29 research outputs found
Nonsense‐mediated mRNA decay: The challenge of telling right from wrong in a complex transcriptome
SMG6 Cleavage Generates Metastable Decay Intermediates from Nonsense-Containing β-Globin mRNA
Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay
Many gene expression factors contain repetitive phosphorylation sites for single kinases, but the functional significance is poorly understood. Here we present evidence for hyperphosphorylation as a mechanism allowing UPF1, the central factor in nonsense-mediated decay (NMD), to increasingly attract downstream machinery with time of residence on target mRNAs. Indeed, slowing NMD by inhibiting late-acting factors triggers UPF1 hyperphosphorylation, which in turn enhances affinity for factors linking UPF1 to decay machinery. Mutational analyses reveal multiple phosphorylation sites contributing to different extents to UPF1 activity with no single site being essential. Moreover, the ability of UPF1 to undergo hyperphosphorylation becomes increasingly important for NMD when downstream factors are depleted. This hyperphosphorylation-dependent feedback mechanism may serve as a molecular clock ensuring timely degradation of target mRNAs while preventing degradation of non-targets, which, given the prevalence of repetitive phosphorylation among central gene regulatory factors, may represent an important general principle in gene expression
The cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3´ end.
8 páginas, 7 figuras -- PAGS nros. 498-505Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3′ end of the mRNPThis work was funded by the Spanish Government (SAF2008-00451 and SAF2011-22988 to O.L.) and the Red Temática de Investigación Cooperativa en Cáncer from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L. and contract to R.M.). O.L. is additionally supported by the Human Frontiers Science Program (RGP39/2008 to O.L.), the Fundación Ramón Areces and the Government from the Autonomous Region of Madrid (S2010-BMD-2316). This work was also supported by the Max Planck Gesellschaft, the Sonderforschungsbereich SFB646, the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft and the Center for Integrated Protein Science Munich (E.C.)Peer reviewe
Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs
Recruitment of the UPF1 nonsense-mediated mRNA decay (NMD) factor to target mRNAs was initially proposed to occur through interaction with release factors at terminating ribosomes. However, recently emerging evidence points toward translation-independent interaction with the 3' untranslated region (UTR) of mRNAs. We mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation in human cells and found that UPF1 preferentially associated with 3' UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition, thus indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination