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Christoph Schweingruber, Paolo Soffientini, Marc-David Ruepp, Angela Bachi, Oliver Mühlemann
Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly...
2016: PloS One
Feng He, Allan Jacobson
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that monitors cytoplasmic mRNA translation and targets mRNAs undergoing premature translation termination for rapid degradation. From yeasts to humans, activation of NMD requires the function of the three conserved Upf factors: Upf1, Upf2, and Upf3. Here, we summarize the progress in our understanding of the molecular mechanisms of NMD in several model systems and discuss recent experiments that address the roles of Upf1, the principal regulator of NMD, in the initial targeting and final degradation of NMD-susceptible mRNAs...
2015: Annual Review of Genetics
Alper Celik, Stephanie Kervestin, Allan Jacobson
Nonsense-mediated mRNA decay (NMD) is one of three regulatory mechanisms that monitor the cytoplasm for aberrant mRNAs. NMD is usually triggered by premature translation termination codons that arise from mutations, transcription errors, or inefficient splicing, but which also occur in transcripts with alternately spliced isoforms or upstream open reading frames, or in the context of long 3'-UTRs. This surveillance pathway requires detection of the nonsense codon by the eukaryotic release factors (eRF1 and eRF3) and the activities of the Upf proteins, but the exact mechanism by which a nonsense codon is recognized as premature, and the individual roles of the Upf proteins, are poorly understood...
July 2015: Biochimie
Zaineb Fourati, Bijoyita Roy, Claudia Millan, Pierre-Damien Coureux, Stéphanie Kervestin, Herman van Tilbeurgh, Feng He, Isabel Usón, Allan Jacobson, Marc Graille
Upf1, Upf2, and Upf3 are the principal regulators of nonsense-mediated mRNA decay (NMD), a cytoplasmic surveillance pathway that accelerates the degradation of mRNAs undergoing premature translation termination. These three proteins interact with each other, the ribosome, the translation termination machinery, and multiple mRNA decay factors, but the precise mechanism allowing the selective detection and degradation of nonsense-containing transcripts remains elusive. Here, we have determined the crystal structure of the N-terminal mIF4G domain from Saccharomyces cerevisiae Upf2 and identified a highly conserved region in this domain that is essential for NMD and independent of Upf2's binding sites for Upf1 and Upf3...
November 11, 2014: Journal of Molecular Biology
Farkas Kerényi, Izabela Wawer, Pawel J Sikorski, Joanna Kufel, Dániel Silhavy
Nonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants...
December 2013: Plant Journal: for Cell and Molecular Biology
Feng He, Robin Ganesan, Allan Jacobson
RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine- and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains...
December 2013: Molecular and Cellular Biology
Stefanie Metze, Veronika A Herzog, Marc-David Ruepp, Oliver Mühlemann
Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin μ and β-Globin) with or without an intron downstream from the PTC...
October 2013: RNA
Ei Ei Min, Bijoyita Roy, Nadia Amrani, Feng He, Allan Jacobson
The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins...
August 2013: RNA
Verena Geißler, Simone Altmeyer, Benjamin Stein, Heike Uhlmann-Schiffler, Hans Stahl
Non-sense-mediated mRNA decay (NMD) is a mechanism of translation-dependent mRNA surveillance in eukaryotes: it degrades mRNAs with premature termination codons (PTCs) and contributes to cellular homeostasis by downregulating a number of physiologically important mRNAs. In the NMD pathway, Upf proteins, a set of conserved factors of which Upf1 is the central regulator, recruit decay enzymes to promote RNA cleavage. In mammals, the degradation of PTC-containing mRNAs is triggered by the exon-junction complex (EJC) through binding of its constituents Upf2 and Upf3 to Upf1...
September 2013: Nucleic Acids Research
Simone C Rufener, Oliver Mühlemann
Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 implies a similar basic mechanism of PTC recognition in all eukaryotes. However, while PTC-containing mRNAs in yeast seem to be available to NMD at each round of translation, mammalian NMD has been reported to be restricted to cap-binding complex (CBC)-bound mRNAs during the pioneer round of translation...
June 2013: Nature Structural & Molecular Biology
Elena Garre, Lorena Romero-Santacreu, Manuela Barneo-Muñoz, Ana Miguel, José E Pérez-Ortín, Paula Alepuz
The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs...
