26 results on '"Singer, Robert H."'
Search Results
2. ZBP1 recognition of beta-actin zipcode induces RNA looping
- Author
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Chao, Jeffrey A., Patskovsky, Yury, Patel, Vivek, Levy, Matthew, Almo, Steven C., and Singer, Robert H.
- Subjects
Binding proteins -- Research ,Genetic translation -- Analysis ,X-ray crystallography -- Usage ,Biological sciences - Published
- 2010
3. Translation of ASH1 mRNA is repressed by Puf6p-Fun12p/eIF5B interaction and released by CK2 phosphorylation
- Author
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Deng, Yingfeng, Singer, Robert H., and Wei Gu
- Subjects
Gene expression -- Analysis ,Messenger RNA -- Research ,Phosphorylation -- Analysis ,Genetic translation -- Analysis ,Yeast fungi -- Genetic aspects ,Biological sciences - Abstract
Yeast based in vitro translation assays are used to characterize the mechanism of Puf6p-mediated translational regulation. Results show that Puf6p suppresses the translation initiation of ASH1 mRNA via interaction with Fun12p during its transport.
- Published
- 2008
4. A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization
- Author
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Wei Gu, Yingfeng Deng, Daniel Zenklusen, and Singer, Robert H.
- Subjects
Proteins -- Research ,Messenger RNA -- Research ,Yeast fungi -- Genetic aspects ,Biological sciences - Abstract
The ASH1 locasome is isolated and the associated proteins by MALDI-TOF are characterized to study the mechanism regulating ASH1 mRNA translation. Puf6p, a protein that functions in the translational control of ASH1 MRNA is proposed and this translational inhibition is necessary before localization can proceed.
- Published
- 2004
5. The cytoplasmic fate of an mRNP is determined cotranscriptionally: exception or rule?
- Author
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Trcek, Tatjana and Singer, Robert H.
- Subjects
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CARRIER proteins , *PROTEIN binding , *YEAST , *CYTOPLASM , *RNA , *MESSENGER RNA , *GENETIC transcription - Abstract
She2p is an RNA-binding protein that recognizes a zipcode on specific mRNAs necessary for the assembly of a protein complex that localizes them to the yeast bud tip. In this issue of Genes & Development, Shen and colleagues (pp. 1914-1926) demonstrate that She2p associates with RNAPII globally, but then recognizes the nascent chain only if it contains a zipcode. This demonstrates yet another case where the mRNA's cytoplasmic fate is determined by the RNAPII complex. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
6. Translation of ASH1 mRNA is repressed by Puf6p--Fun12p/eIF5B interaction and released by CK2 phosphorylation.
- Author
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Yingfeng Deng, Singer, Robert H., and Wei Gu
- Subjects
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MESSENGER RNA , *PHOSPHORYLATION , *PROTEINS , *SACCHAROMYCES cerevisiae , *PROTEIN kinases - Abstract
Translational repression during mRNA transport is essential for spatial restriction of protein production. In the yeast Saccharomyces cerevisae, silencing of ASH1 mRNA before it is localized to the bud cortex in late anaphase is critical for asymmetric segregation of Ash1p to the daughter cell nucleus. Puf6p, an ASH1 mRNA-binding protein, has been implicated in this process as a translational repressor, but the underlying mechanism is unknown. Here, we used yeast extract-based in vitro translation assays, which recapitulate translation and phosphorylation, to characterize the mechanism of Puf6p-mediated translational regulation. We report that Puf6p interferes with the conversion of the 48S complex to the 80S complex during initiation, and this repression by Puf6p is mediated through the general translation factor eIF5B (Fun12p in S. cerevisiae). Puf6p interacts with Fun12p via the PUF domain, and this interaction is RNA-dependent and essential for translational repression by Puf6p. This repression is relieved by phosphorylation of the N-terminal region of Puf6p mediated by protein kinase CK2 (casein kinase II). Inhibition of phosphorylation at Ser31, Ser34, and Ser35 of Puf6p increases its translational repression and results in ASH1 mRNA delocalization. Our results indicate that Puf6p suppresses the translation initiation of ASH1 mRNA via interaction with Fun12p during its transport, and this repression can be released by CK2 phosphorylation in the N-terminal region of Puf6p when the mRNA reaches the bud tip. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
7. Temporal and spatial characterization of nonsense-mediated mRNA decay.
- Author
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Trcek, Tatjana, Sato, Hanae, Singer, Robert H., and Maquat, Lynne E.
