1. Biochemistry and Chemical Biology
  2. Evolutionary Biology

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

  1. Adedeji M Aderounmu
  2. P Joseph Aruscavage
  3. Bryan Kolaczkowski
  4. Brenda L Bass  Is a corresponding author
  1. University of Utah, United States
  2. University of Florida, United States
https://doi.org/10.7554/eLife.85120
Share this article
Cite this article
  1. Adedeji M Aderounmu
  2. P Joseph Aruscavage
  3. Bryan Kolaczkowski
  4. Brenda L Bass
(2023)
Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function
eLife 12:e85120.
https://doi.org/10.7554/eLife.85120
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

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

  1. Adedeji M Aderounmu

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. P Joseph Aruscavage

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Bryan Kolaczkowski

    Department of Microbiology and Cell Science, University of Florida, Gainesville, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. 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.
    ORCID icon "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.

Copyright

© 2023, Aderounmu et al.

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.

Metrics

  • 2,725
    views
  • 251
    downloads
  • 20
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Adedeji M Aderounmu
  2. P Joseph Aruscavage
  3. Bryan Kolaczkowski
  4. Brenda L Bass
(2023)
Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function
eLife 12:e85120.
https://doi.org/10.7554/eLife.85120

Share this article

https://doi.org/10.7554/eLife.85120

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics

    High-resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery

    Conor J Howard, Nathan S Abell ... Nathan B Lubock
    Research Article

    Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

    1. Biochemistry and Chemical Biology

    Coordinated regulation of chemotaxis and resistance to copper by CsoR in Pseudomonas putida

    Meina He, Yongxin Tao ... Wenli Chen
    Research Article

    Copper is an essential enzyme cofactor in bacteria, but excess copper is highly toxic. Bacteria can cope with copper stress by increasing copper resistance and initiating chemorepellent response. However, it remains unclear how bacteria coordinate chemotaxis and resistance to copper. By screening proteins that interacted with the chemotaxis kinase CheA, we identified a copper-binding repressor CsoR that interacted with CheA in Pseudomonas putida. CsoR interacted with the HPT (P1), Dimer (P3), and HATPase_c (P4) domains of CheA and inhibited CheA autophosphorylation, resulting in decreased chemotaxis. The copper-binding of CsoR weakened its interaction with CheA, which relieved the inhibition of chemotaxis by CsoR. In addition, CsoR bound to the promoter of copper-resistance genes to inhibit gene expression, and copper-binding released CsoR from the promoter, leading to increased gene expression and copper resistance. P. putida cells exhibited a chemorepellent response to copper in a CheA-dependent manner, and CsoR inhibited the chemorepellent response to copper. Besides, the CheA-CsoR interaction also existed in proteins from several other bacterial species. Our results revealed a mechanism by which bacteria coordinately regulated chemotaxis and resistance to copper by CsoR.

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics

    Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

    Jiale Zhou, Ding Zhao ... Zhanjun Li
    Research Article

    5-Methylcytosine (m5C) is one of the posttranscriptional modifications in mRNA and is involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we develop a detection method named DRAM (deaminase and reader protein assisted RNA methylation analysis), in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provides transcriptome-wide editing regions which are highly overlapped with the publicly available bisulfite-sequencing (BS-seq) datasets and allows for a more stable and comprehensive identification of the m5C loci. In addition, DRAM system even supports ultralow input RNA (10 ng). We anticipate that the DRAM system could pave the way for uncovering further biological functions of m5C modifications.