Journal: Gene

Article Title: Intron retention is a stress response in sensor genes and is restored by Japanese herbal medicines: A basis for future clinical applications.

doi: 10.1016/j.gene.2022.146496

Figure Lengend Snippet: Fig. 3. Characterization of IncIR events in aging SAMP8 mice (A) IR events increase after aging. Box plots comparing the percent spliced index (PSI) values rep resenting IR levels between R1WC vs. P8WC and R1WL vs. P8WL obtained by rMATS. Box plots show the 25th and 75th percentiles, medians, and whiskers that extend to the minimum and maximum values. ***P < 0.001 (unpaired t-test with R). (B) Genomic features associated with the retained introns. Box plots comparing intron length (in base pairs) and GC content (%) in intron sequences among three groups of introns. The number of loci analyzed for increased IR (IncIR), decreased IR (DecIR), and no IR (NoIR; i.e., correctly spliced) was 36, 47, and 25,3922, respectively. The number of loci for IncIR and DecIR was obtained from the comparison of P8WC (vs. R1WC) in the basal group by rMATS. The number of loci for NoIR was calculated from the number of all expressed genes in the brain. Box plots show the 25th and 75th percentiles, medians, and whiskers that extend to the minimum and maximum values. ***P < 0.001 (unpaired t-test with R). (C) Significantly enriched GO terms for the IncIR genes. The biological process terms from GO for 36 IncIR genes obtained from a comparison of P8WC (vs. R1WC) and 31 IncIR genes from a comparison of P8WL (vs. R1WL), both of which were obtained by rMATS. The GO terms were obtained by using the DAVID tool and a cut-off of P < 0.05. (D) The increase in IR events after aging was confirmed by another method. Box plots comparing the percent spliced index (PSI) values, which represent the level of IR, between R1WC vs. P8WC (138 loci, 71 genes) and R1WL vs. P8WL (50 loci, 29 genes). Box plots show the 25th and 75th percentiles, medians, and whiskers that extend to the minimum and maximum values. ***P < 0.001 (unpaired t-test with R). (E) Significantly enriched GO terms for the IncIR genes obtained by another method. A heatmap showing the significantly enriched biological process terms associated with the 71 IncIR genes (138 loci) in P8WC (vs. R1WC) and 29 IncIR genes (50 loci) in P8WL (vs. R1WL) that were obtained by the PSI method. The enrichment score for each term is represented by its P-value. These data were obtained with the DAVID tool and a P cut-off of P < 0.05. (F) Increased IR during aging that was observed in the normally aging wild-type mice. Scatter plots showing the continuous increase in IR across different ages in the normally aging wild-type mice at 15, 24, and 30 months (M) that were obtained by the PSI method. ***P < 0.001. (G) Significantly enriched GO terms for the IncIR genes during normal aging. A bar chart showing the significantly enriched gene pathways (P < 0.05) for the set of 231 IncIR genes that showed consistent increases in IR in normally aging wild-type mice (Table S5). P-values were obtained using DAVID. ER, endoplasmic reticulum. (H) Some IncIR genes and DEGs were included in a common network. (i) Proteins encoded by two IncIR genes, Polr2i and Tia1 (dark pink), interact with many proteins encoded by DEGs. Data were extracted from a network analysis of the interactions among 36 IncIR genes, 400 up-regulated genes, and 400 down- regulated genes in P8WC (vs. R1WC) of the basal group. (ii) Proteins encoded by two IncIR genes, Mapk7 and Casp3 (dark pink), interact with many proteins encoded by DEGs. Data were extracted from a network analysis of the interactions among 31 IncIR genes, 400 up-regulated genes, and 400 down-regulated genes in P8WL (vs. R1WL) of the LPS-stimulated group. Network analysis was performed on Cytoscape (Shannon, P. et al., 2003) using the STRINGapp (Doncheva, N. et al., 2019). The lengths of the lines between nodes correspond to the combined PPI scores of the nodes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Western blotting was used to determine the protein levels of eight IncIR genes as described previously by our group (Ito et al. 2022) with some additional information as follows: Primary antibodies used in this study were rabbit anti-ATG16L2 (24322–1-AP, Proteintech, IL, USA), rabbit anti-MLXIPL (13256–1-AP, Proteintech), rabbit anti-AMBRA1 (13762–1-AP, Proteintech), rabbit anti-MAPK7 (10036–2-AP, Proteintech), rabbit anti-HPS4 (14627–1-AP, Proteintech), rabbit antiCyclophilin A (PPIA) (14627–1-AP, Proteintech), rabbit anti-Drebrin (DBN1) (10260–1-AP, Proteintech), rabbit anti-RPL23A (16386–1-AP, Proteintech) and mouse anti-GAPDH (sc-32233, Santa Cruz Biotechnology, TX, UAS).

