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Joan A Steitz - One of the best experts on this subject based on the ideXlab platform.
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Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and Nonsense-Mediated Decay
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Andrei Alexandrov, David Colognori, Joan A Steitz, Mei-di ShuAbstract:The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the Nonsense-Mediated Decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association. In vivo depletion of hCWC22, as for yeast Cwc22p, causes a splicing defect, resulting in decreased levels of mature cellular mRNAs. Nonetheless, hCWC22 depletion yields increased levels of spliced RNA from the unusual nonsense codon-containing U22 host gene, which is a natural substrate of NMD. To test whether hCWC22 acts in NMD through coupling splicing to EJC deposition, we searched for mutations in hCWC22 that affect eIF4AIII deposition without affecting splicing. Addition of hCWC22(G168Y) with a mutation at the putative hCWC22/eIF4AIII interface exacerbates the defect in splicing-dependent deposition of eIF4AIII(T334V) with a mutation reported to be in direct contact with mRNA, linking hCWC22 to the process of EJC deposition in vitro. Importantly, the addition of hCWC22(G168Y) affects deposition of eIF4AIII(T334V) without inhibiting splicing or the efficiency of deposition of the endogenous eF4AIII(WT) in the same reaction, demonstrating hCWC22’s specific role in eIF4AIII deposition in addition to its role in splicing. The essential splicing factor CWC22 has, therefore, acquired functions in EJC assembly and NMD during evolution from single-celled to complex eukaryotes.
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Splicing of U12-type introns deposits an exon junction complex competent to induce Nonsense-Mediated mRNA Decay
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Tetsuro Hirose, Mei-di Shu, Joan A SteitzAbstract:Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces Nonsense-Mediated Decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression.
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communication of the position of exon exon junctions to the mrna surveillance machinery by the protein rnps1
Science, 2001Co-Authors: Jens Lykkeandersen, Mei-di Shu, Joan A SteitzAbstract:In mammalian cells, splice junctions play a dual role in mRNA quality control: They mediate selective nuclear export of mature mRNA and they serve as a mark for mRNA surveillance, which subjects aberrant mRNAs with premature termination codons to Nonsense-Mediated Decay (NMD). Here, we demonstrate that the protein RNPS1, a component of the postsplicing complex that is deposited 5' to exon-exon junctions, interacts with the evolutionarily conserved human Upf complex, a central component of NMD. Significantly, RNPS1 triggers NMD when tethered to the 3' untranslated region of beta-globin mRNA, demonstrating its role as a subunit of the postsplicing complex directly involved in mRNA surveillance.
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human upf proteins target an mrna for nonsense mediated Decay when bound downstream of a termination codon
Cell, 2000Co-Authors: Jens Lykkeandersen, Mei-di Shu, Joan A SteitzAbstract:Abstract Nonsense-Mediated Decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons. These are discriminated from true termination codons by downstream cis -elements, such as exon–exon junctions. We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b. While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic. hUpf3a and hUpf3b associate selectively with spliced β-globin mRNA in vivo, and tethering of any hUpf protein to the 3′UTR of β-globin mRNA elicits NMD. These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon–exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.
Sian Ellard - One of the best experts on this subject based on the ideXlab platform.