- Subjects
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MESSENGER RNA , *TRANSFER-messenger RNA , *GENETIC code , *NUCLEOTIDE sequence , *CYTOPLASM - Abstract
Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. T o address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing ß-globin mRNA in intact single cells after the induction of ß-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing ß-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free ß-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
8. Imaging Transcription: Past, Present, and Future.
- Author
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COLEMAN, ROBERT A., ZHE LIU, DARZACQ, XAVIER, TJIAN, ROBERT, SINGER, ROBERT H., and LIONNET, TIMOTHÉE
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CELL imaging , *GENETIC transcription regulation , *GENETIC regulation , *MATHEMATICAL models - Abstract
Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Singlemolecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
9. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.
- Author
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Bin Wu, Miskolci, Veronika, Sato, Hanae, Tutucci, Evelina, Kenworthy, Charles A., Donnelly, Sara K., Yoon, Young J., Cox, Dianne, Singer, Robert H., and Hodgson, Louis
- Subjects
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NUCLEOTIDE sequence , *MESSENGER RNA , *PROTEINS , *FLUORESCENCE resonance energy transfer , *BIOSENSORS - Abstract
Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we showhere a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
10. β-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration.
- Author
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Katz, Zachary B., Wells, Amber L., Hye Yoon Park, Bin Wu, Shenoy, Shailesh M., and Singer, Robert H.
- Subjects
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ACTIN , *MESSENGER RNA , *FOCAL adhesions , *CELL migration , *CELL motility , *WOUND healing - Abstract
Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell's cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports β-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of β-actin mRNA and protein localization by tethering the mRNA to a specific location—in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
11. IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling.
- Author
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Stöhr, Nadine, Köhn, Marcel, Lederer, Marcell, Glaß, Markus, Reinke, Claudia, Singer, Robert H., and Hüttelmaier, Stefan
- Subjects
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CARRIER proteins , *CANCER cells , *CELL migration , *PHOSPHORYLATION , *POLYMERIZATION - Abstract
In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted β-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP3-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
12. Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control.
- Author
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Patel, Vivek L., Mitra, Somdeb, Harris, Richard, Buxbaum, Adina R., Lionnet, Timothée, Brenowitz, Michael, Girvin, Mark, Levy, Matthew, Almo, Steven C., Singer, Robert H., and Chao, Jeffrey A.
- Subjects
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RNA , *CARRIER proteins , *MESSENGER RNA , *HOMOLOGY (Biology) , *BINDING sites - Abstract
How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the β-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 5' element (CGGAC) followed by a variable 3' element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
13. Imaging Organization of RNA Processing within the Nucleus.
- Author
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Biswas J, Li W, Singer RH, and Coleman RA
- Subjects
- Alternative Splicing, RNA, Messenger metabolism, Single Molecule Imaging, Transcription, Genetic, Cell Nucleus metabolism, RNA metabolism
- Abstract
Within the nucleus, messenger RNA is generated and processed in a highly organized and regulated manner. Messenger RNA processing begins during transcription initiation and continues until the RNA is translated and degraded. Processes such as 5' capping, alternative splicing, and 3' end processing have been studied extensively with biochemical methods and more recently with single-molecule imaging approaches. In this review, we highlight how imaging has helped understand the highly dynamic process of RNA processing. We conclude with open questions and new technological developments that may further our understanding of RNA processing., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
14. Fluorescence Imaging Methods to Investigate Translation in Single Cells.
- Author
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Biswas J, Liu Y, Singer RH, and Wu B
- Subjects
- Kinetics, Optical Imaging methods, Protein Biosynthesis, Single-Cell Analysis
- Abstract
Translation is the fundamental biological process that converts the genetic information in messenger RNAs (mRNAs) into functional proteins. Translation regulation allows cells to control when, where, and how many proteins are synthesized. Much of what we know about translation comes from ensemble approaches that measure the average of many cells. The cellular and molecular heterogeneity in the regulation of translation remains largely elusive. Fluorescence microscopy allows interrogation of biological problems with single-molecule, single-cell sensitivity. In recent years, improved design of reagents and microscopy tools has led to improved spatial and temporal resolution of translation imaging. It is now possible to track global translation in specific subcellular compartments and follow the translation dynamics of single transcripts. Highlighted here is the recent progress in translation imaging with emphasis on in vivo translation dynamics. These tools will be invaluable to the study of translation regulation., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