Techniques: Comparison

Journal: Gene

Article Title: Intron retention is a stress response in sensor genes and is restored by Japanese herbal medicines: A basis for future clinical applications.

doi: 10.1016/j.gene.2022.146496

Figure Lengend Snippet: Fig. 6. Restoration of IR genes during aging by medicines is a general phenomenon. (A) An increase in IR events was observed in very old SAMP8 mice and some of these were restored by AD drug candidates. Bar graphs showing the IncIR events in 13 M (vs. 9 M) and the DecIR events in 13 M + drugs (vs. 13 M without drugs) obtained by rMATS. The analysis isolated 31 IncIR genes that were altered with aging in P8-13 M and 39, 31, and 37 DecIR genes that were altered in P8-13 M treated with CAD031 (031), J147 (147), and CMS121 (121), respectively. The bars represent the mean ± SEM. ***P < 0.001 (unpaired t-test with R). (B) Common IR events were recovered by the three AD drug candidates. A Venn diagram showing the genes for which IR was increased by aging in P8-13 M (vs. P8-9 M) and those that were recovered after J147, CMS121, and CAD031 treatments. Inc13M means genes for which IR increased at the age of 13 months. (C) The enriched biological process (BP) terms associated with IR genes recovered after J147, CMS121, and CAD031 treatments. The indicated P-value was obtained with the DAVID tool under the criterion of P < 0.1. (D) A heatmap illustration of the 19 recovered IR genes shown in the Venn diagram in (B). The average number of IJCs and SJCs and the fold change between them (IJCs/SJCs) were calculated for each locus of each gene. 9 M, 13 M, and 13 M + indicate 9 months, 13 months without administration of drugs, and 13 months with the administration of a drug, respectively. (E) The significantly enriched GO terms for the transcriptionally recovered genes after administration of AD drug candidates. A total of 2,018 DEGs were altered with aging in P8-13 M (vs. P8-9 M). In addition, 2,146, 2,481, and 1,321 DEGs were altered by administration of CAD031, CMS121, and J147 (P8-13 M vs. P8-13 M + ), respectively. Significantly enriched GO terms associated with these events are shown (P < 0.05). The enrichment score for each pathway is represented by the –log10P. (F) Transcriptional recovery by administration of chemical compounds in three groups of genes related to (i) mTOR, (ii) translation, and (iii) glycogen biosynthesis. Normalized read counts were used for mRNA expression from brain tissue of genes for which IR was affected by both aging and the treatments with AD drug candidates. The bars represent the mean ± SEM for the normalized read counts. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Table S9; the exact test within edgeR). (G) Common sensors are shared between two different mouse aging models, klotho and SAMP8. A Venn diagram of the 262 IncIR genes from the klotho mice (liver samples), which were obtained by comparison between IncKL– and WT (Okada et al. 2021), and the 44 IncIR genes from SAMP8 mice (brain samples) (see the main text). The 8 IR genes that are found in both samples are shown.

Article Snippet: Western blotting was used to determine the protein levels of eight IncIR genes as described previously by our group (Ito et al. 2022) with some additional information as follows: Primary antibodies used in this study were rabbit anti-ATG16L2 (24322–1-AP, Proteintech, IL, USA), rabbit anti-MLXIPL (13256–1-AP, Proteintech), rabbit anti-AMBRA1 (13762–1-AP, Proteintech), rabbit anti-MAPK7 (10036–2-AP, Proteintech), rabbit anti-HPS4 (14627–1-AP, Proteintech), rabbit antiCyclophilin A (PPIA) (14627–1-AP, Proteintech), rabbit anti-Drebrin (DBN1) (10260–1-AP, Proteintech), rabbit anti-RPL23A (16386–1-AP, Proteintech) and mouse anti-GAPDH (sc-32233, Santa Cruz Biotechnology, TX, UAS).

Techniques: Isolation, Expressing, Comparison