16 results on '"Moore, Melissa J."'
Search Results
2. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex
- Author
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Nott, Ajit, Le Hir, Herve, and Moore, Melissa J.
- Subjects
Gene expression -- Research ,Ribonucleoproteins -- Research ,Metabolism -- Research ,Messenger RNA -- Research ,Mammals -- Genetic aspects ,Mammals -- Research ,Genetic research ,Biological sciences - Abstract
An essential step in eukaryotic gene expression is the removal of introns from nascent transcripts by premRNA splicing. The means by which splicing affects downstream mRNA metabolism is by altering the implement of bound proteins that, together with the mRNA comprise the mRNP.
- Published
- 2004
3. 5' Exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly
- Author
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Reichert, Vienna L., Hir, Herve Le, Jurica, Melissa S., and Moore, Melissa J.
- Subjects
Genetic research -- Analysis ,Messenger RNA -- Genetic aspects ,Proteins -- Genetic aspects ,Genetic engineering -- Research ,Biological sciences - Abstract
Research has been conducted on pre-mRNA splicing which presents a deposition of proton nucleotides unstream of exon-exon junctions on spliced mRNA. The authors have investigated the mechanism and timing of exon junction complex assembly, and have discussed the results.
- Published
- 2002
4. Translation-dependent and -independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T cell activation.
- Author
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Mercier BC, Labaronne E, Cluet D, Guiguettaz L, Fontrodona N, Bicknell A, Corbin A, Wencker M, Aube F, Modolo L, Jouravleva K, Auboeuf D, Moore MJ, and Ricci EP
- Subjects
- Animals, Mice, CD4-Positive T-Lymphocytes metabolism, Mice, Inbred C57BL, T-Lymphocytes metabolism, Ribosomes metabolism, Protein Biosynthesis, RNA Stability, Lymphocyte Activation, RNA, Messenger metabolism, RNA, Messenger genetics
- Abstract
mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4
+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells., (© 2024 Mercier et al.; Published by Cold Spring Harbor Laboratory Press.)- Published
- 2024
- Full Text
- View/download PDF
5. Corrigendum: ASC1 and RPS3 : new actors in 18S nonfunctional rRNA decay.
- Author
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Limoncelli KA, Merrikh CN, and Moore MJ
- Published
- 2018
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6. ASC1 and RPS3 : new actors in 18S nonfunctional rRNA decay.
- Author
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Limoncelli KA, Merrikh CN, and Moore MJ
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- Adaptor Proteins, Signal Transducing genetics, GTP-Binding Proteins genetics, RNA, Fungal metabolism, RNA, Ribosomal, 18S metabolism, Ribosomal Proteins genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Adaptor Proteins, Signal Transducing metabolism, GTP-Binding Proteins metabolism, Protein Processing, Post-Translational, RNA Stability, RNA, Fungal genetics, RNA, Ribosomal, 18S genetics, Ribosomal Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
In budding yeast, inactivating mutations within the 40S ribosomal subunit decoding center lead to 18S rRNA clearance by a quality control mechanism known as nonfunctional 18S rRNA decay (18S NRD). We previously showed that 18S NRD is functionally related to No-Go mRNA Decay (NGD), a pathway for clearing translation complexes stalled on aberrant mRNAs. Whereas the NGD factors Dom34p and Hbs1p contribute to 18S NRD, their genetic deletion (either singly or in combination) only partially stabilizes mutant 18S rRNA. Here we identify Asc1p (aka RACK1) and Rps3p, both stable 40S subunit components, as additional 18S NRD factors. Complete stabilization of mutant 18S rRNA in dom34 Δ; asc1 Δ and hbs1 Δ; asc1 Δ strains indicates the existence of two genetically separable 18S NRD pathways. A small region of the Rps3p C-terminal tail known to be subject to post-translational modification is also crucial for 18S NRD. We combine these findings with the effects of mutations in the 5' → 3' and 3' → 5' decay machinery to propose a model wherein multiple targeting and decay pathways kinetically contribute to 18S NRD., (© 2017 Limoncelli et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2017
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7. A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N 1 -methyladenosine modification.