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The position of premature termination codons in the hepatocyte nuclear factor −1 beta gene determines susceptibility to Nonsense-Mediated Decay
Human Genetics, 2005Co-Authors: Lorna W Harries, Andrew T Hattersley, Coralie Bingham, Christine Bellanne-chantelot, Sian EllardAbstract:The Nonsense-Mediated Decay (NMD) pathway is an mRNA surveillance mechanism that detects and degrades transcripts containing premature termination codons. The position of a truncating mutation can govern the resulting phenotype as mutations in the last exon evade NMD. In this study we investigated the susceptibility to NMD of six truncating HNF-1beta mutations by allele-specific quantitative real-time PCR using transformed lymphoblastoid cell lines. Four of six mutations (R181X, Q243fsdelC, P328L329fsdelCCTCT and A373fsdel29) showed evidence of NMD with levels of mutant transcript at 71% ( p =0.009), 24% ( p =0.008), 22% ( p =0.008) and 3% ( p =0.016) of the wild-type allele respectively. Comparable results were derived from lymphoblastoid cells and renal tubule cells isolated from a patient’s overnight urine confirming that cell lines provide a good model for mRNA analysis. Two mutations (H69fsdelAC and P159fsdelT) produced transcripts unexpectedly immune to NMD. We conclude that truncating mutant transcripts of the HNF-1beta gene do not conform to the known rules governing NMD susceptibility, but instead demonstrate a previously unreported 5′ to 3′ polarity. We hypothesise that this may be due to reinitiation of translation downstream of the premature termination codon. Our study suggests that reinitiation of translation may be an important mechanism in the evasion of NMD, but that other factors such as the distance from the native initiation codon may also play a part.
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messenger rna transcripts of the hepatocyte nuclear factor 1α gene containing premature termination codons are subject to nonsense mediated Decay
Diabetes, 2004Co-Authors: Lorna W Harries, Andrew T Hattersley, Sian EllardAbstract:Mutations in the hepatocyte nuclear factor-1α ( HNF-1a ) gene cause maturity-onset diabetes of the young (MODY). Approximately 30% of these mutations generate mRNA transcripts harboring premature termination codons (PTCs). Degradation of such transcripts by the Nonsense-Mediated Decay (NMD) pathway has been reported for many genes. To determine whether PTC mutant transcripts of the HNF-1 α gene elicit NMD, we have developed a novel quantitative RT-PCR assay. We performed quantification of ectopically expressed mutant transcripts relative to normal transcripts in lymphoblastoid cell lines using a coding single nucleotide polymorphism (cSNP) as a marker. The nonsense mutations R171X, I414G415ATCG→CCA, and P291fsinsC showed reduced mutant mRNA expression to 40% ( P = 0.009), P ≤ 0.0001), and 6% ( P = 0.001), respectively, of the normal allele. Transcript levels were restored using the translation inhibitor cycloheximide, indicating that the instability arises from NMD. The missense mutations G207D and R229P did not show NMD although R229P exhibited moderate RNA instability. This study provides the first evidence that HNF-1 α PTC mutations may be subject to NMD. Mutations that result in significant reduction of protein levels due to NMD will not have dominant-negative activity in vivo. Haploinsufficiency is therefore likely to be the most important mutational mechanism of HNF-1 α mutations causing MODY.
Melissa J Moore - One of the best experts on this subject based on the ideXlab platform.
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introns in utrs why we should stop ignoring them
BioEssays, 2012Co-Authors: Alicia A Bicknell, Melissa J Moore, Can Cenik, Hon N Chua, Frederick P RothAbstract:Although introns in 5'- and 3'-untranslated regions (UTRs) are found in many protein coding genes, rarely are they considered distinctive entities with specific functions. Indeed, mammalian transcripts with 3'-UTR introns are often assumed nonfunctional because they are subject to elimination by Nonsense-Mediated Decay (NMD). Nonetheless, recent findings indicate that 5'- and 3'-UTR intron status is of significant functional consequence for the regulation of mammalian genes. Therefore these features should be ignored no longer.
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Splicing enhances translation in mammalian cells: an additional function of the exon junction complex
Genes & development, 2004Co-Authors: Ajit Nott, Hervé Le Hir, Melissa J MooreAbstract:In mammalian cells, spliced mRNAs yield greater quantities of protein per mRNA molecule than do otherwise identical mRNAs not made by splicing. This increased translational yield correlates with enhanced cytoplasmic polysome association of spliced mRNAs, and is attributable to deposition of exon junction complexes (EJCs). Translational stimulation can be replicated by tethering the EJC proteins Y14, Magoh, and RNPS1 or the Nonsense-Mediated Decay (NMD) factors Upf1, Upf2, and Upf3b to an intronless reporter mRNA. Thus, in addition to its previously characterized role in NMD, the EJC also promotes mRNA polysome association. Furthermore, the ability to stimulate translation when bound inside an open reading frame appears to be a general feature of factors required for NMD.
