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Stem Cells International Volume 2017 ,2017-11-23
The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5
Research Article
Abdullah Shah 1 Farooq Rashid 1 Hassaan Mehboob Awan 1 Shanshan Hu 2 , 3 Xiaolin Wang 1 Liang Chen 1 Ge Shan 1
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DOI:10.1155/2017/8596135
Received 2017-05-16, accepted for publication 2017-08-17, Published 2017-08-17
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摘要

DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m6A RNA demethylase. The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.

授权许可

Copyright © 2017 Abdullah Shah et al. 2017
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

图表

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ∗∗∗P<0.001; P values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).

Determination of the binding domain of DDX3 with ALKBH5. (a) Schematic diagram of full-length DDX3 and the corresponding partial deletion constructs; summary of the results of interaction with ALKBH5 was also shown. (b) Deletion of N-terminal domain, Linker domain, Helicase domain, or the C-terminal domain of DDX3 showed interaction with the full-length ALKBH5, as examined with IP and co-IP. (c) The interaction was abolished when ATP domain of DDX3 was deleted, as examined with IP and co-IP. (d) ATP domain of DDX3 alone interacted with full-length ALKBH5, examined with IP and co-IP. All IP and co-IP were performed in triplicates, and representative results were shown.

Determination of the binding domain of DDX3 with ALKBH5. (a) Schematic diagram of full-length DDX3 and the corresponding partial deletion constructs; summary of the results of interaction with ALKBH5 was also shown. (b) Deletion of N-terminal domain, Linker domain, Helicase domain, or the C-terminal domain of DDX3 showed interaction with the full-length ALKBH5, as examined with IP and co-IP. (c) The interaction was abolished when ATP domain of DDX3 was deleted, as examined with IP and co-IP. (d) ATP domain of DDX3 alone interacted with full-length ALKBH5, examined with IP and co-IP. All IP and co-IP were performed in triplicates, and representative results were shown.

Determination of the binding domain of DDX3 with ALKBH5. (a) Schematic diagram of full-length DDX3 and the corresponding partial deletion constructs; summary of the results of interaction with ALKBH5 was also shown. (b) Deletion of N-terminal domain, Linker domain, Helicase domain, or the C-terminal domain of DDX3 showed interaction with the full-length ALKBH5, as examined with IP and co-IP. (c) The interaction was abolished when ATP domain of DDX3 was deleted, as examined with IP and co-IP. (d) ATP domain of DDX3 alone interacted with full-length ALKBH5, examined with IP and co-IP. All IP and co-IP were performed in triplicates, and representative results were shown.

Determination of the binding domain of DDX3 with ALKBH5. (a) Schematic diagram of full-length DDX3 and the corresponding partial deletion constructs; summary of the results of interaction with ALKBH5 was also shown. (b) Deletion of N-terminal domain, Linker domain, Helicase domain, or the C-terminal domain of DDX3 showed interaction with the full-length ALKBH5, as examined with IP and co-IP. (c) The interaction was abolished when ATP domain of DDX3 was deleted, as examined with IP and co-IP. (d) ATP domain of DDX3 alone interacted with full-length ALKBH5, examined with IP and co-IP. All IP and co-IP were performed in triplicates, and representative results were shown.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

DDX3 modulated m6A RNA demethylation. (a) DDX3 knockdown in HEK293T cells, relative DDX3 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (b) ALKBH5 knockdown in HEK293T cells, ALKBH5 mRNA (upper panel, qPCR), and protein levels (lower panel, Western blots) are shown. (c) Left panel: dot-blot analyses of m6A levels of isolated total RNA, mRNA, and miRNA from ALKBH5 knockdown, DDX3 knockdown, and control (NC, siRNA with scrambled sequences) cells. Right panel: methylene blue staining showing equal RNA loading. (d) IP of endogenous DDX3 could co-IP endogenous AGO2, and IP of endogenous AGO2 could co-IP endogenous DDX3 in HEK293T cells. (e) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HEK293T cells. (f) Quantification of cell proliferation (MTT assay) after knockdown of DDX3 or ALKBH5 in HeLa cells. Dot blots, IP, and co-IP were performed in triplicates, and representative results were shown. Scramble siRNA with scrambled sequences. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. P values were determined with two-tailed Student’s t-test. Error bars represent standard deviation (SD).

Working model for the role of ALKBH5 and DDX3 in m6A RNA demethylation. DDX3 interacts with ALKBH5 to regulate mRNA demethylation and additionally, by interacting with AGO2, may modulate microRNA demethylation. Whether there is direct interaction between ALKBH5 and AGO2 requires further investigations.

通讯作者

1. Liang Chen.CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences, University of Science and Technology of China, Hefei, China, ustc.edu.anqingcl@ustc.edu.cn
2. Ge Shan.CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science, School of Life Sciences, University of Science and Technology of China, Hefei, China, ustc.edu.shange@ustc.edu.cn

推荐引用方式

Abdullah Shah,Farooq Rashid,Hassaan Mehboob Awan,Shanshan Hu,Xiaolin Wang,Liang Chen,Ge Shan. The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5. Stem Cells International ,Vol.2017(2017)

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