8 dynamics of gene expression
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Journal Article↗
Biomedical subjects
T Misteli
Publications and source records attributed to T Misteli.
Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo.
Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.
Amino Acid Sequence↗
The cellular organization of gene expression.
Recent cell biological observations have provided new insights into how transcription, pre-mRNA splicing and 3' processing are organized and coordinated with each other in the mammalian cell nucleus. Morphological observations are supported by biochemical evidence that suggests physical interactions between components of the transcription and RNA processing machineries. A working model of the cellular organization of gene expression is now emerging.
Animals↗
Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity.
SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.
Alternative Splicing↗
The dynamics of a pre-mRNA splicing factor in living cells.
Pre-mRNA splicing is a predominantly co-transcriptional event which involves a large number of essential splicing factors. Within the mammalian cell nucleus, most splicing factors are concentrated in 20-40 distinct domains called speckles. The function of speckles and the organization of cellular transcription and pre-mRNA splicing in vivo are not well understood. We have investigated the dynamic properties of splicing factors in nuclei of living cells. Here we show that speckles are highly dynamic structures that respond specifically to activation of nearby genes. These dynamic events are dependent on RNA polymerase II transcription, and are sensitive to inhibitors of protein kinases and Ser/Thr phosphatases. When single genes are transcriptionally activated in living cells, splicing factors leave speckles in peripheral extensions and accumulate at the new sites of transcription. We conclude that one function of speckles is to supply splicing factors to active genes. Our observations demonstrate that the interphase nucleus is far more dynamic in nature than previously assumed.
Cell Compartmentation↗
Applications of the green fluorescent protein in cell biology and biotechnology.
The recent emergence of an autofluorescent protein, the green fluorescent protein (GFP), has opened the door for the convenient use of intact living cells and organisms as experimental systems in fields ranging from cell biology to biomedicine. We present an overview of some of the major applications of GFP, namely its use in protein tagging and in monitoring gene expression as well as its potential in a variety of biological screens.
Animals↗
Molecular mechanisms in the disassembly and reassembly of the mammalian Golgi apparatus during M-phase.
The mitotic disassembly and reassembly of the mammalian Golgi apparatus is an ideal system to study the molecular mechanisms involved in biogenesis and maintenance of membranous organelles. As cells enter M-phase, Golgi stacks are converted into Golgi clusters of small membrane fragments, which are dispersed throughout the cytoplasmic space during metaphase. Disassembly is dependent on the action of cdc2-kinase and at least two distinct pathways contribute to the fragmentation: one involves the budding of COP I-coated vesicles from Golgi cisternae, the other is a less well characterised COP I-independent pathway. During telophase, the Golgi fragments reassemble and fuse into a fully functional Golgi stack, using at least two distinct ATPase-mediated fusion pathways.
Animals↗
The mammalian Golgi apparatus during M-phase.
The Golgi apparatus in mammalian cells disassembles into several thousand vesicles as cells enter M-phase. Disassembly is dependent on the action of cdc2-kinase and at least two pathways contribute to the fragmentation: One involves the budding of COP-coated vesicles from Golgi cisternae with concomitant inhibition of fusion with their target membranes, the other is a less well characterised COP-independent pathway. During telophase, the Golgi fragments reassemble and fuse into a fully functional Golgi stack, using at least two distinct fusion pathways. The morphological changes of the Golgi apparatus during M-phase offer an ideal system to study how cellular organelles are generated and how their structure is maintained during interphase.
Animals↗
Sorting by COP I-coated vesicles under interphase and mitotic conditions.
COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.
Amino Acid Sequence↗
Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors.
HeLa cell nuclei were permeabilized and reconstituted with nuclear extract to identify soluble nuclear factors which play a role in the organization of pre-mRNA splicing factors in the mammalian cell nucleus. Permeabilized nuclei reconstituted with nuclear extract were active in transcription and DNA replication and nuclear speckles containing pre-mRNA splicing factors were maintained over several hours independent of soluble nuclear components. The characteristic rounding up of nuclear speckles in response to inhibition of RNA polymerase II seen in vivo was reproduced in permeabilized cells and was strictly dependent on a catalytic activity present in the nuclear extract. By inhibitor titration experiments and sensitivity to inhibitor 2, this activity was identified as a member of the serine/threonine protein phosphatase 1 family (PP1). Interference with PP1 activity affected the distribution of pre-mRNA splicing factors in transcriptionally active, permeabilized cells, and excess PP1 activity caused increased dephosphorylation of SR proteins in nuclear speckles. These data show that the dynamic reorganization of the mammalian cell nucleus can be studied in permeabilized cells and that PP1 is involved in the rounding up of speckles as well as the overall organization of pre-mRNA splicing factors in the mammalian cell nucleus.