15. Zipcode Binding Protein 1 (ZBP1; IGF2BP1): A Model for Sequence-Specific RNA Regulation.
- Author
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Biswas J, Nunez L, Das S, Yoon YJ, Eliscovich C, and Singer RH
- Abstract
The fate of an RNA, from its localization, translation, and ultimate decay, is dictated by interactions with RNA binding proteins (RBPs). β-actin mRNA has functioned as the classic example of RNA localization in eukaryotic cells. Studies of β-actin mRNA over the past three decades have allowed understanding of how RBPs, such as ZBP1 (IGF2BP1), can control both RNA localization and translational status. Here, we summarize studies of β-actin mRNA and focus on how ZBP1 serves as a model for understanding interactions between RNA and their binding protein(s). Central to the study of RNA and RBPs were technological developments that occurred along the way. We conclude with a future outlook highlighting new technologies that may be used to address still unanswered questions about RBP-mediated regulation of mRNA during its life cycle, within the cell., (© 2019 Biswas et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2019
- Full Text
- View/download PDF
16. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.
- Author
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Wu B, Miskolci V, Sato H, Tutucci E, Kenworthy CA, Donnelly SK, Yoon YJ, Cox D, Singer RH, and Hodgson L
- Subjects
- Animals, Base Sequence genetics, Capsid Proteins genetics, Cell Line, Cells, Cultured, Codon genetics, Humans, Levivirus genetics, Mice, Nucleotide Motifs, Saccharomyces cerevisiae genetics, Gene Expression, Genetic Techniques, Repetitive Sequences, Nucleic Acid genetics
- Abstract
Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts., (© 2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2015
- Full Text
- View/download PDF
17. Reminiscences on my life with RNA: a self-indulgent perspective.
- Author
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Singer RH
- Subjects
- Humans, Narration, RNA genetics, RNA metabolism, RNA chemistry
- Published
- 2015
- Full Text
- View/download PDF
18. Imaging translation in single cells using fluorescent microscopy.
- Author
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Chao JA, Yoon YJ, and Singer RH
- Subjects
- Animals, Humans, Neurons physiology, Puromycin, Gene Expression Regulation physiology, Microscopy, Fluorescence methods, Neurons ultrastructure, Protein Biosynthesis physiology, Synapses physiology
- Abstract
The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.
- Published
- 2012
- Full Text
- View/download PDF
19. Transgenic expression of ZBP1 in neurons suppresses cocaine-associated conditioning.
- Author
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Lapidus KA, Nwokafor C, Scott D, Baroni TE, Tenenbaum SA, Hiroi N, Singer RH, and Czaplinski K
- Subjects
- Animals, Brain drug effects, Brain metabolism, Cocaine administration & dosage, Conditioning, Psychological drug effects, Cues, DNA-Binding Proteins genetics, Dopamine Uptake Inhibitors administration & dosage, Doxycycline pharmacology, Gene Expression Regulation drug effects, Green Fluorescent Proteins genetics, Immunoprecipitation, Male, Mice, Mice, Transgenic, Microarray Analysis, Neurons drug effects, RNA, Messenger metabolism, RNA-Binding Proteins metabolism, Brain cytology, Conditioning, Psychological physiology, DNA-Binding Proteins metabolism, Gene Expression Regulation genetics, Neurons metabolism
- Abstract
To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We took advantage of the absence of ZBP1 expression in the mature brain to examine the effect of expressing ZBP1 on animal behavior. We constructed a transgene conditionally expressing a GFP-ZBP1 fusion protein in a subset of forebrain neurons and compared cocaine-cued place conditioning in these mice versus noninduced littermates. Transgenic ZBP1 expression resulted in impaired place conditioning relative to nonexpressing littermates, and acutely repressing expression of the transgene restored normal cocaine conditioning. To gain insight into the molecular changes that accounted for this change in behavior, we identified mRNAs that specifically immunoprecipitated with transgenic ZBP1 protein from the brains of these mice. These data suggest that RNA-binding proteins can be used as a tool to identify the post-transcriptional regulation of gene expression in the establishment and function of neural circuits involved in addiction behaviors.
