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PubMed1999

Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes.

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Animals↗
PubMed2001

Structure and assembly of intracellular mature vaccinia virus: thin-section analyses.

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.

Bacterial Proteins↗
PubMed2006

A unidirectional energy transfer cascade process in a ruthenium junction self-assembled by alpha- and beta-cyclodextrins.

A Ru(II) complex, Ru(alpha2beta), with a trisbipyridyl core and two different receptor sites, alpha- and beta-cyclodextrins, acts as a junction to recognize photoactive guests based on anthracene, Anth, and Os(II), Os-ada, in aqueous solution. Upon light excitation of the self-assembled system Anth.Ru(alpha2beta).Os-ada, an energy cascade process takes place from the Anth guest to the Ru(alpha2beta) core, and subsequently, the energy is funneled to the Os-ada guest. The rates of the two photoinduced processes are determined by time-resolved emission and transient absorption spectroscopic techniques. The system introduces a directed self-assembly approach to unidirectional wires that can lead to nanosized arrays by judicious selection of the individual components.

Adamantane↗
PubMed2000

Kinetic analysis of human immunodeficiency virus type 1 assembly reveals the presence of sequential intermediates.

The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.

Animals↗
PubMed1993

Effect of retinyl acetate on the assembly of the fibronectin extracellular matrix and the processing of the fibronectin receptor beta subunit of confluent C3H/10T1/2 mouse embryo fibroblasts.

The mouse embryo fibroblast cell line, C3H/10T1/2, synthesized and deposited a large amount of fibronectin especially in the pericellular matrix. Confluent cultures of these cells cultured in the presence of 0.3 micrograms/ml of retinyl acetate released cell surface fibronectin and the extracellular matrix fibronectin fibrils were disorganized. The immunoblot analysis demonstrated that the number of the fibronectin receptor was decreased in the prolonged culturing of retinyl acetate-treated cells. Immunoprecipitation of 35S-methionine pulse-chase labeled cell extracts by antifibronectin receptor antibody indicated that about one-half of the pre-beta subunit was processed and converted to the mature form in control cells, and only about one-fourth of the pre-beta subunit was processed in the retinyl acetate-treated confluent cells. 1-deoxymannojirimycin (MNJ), which is an inhibitor of oligosaccharide processing, induced disorganization of the extracellular matrix fibronectin assembly similar to that observed with retinyl acetate. The results of this study suggest that a mechanism of action of retinyl acetate is inhibition of the glycosylation during processing of the fibronectin receptor, a step necessary for fibronectin binding and for assembly of the extracellular matrix.

Animals↗
PubMed1993

The theoretical analysis of the process of RNA molecule self-assembly.

The Kinetic approach to the problem of the RNA structure prediction based on the analysis of the molecule self-formation is proposed. Re-structurization that occurs during processing is described in terms of Markov processes. A new formalism designating nucleotides by complex numbers is proposed, leading to the complex unitary space of nucleic vectors. Properties of structure and transition matrices are discussed in relation to the analysis of RNA structural formation processes. The non-linear dynamic behavior of secondary structure transition is analyzed. Soliton-like oscillations of RNA and DNA tertiary structures are predicted. The Monte-Carlo simulation of the RNA structure self-formation is used to calculate the ensemble of the secondary structures of the tRNA(Ala) precursor from Bombix mori formed during processing.

Animals↗
PubMed2001

Natural substitutions at highly conserved T1-domain residues perturb processing and functional expression of squid Kv1 channels.

Shaker-type K-channel alpha-subunits (SqKv1A, B, D) expressed in neurons of the squid stellate ganglion differ in the length of their N-termini and in the species of amino acid present at several points in the T1 domain, an intracellular region involved in the tetramerization process during channel assembly. Heterologous expression of wild-type SqKv1A, B, and D in Xenopus oocytes reveals large differences in the level of both functional channels (assayed by whole-oocyte voltage clamp) and total channel protein (assayed by immunoblotting). Functional expression is poorest with SqKv1A and by far the best with SqKv1D. Biophysical properties of the three SqKv1 channels are essentially identical (assayed by cell-attached patch clamp). Site-directed mutagenesis was used to determine whether the observed differences in expression level are impacted by two residues in the T1 domain at which SqKv1A and B (but not D) differ from the consensus sequences found in many other taxa. In SqKv1A, glycine is substituted for arginine in an otherwise universally conserved sequence (FFDR in the T1(B) subdomain). In SqKv1B, glycine replaces serine in a sequence that is conserved within the Kv1 subfamily (SGLR in the T1(A) subdomain). Restoration of the consensus amino acid at these positions largely accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored alpha-subunits suggests that the anomalous amino acids in SqKv1A and B exert their influence during early steps in channel processing and assembly which take place in the endoplasmic reticulum (ER).

Amino Acid Sequence↗
PubMed1990

Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants.

The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression.

Bacterial Proteins↗
PubMed1985

Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification.