- Author
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Cenik C, Chua HN, Singh G, Akef A, Snyder MP, Palazzo AF, Moore MJ, and Roth FP
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- Adenosine genetics, Adenosine metabolism, Codon, Initiator chemistry, Codon, Initiator metabolism, Eukaryotic Initiation Factor-4E genetics, Eukaryotic Initiation Factor-4E metabolism, Exons, Humans, Open Reading Frames, Protein Binding, Ribosomes genetics, Ribosomes metabolism, 5' Untranslated Regions, Adenosine analogs & derivatives, Base Sequence, Introns, Protein Biosynthesis, Sequence Deletion
- Abstract
Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5 ' proximal- i ntron- m inus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N
1 -methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1 -methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC., (© 2017 Cenik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)- Published
- 2017
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8. Twenty years of technology.
- Author
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Moore MJ
- Subjects
- Transcriptome, RNA genetics
- Published
- 2015
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9. Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes.
- Author
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Chen W, Shulha HP, Ashar-Patel A, Yan J, Green KM, Query CC, Rhind N, Weng Z, and Moore MJ
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- Blotting, Northern, Blotting, Western, High-Throughput Nucleotide Sequencing, Humans, Nucleic Acid Conformation, RNA, Small Nuclear metabolism, Schizosaccharomyces metabolism, Spliceosomes metabolism, Introns genetics, RNA Splicing genetics, RNA, Small Nuclear genetics, Schizosaccharomyces genetics, Spliceosomes genetics
- Abstract
Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2·U5·U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.
- Published
- 2014
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10. Interactions between eIF4AI and its accessory factors eIF4B and eIF4H.
- Author
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Rozovsky N, Butterworth AC, and Moore MJ
- Subjects
- Adenylyl Imidodiphosphate chemistry, Binding Sites, DEAD-box RNA Helicases chemistry, Eukaryotic Initiation Factor-4A chemistry, Eukaryotic Initiation Factors chemistry, Humans, Neoplasm Proteins metabolism, Nuclear Proteins metabolism, Protein Footprinting, Protein Interaction Mapping, RNA chemistry, RNA-Binding Proteins, Ribonucleases chemistry, DEAD-box RNA Helicases metabolism, Eukaryotic Initiation Factor-4A metabolism, Eukaryotic Initiation Factors metabolism, RNA metabolism
- Abstract
Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9-10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30-33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.
- Published
- 2008
- Full Text
- View/download PDF
11. Visualizing the splicing of single pre-mRNA molecules in whole cell extract.
- Author
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Crawford DJ, Hoskins AA, Friedman LJ, Gelles J, and Moore MJ
- Subjects
- Base Sequence, HeLa Cells, Humans, Introns, Saccharomyces cerevisiae genetics, RNA Precursors genetics, RNA Splicing, RNA, Fungal genetics, RNA, Messenger genetics
- Abstract
The excision of introns from nascent eukaryotic transcripts is catalyzed by the spliceosome, a highly complex and dynamic macromolecular machine composed of RNA and protein. Because of its complexity, biochemical analysis of the spliceosome has been previously limited to bulk assays in largely unfractionated cell extracts. We now report development of methodologies for studying the splicing of isolated single pre-mRNA molecules in real time. In this system, a fluorescently tagged pre-mRNA is tethered to a glass surface via its 3'-end. Splicing can be observed in Saccharomyces cerevisiae whole cell extract by monitoring loss of intron-specific fluorescence with a multi-wavelength total internal reflection fluorescence (TIRF) microscope. To prolong fluorophore lifetime, two enzyme-based O2 scavenging systems compatible with splicing were also developed. This work provides a powerful new approach for elucidating the mechanisms of spliceosome function and demonstrates the feasibility of utilizing TIRF microscopy for biochemical studies of single molecules in highly complex environments.