- Published
- 2012
- Full Text
- View/download PDF
20. Cotranscriptional effect of a premature termination codon revealed by live-cell imaging.
- Author
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de Turris V, Nicholson P, Orozco RZ, Singer RH, and Mühlemann O
- Subjects
- Cell Line, Cytoplasm genetics, Cytoplasm metabolism, Gene Expression Regulation, Gene Order, Gene Silencing, Humans, Immunoglobulin mu-Chains genetics, In Situ Hybridization, Fluorescence, Nonsense Mediated mRNA Decay, RNA Helicases, RNA Splicing, Telomerase genetics, Telomerase metabolism, Trans-Activators genetics, Trans-Activators metabolism, Codon, Nonsense, RNA, Messenger metabolism, Transcription, Genetic
- Abstract
Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.
- Published
- 2011
- Full Text
- View/download PDF
21. A nucleoporin, Nup60p, affects the nuclear and cytoplasmic localization of ASH1 mRNA in S. cerevisiae.
- Author
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Powrie EA, Zenklusen D, and Singer RH
- Subjects
- Cell Nucleus genetics, Gene Deletion, In Situ Hybridization, Fluorescence, Nuclear Pore Complex Proteins genetics, Poly A genetics, Poly A metabolism, RNA Transport, RNA, Fungal genetics, RNA, Messenger genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Repressor Proteins genetics, Saccharomyces cerevisiae Proteins genetics, Transcription, Genetic, Cell Nucleus metabolism, Cytoplasm metabolism, Nuclear Pore Complex Proteins metabolism, RNA, Fungal metabolism, RNA, Messenger metabolism, Repressor Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The biogenesis of a localization-competent mRNP begins in the nucleus. It is thought that the coordinated action of nuclear and cytoplasmic components of the localization machinery is required for the efficient export and subsequent subcellular localization of these mRNAs in the cytoplasm. Using quantitative poly(A)(+) and transcript-specific fluorescent in situ hybridization, we analyzed different nonessential nucleoporins and nuclear pore-associated proteins for their potential role in mRNA export and localization. We found that Nup60p, a nuclear pore protein located on the nucleoplasmic side of the nuclear pore complex, was required for the mRNA localization pathway. In a Δnup60 background, localized mRNAs were preferentially retained within the nucleus compared to nonlocalized transcripts. However, the export block was only partial and some transcripts could still reach the cytoplasm. Importantly, downstream processes were also affected. Localization of ASH1 and IST2 mRNAs to the bud was impaired in the Δnup60 background, suggesting that the assembly of a localization competent mRNP ("locasome") was inhibited when NUP60 was deleted. These results demonstrate transcript specificity of a nuclear mRNA retention defect and identify a specific nucleoporin as a functional component of the localization pathway in budding yeast.
- Published
- 2011
- Full Text
- View/download PDF
22. RNA processing and export.
- Author
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Hocine S, Singer RH, and Grünwald D
- Subjects
- Active Transport, Cell Nucleus physiology, DNA-Binding Proteins metabolism, Cell Nucleus metabolism, Gene Expression Regulation physiology, RNA Processing, Post-Transcriptional physiology, RNA, Messenger metabolism, Transcription, Genetic physiology
- Abstract
Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression.
- Published
- 2010
- Full Text
- View/download PDF
23. Myo2p, a class V myosin in budding yeast, associates with a large ribonucleic acid-protein complex that contains mRNAs and subunits of the RNA-processing body.
- Author
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Chang W, Zaarour RF, Reck-Peterson S, Rinn J, Singer RH, Snyder M, Novick P, and Mooseker MS
- Subjects
- Actins metabolism, Adenosine Triphosphatases metabolism, Base Sequence, DNA Primers genetics, Macromolecular Substances, Myosin Heavy Chains genetics, Myosin Type V genetics, Oligonucleotide Array Sequence Analysis, Organelles metabolism, Polyribosomes metabolism, RNA Processing, Post-Transcriptional, RNA, Fungal chemistry, RNA, Fungal genetics, RNA, Messenger chemistry, RNA, Messenger genetics, Ribonucleoproteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Secretory Vesicles metabolism, Vacuoles metabolism, Myosin Heavy Chains chemistry, Myosin Heavy Chains metabolism, Myosin Type V chemistry, Myosin Type V metabolism, RNA, Fungal metabolism, RNA, Messenger metabolism, Ribonucleoproteins chemistry, Ribonucleoproteins metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Myo2p is an essential class V myosin in budding yeast with several identified functions in organelle trafficking and spindle orientation. The present study demonstrates that Myo2p is a component of a large RNA-containing complex (Myo2p-RNP) that is distinct from polysomes based on sedimentation analysis and lack of ribosomal subunits in the Myo2p-RNP. Microarray analysis of RNAs that coimmunoprecipitate with Myo2p revealed the presence of a large number of mRNAs in this complex. The Myo2p-RNA complex is in part composed of the RNA processing body (P-body) based on coprecipitation with P-body protein subunits and partial colocalization of Myo2p with P-bodies. P-body disassembly is delayed in the motor mutant, myo2-66, indicating that Myo2p may facilitate the release of mRNAs from the P-body.
