DDX6 Regulates mRNA Transcription & Translation Pathways

DDX6 (RCK/p54) was first isolated from the RC-K8 B-cell lymphoma line over 30 years ago. This 472 amino acid, 53.2 kDa “DEAD-Box” ATP-dependent RNA helicase bears the signature Asp-Glu-Ala-Asp motif, characteristic of all DEAD box proteins. Dead box proteins contain both ATPase and helicase activity, and are known for their particular protein-protein and RNA-protein interactions that regulate many RNA functions.²

DDX6 interacts with partner proteins, mRNAs, and miRNAs to regulate transcript availability and expression to preserve homeostasis. It is sought after as a drug target for many disease states, including cancers, viruses, autoimmune diseases, neurological diseases, and aging.

Assembly of the DDX6 Protein

DDX6 is dominated by two central Rec A-like domains, separated by a hinge region, that are flanked by distinctive N and C terminal sequences.¹ While both Rec A regions cooperate in ATP binding/hydrolysis and RNA binding/unwinding, the N-terminal Rec A-like domain largely controls ATP interactions, while the C-terminal Rec A-like domain principally administers RNA contacts and interactions with protein partners. Unlike most helicases, DDX6 can avidly bind RNA even in the absence of ATP.²

While there are putative nuclear export and nuclear localization patches in the N-terminal region, these have been shown to be structurally masked by folding. Since DDX6 can shuttle between the nucleus and cytoplasm, new work has advanced a model of this phenomenon. DDX6 utilizes a C-terminal sequence to “hitchhike” on mitotic chromosomes in proliferating cells. There is also evidence to support a “piggyback” association between DDX6 and 4E-T that shuttles DDX6 into the nucleus (via Importins) and out of the nucleus (via CRM1).³

Regulating mRNA Transcription and Translation

The main functions of DDX6 center around enhancing mRNA decapping and repressing translation via ribonucleoprotein (RNP) condensates. Although mRNA decapping usually precedes its decay, in certain circumstances, decapping facilitates translation. Interacting with CNOT1, DDX6 enzymatically drives translational repression as part of the miRNA-induced silencing complex (miRISC) that both deadenylates and decaps mRNA. Some miRNAs can, in turn, repress DDX6.

In concert with key binding partners, DDX6 is crucial for the formation of processing bodies (PBs) and stress granules (SGs) from mRNA and RNA binding protein-rich RNPs that sequester intact transcripts. These structures serve as storage depots for transcripts that may be necessary for appropriate cellular responses to diverse signaling inputs or stresses, ultimately parsing the translation or destruction of their contents. Lack of sufficient DDX6 subsequently leads to the dissolution of PBs and the release of retained mRNA. Likewise, inappropriate accumulation of PBs or SGs can withhold mRNA species from translation and interfere with cellular homeostasis. Intact, wild type DDX6 effectively mediates these processes in response to signaling. DDX6 that lacks ATPase function constitutively forms SGs, even in the absence of any inducements. So, for the formation of SGs, lack of intracellular ATP, a form of stress, is a key activator of DDX6.⁴

Recently, DDX6 has also been found to inhibit A-to-I RNA editing by the ADAR system in neuronal cells, promoting differentiation. DDX6’s C-terminal region is required for this function, but its ATPase activity is not.⁵

Applications in Health and Disease

Overexpression of DDX6 has been found in colorectal, gastric, and lung cancer, causing upregulation of c-Myc, HER2, and FGFR2 expression and abetting epithelial-mesenchymal transition (EMT).^6,7,8 Generally, in cancer, DDX6’s interaction with various regulatory miRNAs has been impaired.⁹

DDX6 additionally plays several roles in innate immunity. It serves as a co-sensor for viral RNA and an enhancer of RIG-I-mediated IFN induction.¹⁰ However, it also restricts aberrant expression of interferon stimulated genes in the absence of infection and is, therefore, becoming an important player in autoimmunity.¹¹ Nonetheless, certain pathogens, such as the Hepatitis C and Zika viruses, have evolved to take advantage of DDX6 to increase their virulence ^12,13

Several de novo, C-terminal mutations in DDX6 have profound influence on proper neurodevelopment, leading to intellectual disability.¹⁴ In Alzheimer’s disease, pathological tau variants associate with DDX6 to impair its regulatory function. Proper DDX6/tau binding normally increases miRISC-mediated gene silencing activity by let-7a and miRNAs.¹⁵

In aging, an increase in PB accumulation and decrease in translation can extend lifespan, depending on the detailed makeup of the PBs and the resilience of the host stress response system.¹⁶ Due to its pivotal role in PB and SG formation, DDX6 could significantly affect lifespan and health span.¹⁷

Identify & Characterize DEAD-Box Helicase Modulators with Transcreener Assays

Because of the protein’s ever-growing implications in human health and longevity, discovery of DDX6 modulators shows promise for many therapeutic treatments. The Transcreener ADP² Assay is a robust, high-throughput screening (HTS) method designed to identify and characterize ATPase/Helicase modulators, such as DEAD-Box proteins. Our DDX3 application provides an excellent example of using this assay to study DEAD-Box proteins.

View the DDX3 Application Assay

References

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4. Majerciak V., Zhou T. and Zheng, Z-M. (2021) RNA helicase DDX6 in P-bodies is essential for the assembly of stress granules. bioRxiv 2021.09.24.461736. https://doi.org/10.1101/2021.09.24.461736
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10. Nunez, R.D. et al. (2018) The RNA Helicase DDX6 Associates with RIG-I to Augment Induction of Antiviral Signaling. Int. J. Mol. Sci., 19(7), 1877. https://doi.org/10.3390/ijms19071877
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13. Michalski, D. et al. (2019) Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection. J. Biol. Chem., 294(44) 16282–16296. https://doi.org/10.1074/jbc.RA119.009129
14. Weil, D. et al. (2020) Mutations in genes encoding regulators of mRNA decapping and translation initiation: links to intellectual disability. Biochem. Soc. Trans., 48 (3): 1199–1211. Review. https://doi.org/10.1042/BST20200109
15. da Costa, P.J. (2022) Tau mRNA Metabolism in Neurodegenerative Diseases: A Tangle Journey. Biomedicines, 10(2), 241. Review. https://doi.org/10.3390/biomedicines10020241
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