Amanitins↗
Evidence for recycling of the resident medial/trans Golgi enzyme, N-acetylglucosaminyltransferase I, in ldlD cells.
ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc-Glc-NAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-Glc-NAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.
Animals↗
Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system.
Rat liver Golgi stacks were incubated with mitotic cytosol for 30 min at 37 degrees C to generate mitotic Golgi fragments comprising vesicles, tubules, and cisternal remnants. These were isolated and further incubated with rat liver cytosol for 60 min. The earliest intermediate observed by electron microscopy was a single, curved cisterna with tubular networks fused to the cisternal rims. Elongation of this cisterna was accompanied by stacking and further growth at the cisternal rims. Stacks also fused laterally so that the typical end product was a highly curved stack of 2-3 cisternae mostly enclosing an electron-lucent space. Reassembly occurred in the presence of nocodazole or cytochalasin B but not at 4 degrees C or in the absence of energy supplied in the form of ATP and GTP. Pretreatment of the mitotic fragments and cytosol with N-ethylmaleimide (NEM) also prevented reassembly. GTP gamma S and A1F prevented reassembly when added during fragmentation but not when added to the reassembly mixture. In fact, GTP gamma S stimulated reassembly such that all cisternae were stacked at the end of the incubation and comprised 40% of the total membrane. In contrast, microcystin inhibited stacking so that only single cisternae accumulated. Together these results provide a detailed picture of the reassembly process and open up the study of the architecture of the Golgi apparatus to a combined morphological and biochemical analysis.
Animals↗
A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system.
Golgi stacks were previously shown to be converted into tubular networks when incubated in mitotic cytosol depleted of the coatomer subunit of COP I coats (Misteli and Warren, 1994). Similar, though smaller, networks are now shown to be an early intermediate on the Golgi fragmentation pathway both in vitro and in vivo. Their appearance mirrors the disappearance of Golgi cisternae and at their peak they constitute 35% of total Golgi membrane. They are consumed by two pathways, the first involving the budding of COP I-coated vesicles described previously (Misteli and Warren, 1994). The second involves a COP I-independent mechanism that leads eventually to a vesicle fraction that is larger in size and more heterogeneous than that produced by the COP I-mechanism. We suggest that both pathways operate concurrently at the onset of mitotic fragmentation. The COP I-independent pathway converts cisternae into tubular networks that then fragment. The COP I-dependent pathway partially consumes first the cisternae at the beginning of the incubation and then the tubular networks that form from them.
Animals↗
Mitotic disassembly and reassembly of the Golgi apparatus.
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Animals↗
Mitotic disassembly of the Golgi apparatus in vivo.
Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50-70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.
Animals↗
COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system.
Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional length of Golgi stacks fell in the presence of mitotic cytosol by approximately 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was matched by the appearance of both small (50-100 nm in diameter) and large (100-200 nm in diameter) vesicular profiles. Small vesicular profiles constituted more than 50% of the total membrane after 60 min of incubation and they were shown to be vesicles or very short tubules by serial sectioning. In the presence of GTP gamma S all of the small vesicles were COP-coated and both the extent and the rate at which they formed were sufficient to account for the production of small vesicles during mitotic incubation. The involvement of the COP-mediated budding mechanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer formed but highly fenestrated networks appeared, an effect reversed by the readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of the Golgi apparatus during mitosis in animal cells is caused by continued budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.
Animals↗
DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.
The temperature-sensitive Chinese hamster ovary (CHO) cell mutant DS28-6 has been previously shown to be pleiotropically defective in protein secretion. We have examined the mutant cells to determine the intracellular site of the block in secretion. By transmission electron microscopy a time-dependent disassembly of the Golgi apparatus was found under nonpermissive temperature, which resulted in the loss of the cisternal stack. Complete reorganization of the Golgi apparatus occurred after shift to permissive temperature. Under nonpermissive temperature, a microtubule- and energy-dependent redistribution of Golgi mannosidase II and galactosyltransferase into a pattern characteristic of the endoplasmic reticulum (ER) was observed. Inhibition of protein synthesis by cycloheximide had no influence on Golgi mannosidase II redistribution. Evidence for Golgi apparatus-associated processing of oligosaccharides in the ER was obtained by lectin-gold cytochemistry revealing the presence of the galactose (beta 1----4)N-acetylglucosamine sequence and sialic acid residues. Furthermore, 7-nitrobenz-2-oxa-1,3-diazol-4-yl-tagged ceramide, a lipidic trans-Golgi apparatus marker in CHO cells, exhibited an energy-dependent redistribution into the ER. These effects were fully reversible upon shift to permissive temperature. Thus, mutant DS28-6 cells exhibit key features of the brefeldin A phenotype, which suggests that the observed brefeldin A effects result from interference with a normally occurring cellular process.
Animals↗