Structural and biochemical insights into the recognition of RNA helicase CGH-1 by CAR-1 in C. elegans

Zhang, Yong; Lv, Mengqi; Li, Fudong; Li, Meili; Zhang, Jiahai; Shi, Yunyu; Hong, Ju

doi:10.1016/j.bbrc.2021.02.119

Cited by 1 publication

(14 citation statements)

References 17 publications

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“…Plasmids and constructs. Expression plasmids regarding CGH-1 and CAR-1 were constructed as previously described 17 . In brief, the cDNA fragments encoding CGH-1 RecA2 (residues 248-420), CAR-1 FDF-TFG (residues 184-268) were amplified by PCR from C. elegans genome library and cloned into the NdeI and XhoI site of a modified pET-28a (Novagen) vector (p28a), in which a thrombin protease cleavage site was removed.…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression and purification. CGH-1 protein expression and purification were performed described as in our most recent paper 17 . In brief, proteins were expressed in E. coli Gold (DE3) strain (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…ITC assays were performed as previously described 17 . In brief, it was carried out on a MicroCal PEAQ-ITC (Malvern) at 20 °C with the following settings: reference power, 5 μcal/s; initial delay, 60 s; stir speed, 750 rpm; spacing time, 120 s. Proteins (or its mutants) and peptides were adjusted to 0.05 mM and 0.5 mM, respectively.…”

Section: Isothermal Titration Calorimetrymentioning

confidence: 99%

“…1A). In our most recent study, we have demonstrated in vitro that the ATPase activity of CGH-1 can be robustly stimulated by the MIF4G domain of NTL-1a (Not1 in yeast and CNOT1 in humans) in the presence of poly(U) RNA and ATP 17 . The Ccr4-Not deadenylase complex is responsible for the main ploy(A) removal activity in C. elegans 22 .…”

Section: Cgh-1 Reca2 Interacts With Edc-3 Fdf-fek Domain In Vitromentioning

confidence: 99%

“…The Ccr4-Not deadenylase complex is responsible for the main ploy(A) removal activity in C. elegans 22 . We have also investigated in vitro that C. elegans CGH-1/CAR-1 (Scd6p in yeast and LSm14A in humans) interface 17 .…”

Section: Cgh-1 Reca2 Interacts With Edc-3 Fdf-fek Domain In Vitromentioning

confidence: 99%

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Insight into the interaction between the RNA helicase CGH-1 and EDC-3 and its implications

Zhang

Wang

Yang

et al. 2021

Sci Rep

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Previous studies indicated that the P-body components, CGH-1 and EDC-3 may play a crucial role in the regulation of lifespan in Caenorhabditis elegans. Homo sapiens DDX6 or Saccharomyces cerevisiae Dhh1p (CGH-1 in C. elegans) could form complexes with EDC3 (Edc3p in yeast), respectively, which is significant for translation inhibition and mRNA decay. However, it is currently unclear how CGH-1 can be recognized by EDC-3 in C. elegans. Here, we provided structural and biochemical insights into the interaction between CGH-1 and EDC-3. Combined with homology modeling, mutation, and ITC assays, we uncovered an interface between CGH-1 RecA2 domain and EDC-3 FDF-FEK. Additionally, GST-pulldown and co-localization experiments confirmed the interaction between CGH-1 and EDC-3 in vitro and in vivo. We also analyzed PATR-1-binding interface on CGH-1 RecA2 by ITC assays. Moreover, we unveiled the similarity and differences of the binding mode between EDC-3 and CAR-1 or PATR-1. Taken together, these findings provide insights into the recognition of DEAD-box protein CGH-1 by EDC-3 FDF-FEK motif, suggesting important functional implications.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Isothermal Titration Calorimetrymentioning

confidence: 99%

Section: Cgh-1 Reca2 Interacts With Edc-3 Fdf-fek Domain In Vitromentioning

confidence: 99%

Section: Cgh-1 Reca2 Interacts With Edc-3 Fdf-fek Domain In Vitromentioning

confidence: 99%

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