Rehman S et al. (2024), The mRNA N6-Methyladenosine Response to Dehydra...

XB-ART-61096

Animals (Basel) 2024 Nov 15;1422:. doi: 10.3390/ani14223288.

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The mRNA N6-Methyladenosine Response to Dehydration in Xenopus laevis.

Rehman S , Parent M , Storey KB .

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The African clawed frog, Xenopus laevis, exhibits remarkable adaptations to survive in its arid habitat, including behavioral and metabolic changes during periods of drought. During extreme dehydration, X. laevis undergoes estivation, a state characterized by increased urea and ammonia levels, depression of the metabolic rate, and tissue hypoxia. To understand the molecular mechanisms underlying these adaptations, we investigated the potential role of N6-methyladenosine (m6A), a widespread mRNA modification, in X. laevis during extreme dehydration. We analyzed the protein levels of key components in the m6A pathway, including writers (METTL3, METTL14, and WTAP), erasers (ALKBH5 and FTO), and readers (SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a), in the liver and kidneys of control frogs and frogs that had lost 35 ± 0.93% of their total body water. The relative protein levels generally decreased or remained unchanged, with the exception of YTHDF3, which depicted a protein level increase in the liver. Notable changes included eIF3a, which was downregulated by 26 ± 8% and 80 ± 8% in the dehydrated liver and kidney tissues, respectively. Additionally, the total m6A increased by 353 ± 30% and 177 ± 17% in dehydrated liver and kidney RNA samples, respectively. This study highlights the importance of epigenetic mechanisms in stress tolerance and provides a foundation for further exploration of the role of epigenetics in dehydration tolerance.

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Species referenced: Xenopus laevis
Genes referenced: alkbh5 eif3a fto mettl14 mettl3 srsf3 wtap ythdf1 ythdf2 ythdf3

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	Figure 1. Relative protein expression levels of METTL3, METTL14, and WTAP in livers of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4–5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are significantly different from the corresponding control (p < 0.05).
	Figure 2. Relative protein expression levels of METTL3, METTL14, and WTAP in kidneys from control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are significantly different from controls (p < 0.05).
	Figure 3. Relative protein expression levels of ALKBH5 and FTO in livers from control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are statistically significantly different from controls (p < 0.05).
	Figure 4. Relative protein expression levels of ALKBH5 and FTO in kidneys of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed a Student’s t-test; asterisks indicate that are statistically significantly different from controls (p < 0.05).
	Figure 5. Relative protein expression levels of SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a in livers of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4–5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).
	Figure 6. Relative protein expression levels of SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a in kidneys from control and dehydrated of X. laevis, as determined by Western blots. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).
	Figure 7. Relative m6A demethylase enzyme activity in liver and kidney tissues of X. laevis from control and high-dehydration conditions. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Independent trials were taken as the mean of duplicate measurements. Data were analyzed using a Student’s t-test.
	Figure 8. Relative total m6A content in total RNA samples from X. laevis liver and kidney tissues comparing control vs. high-dehydration conditions. Data are mean ± SEM, n = 3 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).
	Figure 1. Relative protein expression levels of METTL3, METTL14, and WTAP in livers of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4–5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are significantly different from the corresponding control (p < 0.05).
	Figure 2. Relative protein expression levels of METTL3, METTL14, and WTAP in kidneys from control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are significantly different from controls (p < 0.05).
	Figure 3. Relative protein expression levels of ALKBH5 and FTO in livers from control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks indicate values that are statistically significantly different from controls (p < 0.05).
	Figure 4. Relative protein expression levels of ALKBH5 and FTO in kidneys of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Data were analyzed a Student’s t-test; asterisks indicate that are statistically significantly different from controls (p < 0.05).
	Figure 5. Relative protein expression levels of SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a in livers of control and dehydrated X. laevis, as determined by Western blotting. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 4–5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).
	Figure 6. Relative protein expression levels of SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a in kidneys from control and dehydrated of X. laevis, as determined by Western blots. Corresponding bands from immunoblots are shown below the histogram. Data are mean ± SEM, n = 5 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).
	Figure 7. Relative m6A demethylase enzyme activity in liver and kidney tissues of X. laevis from control and high-dehydration conditions. Data are mean ± SEM, n = 4 independent trials on samples from different animals. Independent trials were taken as the mean of duplicate measurements. Data were analyzed using a Student’s t-test.
	Figure 8. Relative total m6A content in total RNA samples from X. laevis liver and kidney tissues comparing control vs. high-dehydration conditions. Data are mean ± SEM, n = 3 independent trials on samples from different animals. Data were analyzed using a Student’s t-test; asterisks represent values that have statistically significant changes compared with controls (p < 0.05).