- Published
- 2008
- Full Text
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12. Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay.
- Author
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Shibuya T, Tange TØ, Stroupe ME, and Moore MJ
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Amino Acid Motifs, Amino Acid Sequence, Base Sequence, Binding Sites genetics, Codon, Nonsense, Eukaryotic Initiation Factor-4A chemistry, Eukaryotic Initiation Factor-4A metabolism, Exons, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, HeLa Cells, Humans, In Vitro Techniques, Macromolecular Substances, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, RNA Precursors chemistry, RNA Precursors genetics, RNA Precursors metabolism, RNA Splicing, RNA Stability, RNA, Messenger chemistry, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Eukaryotic Initiation Factor-4A genetics, Mutation, RNA, Messenger genetics, RNA, Messenger metabolism
- Abstract
The exon junction complex (EJC) is deposited on mRNAs by the process of pre-mRNA splicing and is a key effector of downstream mRNA metabolism. We previously demonstrated that human eIF4AIII, which is essential for nonsense-mediated mRNA decay (NMD), constitutes at least part of the RNA-binding platform anchoring other EJC components to the spliced mRNA. To determine the regions of eIF4AIII that are functionally important for EJC formation, for binding to other EJC components, and for NMD, we now report results of an extensive mutational analysis of human eIF4AIII. Using GFP-, GST- or Flag-fusions of eIF4AIII versions containing site-specific mutations or truncations, we analyzed subcellular localizations, protein-protein interactions, and EJC formation in vivo and in vitro. We also tested whether mutant proteins could rescue NMD inhibition resulting from RNAi depletion of endogenous eIF4AIII. Motifs Ia and VI, which are conserved among the eIF4A family of RNA helicases (DEAD-box proteins), are crucial for EJC formation and NMD, as is one eIF4AIII-specific region. An additional eIF4AIII-specific motif forms part of the binding site for MLN51, another EJC core component. Mutations in the canonical Walker A and B motifs that eliminate RNA-dependent ATP hydrolysis by eIF4AIII in vitro are of no detectable consequence for EJC formation and NMD activation. Implications of these findings are discussed in the context of other recent results and a new structural model for human eIF4AIII based on the known crystal structure of Saccharomyces cerevisiae eIF4AI.
- Published
- 2006
- Full Text
- View/download PDF
13. Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core.
- Author
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Tange TØ, Shibuya T, Jurica MS, and Moore MJ
- Subjects
- Cell Nucleus chemistry, Cell Nucleus metabolism, Chromatography, Liquid, Co-Repressor Proteins, Cytoplasm chemistry, Eukaryotic Initiation Factor-4A genetics, Eukaryotic Initiation Factor-4A metabolism, Gene Expression, HeLa Cells, Humans, Mass Spectrometry, Models, Biological, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, RNA, Messenger metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Ribonucleoproteins chemistry, Ribonucleoproteins genetics, Carrier Proteins genetics, Carrier Proteins metabolism, Exons, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA Splicing, Ribonucleoproteins metabolism
- Abstract
The multiprotein exon junction complex (EJC) is deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. In mammalian cells, this complex serves as a key modulator of spliced mRNA metabolism. To date, neither the complete composition nor the exact assembly pathway of the EJC has been entirely elucidated. Using in vitro splicing and a two-step chromatography procedure, we have purified the EJC and analyzed its components by mass spectrometry. In addition to finding most of the known EJC factors, we identified two novel EJC components, Acinus and SAP18. Heterokaryon analysis revealed that SAP18 is a shuttling protein whereas Acinus is restricted to the nucleus. In MS2 tethering assays Acinus stimulated gene expression at the RNA level, while MLN51, another EJC factor, stimulated mRNA translational efficiency. Using tandem affinity purification (TAP) of proteins overexpressed in HeLa cells, we demonstrated that Acinus binds directly to another EJC component, RNPS1, while stable association of SAP18 to form the trimeric apoptosis and splicing associated protein (ASAP) complex requires both Acinus and RNPS1. Using the same methodology, we further identified what appears to be the minimal stable EJC core, a heterotetrameric complex consisting of eIF4AIII, Magoh, Y14, and MLN51.
