ABSTRACT In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel... more ABSTRACT In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the “ultrasonic emulsification method”. It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA–RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA–BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA–BSA complex are reported.
Physical review. E, Statistical, nonlinear, and soft matter physics, 2015
The stochastic process of gene expression is commonly controlled at the level of RNA transcriptio... more The stochastic process of gene expression is commonly controlled at the level of RNA transcription. The synthesis of messenger RNA (mRNA) is a multistep process, performed by RNA polymerase II and controlled by many transcription factors. Although mRNA transcription is intensively studied, real-time in vivo dynamic rates of a single transcribing polymerase are still not available. A popular method for examining transcription kinetics is the fluorescence recovery after photobleaching (FRAP) approach followed by kinetic modeling. Such analysis has yielded a surprisingly broad range of transcription rates. As transcription depends on many variables such as the chromatin state, binding and unbinding of transcription factors, and cell phase, transcription rates are stochastic variables. Thus, the distribution of rates is expected to follow Poissonian statistics, which does not coincide with the wide range of transcription rate results. Here we present an approach for analyzing FRAP data ...
The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limita... more The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.
Technical advances in the field of live-cell imaging have introduced the cell biologist to a new,... more Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time.
Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed loc... more Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed locally by the mesenchymal component of the hemopoietic microenvironment. Its expression is regulated on the mRNA level by different cytokines, and the biological activity of the protein is tightly controlled by several inhibitory molecules. Activin A affects hemopoietic cells of various lineages, as evidenced by in vitro studies of leukemia and lymphoma cell lines, which were used to elucidate the mechanism of its action. In the B-cell lineage, activin A is a cell cycle inhibitor, a mediator of apoptosis, and a cytokine antagonist. Limited information is available on the effects of activin A on normal hemopoietic cells. Recent studies suggest that it might be a negative regulator of normal B lymphopoiesis. Whereas the functions of activin A in vitro are well established, further research tools are needed to elucidate its role within specific hemopoietic microenvironments in vivo.
A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a nove... more A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a novel mouse monoclonal antibody (B92) raised against stromal cells. The protein was found in the immunizing cells using a sensitive method. However, its detection in the bone marrow by the B92 antibody seemed to stem from the abundance of p49 in immature cells of the myeloid lineage. Study of the bone marrow following in vivo irradiation or 5-fluorouracil (5-FU) treatment, in vitro culture with differentiation-inducing factors and long-term culture, and cell sorting all pointed in the same direction: the protein was found in early myeloid cells and in hematopoietic precursor cells. These results were in accordance with the specific presence of p49 in primary radiation-induced myeloid leukemia and its absence in spontaneous B lymphoma. Immunofluorescent staining using B92 antibody detected a nuclear antigen forming a dotted pattern in early myeloid cells and day 12 colony-forming units-spleen...
Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the c... more Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.
The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore mea... more The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods. In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells. Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells. Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest. The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed. To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs. In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.
RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mat... more RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.
RNAs are exported from the nucleus to the cytoplasm, where they undergo translation and produce p... more RNAs are exported from the nucleus to the cytoplasm, where they undergo translation and produce proteins needed for the cellular life cycle. Some mRNAs are targeted by different RNA decay mechanisms and thereby undergo degradation. The 5'-->3' degradation machinery localizes to cytoplasmic complexes termed P bodies (PBs). They function in RNA turnover, translational repression, RNA-mediated silencing, and RNA storage. A quantitative live-cell imaging approach to study the dynamic aspects of PB trafficking in the cytoplasm revealed that PB movements are rather confined and dependent on an existing microtubule network. Microtubule depolymerization led to a drastic decrease in PB mobility, as well as a release of regulation on PB assembly and a dramatic increase in PB numbers. The different aspects of PB trafficking and encounters with mRNA molecules in the cytoplasm are discussed.
The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm thro... more The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm through the nuclear pore complex (NPC) is an important basic cellular process. Originally, in order to visualize this process, single mRNP export was examined using electron microscopy (EM) in fixed Chironomus tentans specimens. These studies described the nucleocytoplasmic translocation of huge mRNPs (~30 kb) transcribed from the Balbiani-ring genes. However, knowledge of the in vivo mRNP kinetics in cell compartments remained poor up until recently. The current use of unique fluorescent protein tags, which are able to bind to mRNA transcripts, has allowed the detection and measurements of single mRNP kinetics in living cells. This has demonstrated that mRNP movement is affected by the size of the transcript and the splicing process. It was found that mRNP rates of translocation are slower in the nucleus compared to the cytoplasm and that the cell nucleus contains interchromatin tracks in which mRNPs diffuse. In order to track single mRNP movement in living cells, it is important to be able to identify single mRNP molecules transcribed from a certain gene, at the single-cell level. Single-molecule analysis of gene expression requires advanced imaging systems and analytical software in order to detect and follow the movement of single mRNPs. In this chapter we describe the methods required for the detection and tracking of single mRNP movement in living mammalian cells.
