Mapping interactions within the NMD decapping complex reveals how Upf1 recruits Dcp2 onto mRNA targets

Abstract

Upf1 RNA helicase is a pivotal factor in the conserved nonsense-mediated mRNA decay (NMD) process. Upf1 is responsible for coordinating the recognition of premature termination codons (PTC) in a translation-dependent manner and subsequently triggering mRNA degradation. Multiple factors assist Upf1 during these two consecutive steps. In Saccharomyces cerevisiae, Upf2 and Upf3 associated to Upf1 (Upf1-2/3) contribute to PTC recognition but are absent from the Upf1-decapping complex that includes Nmd4, Ebs1, Dcp1, and Dcp2. Despite their importance for NMD, the organization and dynamics of these Upf1-containing complexes remains unclear. Here, we show how distinct domains of Upf1 make direct contacts with Dcp1/Dcp2, Nmd4 and Ebs1 using recombinant proteins. These proteins also bind to each other forming an extended network of interactions within the Upf1-decapping complex. Dcp2 and Upf2 are shown to compete for the same binding site in the Upf1 N-terminal CH domain. This finding accounts for the isolation of two mutually exclusive Upf1-containing complexes from cell lysates. Furthermore, we show how Nmd4-assisted recruitment of Upf1 promotes the anchoring of the decapping protein to the mRNA.