Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function

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Abstract

Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer’s helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was undetectable in the vertebrate Dicer-1 ancestor, which had even lower dsRNA affinity. Reverting residues in the ATP hydrolysis pocket was insufficient to rescue hydrolysis, but additional substitutions distant from the pocket rescued vertebrate Dicer-1's ATPase function. Our work suggests Dicer lost ATPase function in the vertebrate ancestor due to loss of ATP affinity, involving motifs distant from the active site, important for coupling dsRNA binding to the active conformation. By competing with Dicer for viral dsRNA, RIG-I-like receptors important for interferon signaling may have allowed, or actively caused, loss of ATPase function.

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All data generated or analyzed during this study are included in the manuscript and supporting files. Source Data files have been provided for all gels displayed in Figures 2, 3 and 4. Phylogenetic and ancestral protein reconstruction data are provided as Supplementary Files.

Article and author information

Author details

Adedeji M Aderounmu

Department of Biochemistry, University of Utah, Salt Lake City, United States

Competing interests
The authors declare that no competing interests exist.
P Joseph Aruscavage

Department of Biochemistry, University of Utah, Salt Lake City, United States

Competing interests
The authors declare that no competing interests exist.
Bryan Kolaczkowski

Department of Microbiology and Cell Science, University of Florida, Gainesville, United States

Competing interests
The authors declare that no competing interests exist.
Brenda L Bass

Department of Biochemistry, University of Utah, Salt Lake City, United States

For correspondence
bbass@biochem.utah.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-1728-2254

Funding

School of Medicine (Graduate Student Fellowship)

Adedeji M Aderounmu

National Institute of General Medical Sciences (R35GM141262)

Brenda L Bass

National Cancer Institute (R01CA260414)

Brenda L Bass

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.