ABSTRACT In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel... more ABSTRACT In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the “ultrasonic emulsification method”. It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA–RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA–BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA–BSA complex are reported.
Physical review. E, Statistical, nonlinear, and soft matter physics, 2015
The stochastic process of gene expression is commonly controlled at the level of RNA transcriptio... more The stochastic process of gene expression is commonly controlled at the level of RNA transcription. The synthesis of messenger RNA (mRNA) is a multistep process, performed by RNA polymerase II and controlled by many transcription factors. Although mRNA transcription is intensively studied, real-time in vivo dynamic rates of a single transcribing polymerase are still not available. A popular method for examining transcription kinetics is the fluorescence recovery after photobleaching (FRAP) approach followed by kinetic modeling. Such analysis has yielded a surprisingly broad range of transcription rates. As transcription depends on many variables such as the chromatin state, binding and unbinding of transcription factors, and cell phase, transcription rates are stochastic variables. Thus, the distribution of rates is expected to follow Poissonian statistics, which does not coincide with the wide range of transcription rate results. Here we present an approach for analyzing FRAP data ...
The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limita... more The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.
Technical advances in the field of live-cell imaging have introduced the cell biologist to a new,... more Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time.
Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed loc... more Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed locally by the mesenchymal component of the hemopoietic microenvironment. Its expression is regulated on the mRNA level by different cytokines, and the biological activity of the protein is tightly controlled by several inhibitory molecules. Activin A affects hemopoietic cells of various lineages, as evidenced by in vitro studies of leukemia and lymphoma cell lines, which were used to elucidate the mechanism of its action. In the B-cell lineage, activin A is a cell cycle inhibitor, a mediator of apoptosis, and a cytokine antagonist. Limited information is available on the effects of activin A on normal hemopoietic cells. Recent studies suggest that it might be a negative regulator of normal B lymphopoiesis. Whereas the functions of activin A in vitro are well established, further research tools are needed to elucidate its role within specific hemopoietic microenvironments in vivo.
A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a nove... more A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a novel mouse monoclonal antibody (B92) raised against stromal cells. The protein was found in the immunizing cells using a sensitive method. However, its detection in the bone marrow by the B92 antibody seemed to stem from the abundance of p49 in immature cells of the myeloid lineage. Study of the bone marrow following in vivo irradiation or 5-fluorouracil (5-FU) treatment, in vitro culture with differentiation-inducing factors and long-term culture, and cell sorting all pointed in the same direction: the protein was found in early myeloid cells and in hematopoietic precursor cells. These results were in accordance with the specific presence of p49 in primary radiation-induced myeloid leukemia and its absence in spontaneous B lymphoma. Immunofluorescent staining using B92 antibody detected a nuclear antigen forming a dotted pattern in early myeloid cells and day 12 colony-forming units-spleen...
Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the c... more Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.
The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore mea... more The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods. In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells. Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells. Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest. The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed. To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs. In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.
RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mat... more RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.
RNAs are exported from the nucleus to the cytoplasm, where they undergo translation and produce p... more RNAs are exported from the nucleus to the cytoplasm, where they undergo translation and produce proteins needed for the cellular life cycle. Some mRNAs are targeted by different RNA decay mechanisms and thereby undergo degradation. The 5'-->3' degradation machinery localizes to cytoplasmic complexes termed P bodies (PBs). They function in RNA turnover, translational repression, RNA-mediated silencing, and RNA storage. A quantitative live-cell imaging approach to study the dynamic aspects of PB trafficking in the cytoplasm revealed that PB movements are rather confined and dependent on an existing microtubule network. Microtubule depolymerization led to a drastic decrease in PB mobility, as well as a release of regulation on PB assembly and a dramatic increase in PB numbers. The different aspects of PB trafficking and encounters with mRNA molecules in the cytoplasm are discussed.
The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm thro... more The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm through the nuclear pore complex (NPC) is an important basic cellular process. Originally, in order to visualize this process, single mRNP export was examined using electron microscopy (EM) in fixed Chironomus tentans specimens. These studies described the nucleocytoplasmic translocation of huge mRNPs (~30 kb) transcribed from the Balbiani-ring genes. However, knowledge of the in vivo mRNP kinetics in cell compartments remained poor up until recently. The current use of unique fluorescent protein tags, which are able to bind to mRNA transcripts, has allowed the detection and measurements of single mRNP kinetics in living cells. This has demonstrated that mRNP movement is affected by the size of the transcript and the splicing process. It was found that mRNP rates of translocation are slower in the nucleus compared to the cytoplasm and that the cell nucleus contains interchromatin tracks in which mRNPs diffuse. In order to track single mRNP movement in living cells, it is important to be able to identify single mRNP molecules transcribed from a certain gene, at the single-cell level. Single-molecule analysis of gene expression requires advanced imaging systems and analytical software in order to detect and follow the movement of single mRNPs. In this chapter we describe the methods required for the detection and tracking of single mRNP movement in living mammalian cells.
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Papers by Yaron Shav-tal