Fig. 4: MaTAR25 is a positive upstream regulator of Tns1.

a Cell fractionation was performed to isolate cytoplasmic, nucleoplasmic, and chromatin-associated RNA. qRT-PCR was used to determine the subcellular localization ratio of MaTAR25 transcripts. β-actin and Malat1 were used as marker RNAs for quality control. Data are presented as mean values ± SD (n = 3 independent experiments). b Schematic diagram showing the targeting of biotin-labeled oligonucleotides binding MaTAR25 transcripts for Chromatin Isolation by RNA Purification (ChIRP)-seq. Odd and even oligo pools (seven oligos in each pool) were used, and qRT-PCR was performed to check the RNA purification efficiency. Data are presented as mean values ± SD (n = 3 independent experiments). c Venn diagram showing differentially expressed genes in MaTAR25 KO cells identified from RNA-Seq overlapped with MaTAR25 ChIRP-seq data. The top candidate genes are listed. Statistics were determined using DESeq2, and a FDR-adjusted P value of <0.1 was set as a threshold for statistical significance. d Validation of Tns1 as a MaTAR25-targeted gene by qRT-PCR and immunoblotting in 4T1 control and MaTAR25 KO cells (n = 2 independent experiments). e The RNA expression level of Tns1 is rescued upon ectopic expression of MaTAR25 in MaTAR25 KO cells as determined by qRT-PCR (n = 2 independent experiments). f CRISPR/Cas9 targeting was used in 4T1 cells to generate Tns1 knockout clones. The upper panel shows expression levels of Tns1 in 4T1 control, Tns1 KO clone1, and Tns1 KO clone2 by immunoblotting. The lower panel shows the cell counting viability assay result of 4T1 control1, Tns1 KO1, and Tns1 KO2. Results are mean ± SD (n = 3 independent experiments). *P < 0.05 (paired Student’s t test; two-tailed). g Ectopic expression of Tns1 in MaTAR25 KO cells rescues the cell viability defect. The top panel shows expression levels of Tns1 in 4T1 control1, MaTAR25 KO1, MaTAR25 KO1 with Tns1 ectopic expression clone3 by qRT-PCR. Results are mean ± SD (n = 3 independent experiments). *P < 0.05 (paired Student’s t test; two-tailed). The bottom panel shows the cell counting viability assay results of 4T1 control1, MaTAR25 KO1, MaTAR25 KO1 with MaTAR25 ectopic expression, and MaTAR25 KO1 with Tns1 ectopic expression clone1-3. Results are mean ± SD (n = 2 independent experiments).