Structural dynamics of detector and effector NMD complexes – DEFineNMD

The employed strategies include three main axes: the study of purified complexes, the reconstitution and structural and functional characterization of complexes in vitro and the study of the role of sub-complexes or individual components in NMD.

Preliminary results indicate novel features of the Upf1 RNA helicase, a core NMD factor, and established structures for an NMD cofactor alone or in association with Upf1.

The preliminary results will be validated and should allow the publication of scientific articles.

Work in progress.

Nonsense-mediated mRNA decay (NMD) is a major surveillance pathway that degrades mRNAs carrying premature termination codons (PTC) and encoding undesired truncated proteins. The true diversity of NMD substrates became apparent in recent years and is much larger than initially thought. NMD literally shapes eukaryotic transcriptomes as perva­sive transcription, non-coding RNAs synthesis, splicing inefficiency and transcription start site selec­tion imprecision, all generate NMD substrates. Essential for development in mammals, NMD is linked with telomere maintenance, telomerase activity regulation as well as with human genetic diseases and tumorigenesis. Despite the importance of NMD and the conservation of the major core factors Upf1, Upf2 and Upf3 from yeast to humans, current NMD models are still incomplete and depend on factors that are not universally present in eukaryotes. Moreover, they explain only partially how translation-dependent PTC recognition is linked with RNA degradation in molecular terms.
By combining fast affinity purification and quantitative mass spectrometry, we recently made a breakthrough in understanding NMD mechanisms. Our approach unambigu­ously demonstrated the existence of two mutually exclusive and successive protein complexes that both contain Upf1. We named the complex composed of the three core Upf factors, “Detector” and the com­plex composed of Upf1 and factors related to RNA degradation “Effec­tor”. These results highlight a pivotal role for Upf1 whose ATP-dependent RNA helicase activities are essential for NMD, and suggest that the protein must accommodate two com­pletely different sets of proteins during NMD. Our data give support to an NMD model that only consists of universally conserved factors and challenges current models but also leads to new hypotheses about NMD mechanisms, notably concerning the switch between the detection and degradation phases.
The DEFineNMD project originates from our Detector/Effector NMD model and from recent conceptual advances in the field. Based on their respective expertise, the four partners involved in this project will combine biochemistry, crystallography, biophysics and functional genomics to characterize both the structure and the function of the Detector and Effector complexes. We will structurally characterize the subcomplexes formed around Upf1 both in vivo and in vitro with reconstituted RNA-protein complexes. The role of Upf1 helicase activities in both the assembly and the remodeling of Detector and Effector complexes will be studied at a single molecule scale with biophysical methods using magnetic tweezers. We will use quantitative strategies based on mass-spectrometry and chemical protein modification to investigate the composi­tion and conformation of NMD complexes isolated from yeast cells. Our biophysical and structural data will be complemented by functional analysis in yeast. Finally, the main predictions of the yeast De­tector/Effector model will be tested in mammalian cells to identify the similarities and differences be­tween NMD mechanisms that evolved in highly divergent species.
This project should provide structural and functional data on the molecular events that underlie NMD, to allow mechanism-based predictions of the best and worse RNA substrates. The obtained results will serve to better understand RNA turnover and its constraints on genome sequence evolu­tion in relation with gene expression. It will also give a molecular basis for the involvement of NMD in many cellular pathways, including telomere maintenance, host-virus interactions and carcinogenesis.