- Published
- 2005
- Full Text
- View/download PDF
14. Association of ribosomal proteins with nascent transcripts in S. cerevisiae.
- Author
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Schroder PA and Moore MJ
- Subjects
- Blotting, Western, Centrifugation, Density Gradient, Chromatin Immunoprecipitation, Genes, Fungal, Haploidy, Plasmids, Protein Biosynthesis, RNA, Fungal metabolism, RNA, Ribosomal metabolism, Ribonuclease T1 pharmacology, Ribonuclease, Pancreatic pharmacology, Ribosomal Proteins genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Ribosomal Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Transcription, Genetic
- Abstract
Although it is generally accepted that transcription and translation are spatially separated in eukaryotes, a number of recent observations have called this belief into question. In particular, several studies have shown that parts of the translation machinery, including ribosomal proteins, can be found associated with sites of active transcription in metazoans. Here we describe results of chromatin immunoprecipitation (ChIP) experiments designed to determine whether ribosomal proteins associate with nascent transcripts in Saccharomyces cerevisiae and whether this association reflects a functional engagement of the translation machinery. We find that HAT-tagged ribosomal proteins can be detected in association with nascent RNAs in budding yeast. However, our data clearly indicate that this binding is independent of transcript translatability, so is therefore not indicative of nuclear translation.
- Published
- 2005
- Full Text
- View/download PDF
15. A quantitative analysis of intron effects on mammalian gene expression.
- Author
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Nott A, Meislin SH, and Moore MJ
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- Animals, Anthozoa enzymology, Anthozoa genetics, Cell Nucleus metabolism, Cytoplasm metabolism, Genes, T-Cell Receptor beta, HeLa Cells, Humans, Luciferases genetics, Luciferases metabolism, Protein Biosynthesis, RNA Splicing, RNA Stability, RNA, Catalytic metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Gene Expression, Introns
- Abstract
In higher eukaryotes, intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles. Introns and the act of their removal by the spliceosome can affect gene expression at many different levels, including transcription, polyadenylation, mRNA export, translational efficiency, and the rate of mRNA decay. However, the extent to which each of these steps contributes to the overall effect of any one intron on gene expression has not been rigorously tested. Here we report construction and initial characterization of a luciferase-based reporter system for monitoring the effects of individual introns and their position within the gene on protein expression in mammalian cells. Quantitative analysis of constructs containing human TPI intron 6 at two different positions within the Renilla luciferase open reading frame revealed that this intron acts primarily to enhance mRNA accumulation. Spliced mRNAs also exhibited higher translational yields than did intronless transcripts. However, nucleocytoplasmic mRNA distribution and mRNA stability were largely unaffected. These findings were extended to two other introns in a TCR-beta minigene.
- Published
- 2003
- Full Text
- View/download PDF
16. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.
- Author
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Jurica MS, Licklider LJ, Gygi SR, Grigorieff N, and Moore MJ
- Subjects
- Adenosine Triphosphatases chemistry, DEAD-box RNA Helicases, Fungal Proteins chemistry, Mass Spectrometry, Microscopy, Electron, RNA Helicases, RNA Precursors chemistry, RNA Splicing, RNA Splicing Factors, RNA, Small Nuclear chemistry, RNA-Binding Proteins chemistry, Ribonuclease H chemistry, Ribonuclease H metabolism, Cell Cycle Proteins, DNA-Binding Proteins, Ribonucleoproteins, Small Nuclear, Saccharomyces cerevisiae Proteins, Spliceosomes chemistry
- Abstract
We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.
- Published
- 2002
- Full Text
- View/download PDF
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