- Published
- 2008
- Full Text
- View/download PDF
24. Dynamic association and localization of human H/ACA RNP proteins.
- Author
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Kittur N, Darzacq X, Roy S, Singer RH, and Meier UT
- Subjects
- Active Transport, Cell Nucleus, Amino Acid Sequence, Binding Sites, Cells, Cultured, Dyskeratosis Congenita genetics, Humans, Molecular Sequence Data, Nuclear Localization Signals, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA, Small Nuclear genetics, Telomerase genetics, Telomerase metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, RNA, Small Nuclear metabolism, Ribonucleoproteins, Small Nucleolar genetics, Ribonucleoproteins, Small Nucleolar metabolism
- Abstract
Mammalian H/ACA RNPs are essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. To form mature RNA-protein complexes, one H/ACA RNA associates with four core proteins. In the cell, this process is assisted by at least one nuclear assembly factor, NAF1. Here we report several unanticipated dynamic aspects of H/ACA RNP proteins. First, when overexpressed, NAF1 delocalizes to the cytoplasm. However, its nucleocytoplasmic shuttling properties remain unaffected. These observations demonstrate a subtle equilibrium between NAF1 expression levels and the availability of NAF1 nuclear binding sites. Second, although NAF1 is excluded from mature RNPs in nucleoli and Cajal bodies, NAF1 associates with mature H/ACA RNA in cell lysates. This association occurs post-lysis because it is observed even when NAF1 and the H/ACA RNA are expressed in separate cells. This documents a protein-RNP association in cell lysates that is absent from intact cells. Third, in similar experiments, all H/ACA core proteins, except NAP57, exchange with their exogenous counterparts, portraying an unexpected dynamic picture of H/ACA RNPs. Finally, the irreversible association of only NAP57 with H/ACA RNA and the conundrum that only NAP57 is mutated in X-linked dyskeratosis congenita (even though most core proteins are required for maintaining H/ACA RNAs) may be more than a coincidence.
- Published
- 2006
- Full Text
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25. A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization.
- Author
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Gu W, Deng Y, Zenklusen D, and Singer RH
- Subjects
- 3' Untranslated Regions, DNA-Binding Proteins biosynthesis, Gene Expression Regulation, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins physiology, Repressor Proteins biosynthesis, Ribonucleoproteins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transcription Factors biosynthesis, DNA-Binding Proteins genetics, Drosophila Proteins, Protein Biosynthesis, Repressor Proteins genetics, Repressor Proteins physiology, Saccharomyces cerevisiae Proteins physiology, Transcription Factors genetics
- Abstract
In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p-bound PUF consensus sequences in the 3'UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro. In the puf6 Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed.
- Published
- 2004
- Full Text
- View/download PDF
26. An active precursor in assembly of yeast nuclear ribonuclease P.
- Author
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Srisawat C, Houser-Scott F, Bertrand E, Xiao S, Singer RH, and Engelke DR
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Nucleolus metabolism, Endoribonucleases genetics, Enzyme Precursors genetics, Molecular Sequence Data, Protein Subunits, RNA, Catalytic genetics, RNA, Fungal metabolism, Ribonuclease P, Ribonucleoproteins genetics, Saccharomyces cerevisiae Proteins genetics, Sequence Homology, Amino Acid, Endoribonucleases metabolism, Enzyme Precursors metabolism, RNA, Catalytic metabolism, Ribonucleoproteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Yeasts metabolism
- Abstract
The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.
- Published
- 2002
- Full Text
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