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eIF4AIII binds spliced mRNA in the exon junction complex and is essential for Nonsense-Mediated Decay
Nature Structural and Molecular Biology, 2004Co-Authors: Toshiharu Shibuya, Thomas Tange, Nahum Sonenberg, Melissa J MooreAbstract:The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for Nonsense-Mediated mRNA Decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs.
Romão Luísa - One of the best experts on this subject based on the ideXlab platform.
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How mRNA translation can modulate Nonsense-Mediated Decay
2025Co-Authors: Romão LuísaAbstract:About one third of the gene mutations found in human genetic disorders, including cancer, result in premature translation-termination codons (PTCs) and the rapid degradation of the corresponding mRNAs by Nonsense-Mediated Decay (NMD). However, we have found that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC) can substantially escape NMD, which contradicts the current models for this mechanism. In fact, our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eukaryotic release factor 3 (eRF3) with a consequent enhancement of the termination reaction and repression of the NMD response. Here, I will provide strong evidence that the eukaryotic initiation factor 3 (eIF3) is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC, and I will dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit.FCT/PTDC/BIMONC/4890/2014N/
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How mRNA translation is involved in modulating Nonsense-Mediated Decay in transcripts with AUG-proximal nonsense mutations
2025Co-Authors: Romão LuísaAbstract:Nonsense-Mediated mRNA Decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.N/
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The interface between mRNA translation and Nonsense-Mediated Decay in AUG-proximal nonsense-mutated transcripts
2025Co-Authors: Onofre Claudia, Menezes Juliane, Peixeiro Isabel, Barbosa Cristina, Duarte Andreia, Costa Paulo, Romão LuísaAbstract:Nonsense-Mediated mRNA Decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.This work was partially supported by Fundação para a Ciência e a Tecnologia (PEst-OE/BIA/UI4046/2011, FCT/PTDC/BIM-MED/0352/2012 and PTDC/BIM-MEC/3749/2014).N/
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The interplay between mRNA translation and Nonsense-Mediated Decay in transcripts with short open reading frames
2025Co-Authors: Onofre Claudia, Menezes Juliane, Peixeiro Isabel, Barbosa Cristina, Romão LuísaAbstract:Mammalian Nonsense-Mediated mRNA Decay (NMD) is a splicing- and translation-dependent surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. In general, the location of a PTC greater than 50 nucleotides upstream to the last exon-exon junction constitutes a major determinant of NMD. However, we have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we present strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.This work was partially supported by Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC).N/
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The interaction between mRNA translation and Nonsense-Mediated Decay in AUG-proximal nonsense-mutated transcripts
2025Co-Authors: Onofre Claudia, Menezes Juliane, Peixeiro Isabel, Romão LuísaAbstract:Nonsense-Mediated mRNA Decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.This work was partially supported by Fundação para a Ciência e a Tecnologia (PEst-OE/BIA/UI4046/2011 and FCT/PTDC/BIM-MED/0352/2012).N/
Shagun Aggarwal - One of the best experts on this subject based on the ideXlab platform.
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a splice site mutation in herc1 leads to syndromic intellectual disability with macrocephaly and facial dysmorphism further delineation of the phenotypic spectrum
American Journal of Medical Genetics Part A, 2016Co-Authors: Shagun Aggarwal, Aneek Das Bhowmik, Vedam L Ramprasad, Sakthivel Murugan, Ashwin DalalAbstract:We report on a sib pair of Indian origin presenting with intellectual disability, dysmorphism, and macrocephaly. Exome sequencing revealed a homozygous splice site HERC1 mutation in both probands. Functional analysis revealed use of an alternate splice site resulting in formation of a downstream stop codon and nonsense mediated Decay. In the light of recent reports of HERC1 mutations in two families with a similar phenotypic presentation, this report reiterates the pathogenic nature and clinical consequences of HERC1 disruption. © 2016 Wiley Periodicals, Inc.
