Crystal Structure of the Nmd4-Upf1 Complex Reveals a Conserved Binding Motif Critical for Nonsense-Mediated mRNA Decay

Core Concepts

The interaction between the yeast Nmd4 protein and the helicase domain of Upf1, a central factor in the nonsense-mediated mRNA decay (NMD) pathway, is mediated by a conserved C-terminal "arm" region of Nmd4 and is important for Upf1's ATPase activity and the degradation of NMD substrates.

Abstract

The content describes the crystal structure of the complex formed between the yeast Nmd4 protein and the helicase domain of the central NMD factor Upf1. The key findings are: Nmd4 interacts with Upf1 helicase domain primarily through its C-terminal "arm" region, forming an extensive interface. The "arm" region is necessary and sufficient for the Nmd4-Upf1 interaction. The Nmd4-Upf1 interaction stimulates the ATPase activity of Upf1 and increases its affinity for RNA, suggesting the interaction impacts Upf1's enzymatic properties. Mutations in the conserved "arm" region of Nmd4 disrupt the Nmd4-Upf1 interaction and impair Nmd4's ability to stimulate Upf1 ATPase activity. In yeast, the Nmd4-Upf1 interaction is important for the degradation of NMD substrates, particularly when the Upf1 N-terminal domain is absent. A similar conserved "arm-like" motif is identified in the human SMG6 protein, a key NMD factor. This motif is also important for the interaction between SMG6 and the UPF1 helicase domain, as well as for the optimal degradation of endogenous NMD substrates in human cells. These results support the existence of a conserved molecular mechanism for NMD across eukaryotes, centered around the interaction between Upf1/UPF1 and Nmd4/SMG6.

Stats

The Nmd4 protein interacts with Upf1 helicase domain with a Kd of 2.1 μM. The Nmd4 "arm" region interacts with Upf1 helicase domain with a Kd of 1.96 μM. The Nmd4 PIN domain does not interact with Upf1 helicase domain. Nmd4 stimulates the ATPase activity of Upf1 helicase domain. The Nmd4 "arm" region and PIN domain synergistically enhance Upf1 ATPase activity. Nmd4 increases the affinity of Upf1 helicase domain for RNA.

Quotes

"The Nmd4 protein wraps around Upf1 RecA1 domain." "Nmd4 stimulates Upf1 ATPase activity." "The interaction of Nmd4 with Upf1 is important for NMD in vivo." "An arm-like region of human SMG6 contributes to its binding to UPF1 and its role in NMD."

Key Insights Distilled From

Identification of an evolutionary conserved binding motif responsible for the recruitment of NMD factors to the UPF1 helicase

by Barbarin-Boc... at www.biorxiv.org 03-03-2024

https://www.biorxiv.org/content/10.1101/2024.02.27.582253v1

Deeper Inquiries

How do the interactions between Nmd4/SMG6 and Upf1/UPF1 influence the recruitment and regulation of other NMD factors, such as the decapping and deadenylation complexes

The interactions between Nmd4/SMG6 and Upf1/UPF1 play a crucial role in the recruitment and regulation of other NMD factors, such as the decapping and deadenylation complexes. The Nmd4/SMG6 proteins interact with the helicase domain of Upf1/UPF1, which is a central ATP-dependent RNA helicase in the NMD pathway. This interaction stimulates the ATPase activity of Upf1/UPF1, leading to the destabilization of NMD substrates. The Nmd4/SMG6 "arm" region, which interacts with Upf1/UPF1, is essential for this process. By enhancing the ATPase activity of Upf1/UPF1, Nmd4/SMG6 contributes to the tagging of premature stop codons and subsequent recruitment of RNA decay factors. This interaction also strengthens the binding of Upf1/UPF1 to RNA, facilitating the recognition and degradation of aberrant mRNAs. Additionally, the Nmd4/SMG6-Upf1/UPF1 complex may serve as a platform for the recruitment and coordination of other NMD factors, such as the decapping and deadenylation complexes, to ensure efficient mRNA decay.

What are the structural and mechanistic details of how the Nmd4/SMG6 "arm" region modulates the enzymatic activities of Upf1/UPF1, beyond the observed effects on ATPase activity

The Nmd4/SMG6 "arm" region modulates the enzymatic activities of Upf1/UPF1 beyond the observed effects on ATPase activity through its interaction with the helicase domain. The structural details of this interaction reveal that the "arm" region of Nmd4/SMG6 interacts with specific residues in the helicase domain of Upf1/UPF1, forming hydrogen bonds, salt bridges, and hydrophobic interactions. This interaction stabilizes Upf1/UPF1 on RNA and enhances its RNA binding affinity, contributing to the efficient recognition and degradation of NMD substrates. The "arm" region acts as a critical mediator in the recruitment and regulation of other NMD factors by Upf1/UPF1, such as the decapping and deadenylation complexes. This mechanism ensures the coordinated and timely degradation of aberrant mRNAs in the NMD pathway.

Given the conservation of this "arm" motif, are there any species-specific adaptations or additional regulatory mechanisms that have evolved around the core Nmd4/SMG6-Upf1/UPF1 interaction across different eukaryotic lineages

The conservation of the "arm" motif in Nmd4/SMG6 across different eukaryotic lineages suggests a fundamental role in the NMD pathway. While the core interaction between Nmd4/SMG6 and Upf1/UPF1 is conserved, there may be species-specific adaptations and additional regulatory mechanisms that have evolved around this interaction. For example, in metazoans, the presence of a 14-3-3 domain in SMG6 proteins adds an additional layer of regulation through phosphorylation-dependent interactions with UPF1. This phosphorylation-dependent interaction may fine-tune the recruitment and activity of NMD factors in response to cellular signaling pathways. Additionally, the evolution of specific protein domains or motifs in different organisms may reflect adaptations to the complexity of mRNA processing and degradation systems. These adaptations could enhance the efficiency and specificity of the NMD pathway in different eukaryotic lineages, while maintaining the core functionality of the Nmd4/SMG6-Upf1/UPF1 interaction in mRNA decay.