2013: PloS One
Ana M Matia-González, Ayesha Hasan, Gøril H Moe, Juan Mata, Miguel A Rodríguez-Gabriel
Nonsense-mediated mRNA decay (NMD) is a highly conserved mechanism of mRNA degradation. NMD eliminates mRNAs containing premature termination codons (PTCs), preventing the production of truncated proteins with possible deleterious effects. However, there is mounting evidence that NMD factors, like Upf1, Upf2 and Upf3, participate in general regulation of gene expression, affecting the expression of genes lacking PTCs. We have used the fission yeast Schizosaccharomyces pombe to identify mRNAs directly regulated by NMD...
June 2013: RNA Biology
Francesca Fiorini, Marc Boudvillain, Hervé Le Hir
The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form...
February 1, 2013: Nucleic Acids Research
Zsuzsanna Mérai, Anna H Benkovics, Tünde Nyikó, Mónika Debreczeny, László Hiripi, Zoltán Kerényi, Éva Kondorosi, Dániel Silhavy
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation...
January 2013: Plant Journal: for Cell and Molecular Biology
Francesca Fiorini, Fabien Bonneau, Hervé Le Hir
Degradation of eukaryotic mRNAs harboring a premature translation termination codon is ensured by the process of nonsense-mediated mRNA decay (NMD). The main effector of this quality-control pathway is the conserved RNA helicase UPF1 that forms a surveillance complex with the proteins UPF2 and UPF3. In all the organisms tested, the ATPase activity of UPF1 is essential for NMD. Here, we describe the expression of active recombinant UPF proteins and the reconstitution of the surveillance complex in vitro. To understand how UPF1 is regulated during NMD, we developed different biochemical approaches...
2012: Methods in Enzymology
G A Zhuravleva, V A Gryzina
Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae, the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that deletion of either UPF1 or UPF2 increased viability of sup45 mutants, while effect of UPF3 deletion is allele-specific...
March 2012: Molekuliarnaia Biologiia
Roberto Melero, Gretel Buchwald, Raquel Castaño, Monika Raabe, David Gil, Melisa Lázaro, Henning Urlaub, Elena Conti, Oscar Llorca
Nonsense-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...
May 2012: Nature Structural & Molecular Biology
Stephanie Kvas, Gregory B Gloor, Christopher J Brandl
BACKGROUND: Tra1 is an essential protein in Saccharomyces cerevisiae. It was first identified in the SAGA and NuA4 complexes, both with functions in multiple aspects of gene regulation and DNA repair, and recently found in the ASTRA complex. Tra1 belongs to the PIKK family of proteins with a C-terminal PI3K domain followed by a FATC domain. Previously we found that mutation of leucine to alanine at position 3733 in the FATC domain of Tra1 (tra1-L3733A) results in transcriptional changes and slow growth under conditions of stress...
2012: BMC Genetics
Chuan Shi, Ian T Baldwin, Jianqiang Wu
Nonsense-mediated mRNA decay (NMD) is an important mRNA quality surveillance pathway in all eukaryotes that eliminates aberrant mRNAs derived from various sources. Three NMD factor proteins, UPF1, UPF2, and UPF3 are required for the NMD process and were found to be also involved in certain stress responses in mammalian and yeast cells. Using Arabidopsis thaliana mutants of UPF1 and UPF3 and UPF2-silenced lines (irUPF2), we examined the involvement of UPF1, UPF2, and UPF3 in development and in response to stresses, wounding and infection by Pseudomonas syringae pv...
February 2012: Journal of Integrative Plant Biology
Chuan Shi, Ian T Baldwin, Jianqiang Wu
Nonsense-mediated mRNA decay (NMD) is an important mRNA quality surveillance pathway in all eukaryotes that eliminates aberrant mRNAs derived from various sources. Three NMD factor proteins, UPF1, UPF2, and UPF3 are required for the NMD process and were found to be also involved in certain stress responses in mammalian and yeast cells. Using Arabidopsis thaliana mutants of UPF1 and UPF3 and UPF2-silenced lines (irUPF2), we examined the involvement of UPF1, UPF2, and UPF3 in development and in responses to stresses, wounding and infection by Pseudomonas syringae pv...
December 14, 2011: Journal of Integrative Plant Biology
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