5). This experiment was done once. Source data and unprocessed gel image are provided in Source Data file.