The in vivo synthesis and assembly of human small nuclear ribonucleoproteins (snRNPs) have been studied using pulse/chase analysis. Antibodies derived from patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) recognize distinguishable subsets of pulse-labeled snRNP peptides. These antibodies were used to immunoprecipitate sucrose gradient fractionated pulse-labeled and pulse/chased snRNP proteins. The results indicate that assembly of the U RNA-containing snRNPs is a multistep process involving prior assembly of an RNA-free 6S core particle. This precursor contains snRNP peptides D, E, F, and G, which are common to all the different U RNA-containing particles. Furthermore, a posttranslational modification of one of the U1 snRNP-specific peptides has been observed, and the kinetics of this process indicates that the modification occurs after particle assembly. Functional and structural implications of a protein core for snRNP particles are discussed.

Antibodies↗
PubMed2004

The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5.

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

Animals↗
PubMed2024

Chemical approaches to probe and engineer AAV vectors.

Adeno-associated virus (AAV) has emerged as the most promising vector for in vivo human gene therapy, with several therapeutic approvals in the last few years and countless more under development. Underlying this remarkable success are several attractive features that AAV offers, including lack of pathogenicity, low immunogenicity, long-term gene expression without genomic integration, the ability to infect both dividing and non-dividing cells, etc. However, the commonly used wild-type AAV capsids in therapeutic development present significant challenges, including inadequate tissue specificity and the need for large doses to attain therapeutic effectiveness, raising safety concerns. Additionally, significant preexisting adaptive immunity against most natural capsids, and the development of such anti-capsid immunity after the first treatment, represent major challenges. Strategies to engineer the AAV capsid are critically needed to address these challenges and unlock the full promise of AAV gene therapy. Chemical modification of the AAV capsid has recently emerged as a powerful new approach to engineer its properties. Unlike genetic strategies, which can be more disruptive to the delicate capsid assembly and packaging processes, "late-stage" chemical modification of the assembled capsid-whether at natural amino acid residues or site-specifically installed noncanonical amino acid residues-often enables a versatile approach to introducing new properties to the capsid. This review summarizes the significant recent progress in AAV capsid engineering strategies, with a particular focus on chemical modifications in advancing the next generation of AAV-based gene therapies.

Dependovirus↗
PubMed2025

Mapler: a pipeline for assessing assembly quality in taxonomically rich metagenomes sequenced with HiFi reads.

SUMMARY: Metagenome assembly seeks to reconstruct the most high-quality genomes from sequencing data of microbial ecosystems. Despite technological advancements that facilitate assembly, such as Hi-Fi long reads, the process remains challenging in complex environmental samples consisting of hundreds to thousands of populations. Mapler is a metagenome assembly and evaluation pipeline with a focus on evaluating the quality of Hi-Fi long read metagenome assemblies. It incorporates several state-of-the-art metrics, as well as novel metrics assessing the diversity that remains uncaptured by the assembly process. Mapler facilitates the comparison of assembly strategies and helps identify methodological bottlenecks that hinder genome reconstruction. AVAILABILITY AND IMPLEMENTATION: Mapler is open source and publicly available under the AGPL-3.0 licence at https://github.com/Nimauric/Mapler. Source code is implemented in Python and Bash as a Snakemake pipeline. A snapshot of the code is available on Software Heritage at swh:1:snp:df4f5f02e22ebbab285ec14af58d4d88436ee5d6. Raw data and results are available at https://entrepot.recherche.data.gouv.fr/dataset.xhtml?persistentId=doi:10.57745/2SA8AB.

Metagenome↗
PubMed2004

Forespore membrane assembly in yeast: coordinating SPBs and membrane trafficking.

In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, sporulation involves de novo synthesis of forespore membrane (FSM) within the cytoplasm of mother cells. The FSM ultimately becomes the plasma membrane of the developing ascospores. Several protein components of the FSM have been identified. Visualization of these proteins has demonstrated the dynamic nature of the genesis and development of the FSM. It begins to develop at the differentiated outer plaque of the spindle pole bodies (SPBs) and extends outwards, encapsulating each of the haploid nuclei produced by meiosis. Several coiled-coil proteins are specifically recruited to the SPBs and play indispensable roles in FSM assembly. Temporal and spatial coordination of meiotic nuclear divisions and membrane assembly is of special importance. Comparison of the processes of FSM assembly in these yeasts shows that the basic mechanism has been conserved, even though the individual proteins involved are often different. Understanding these dynamic aspects of yeast sporulation will help to elucidate a general mechanism for the cellularization of cytoplasm containing multiple nuclei.

Biological Transport↗
PubMed2000

ADP-ribosylation factor 1 and its activation of phospholipase D are important for the assembly of very low density lipoproteins.

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

1-Butanol↗
PubMed1997

Two-step processing is not essential for the import and assembly of functionally active iron-sulfur protein into the cytochrome bc1 complex in Saccharomyces cerevisiae.

The iron-sulfur protein of the cytochrome bc1 complex is one of a small number of proteins that are processed in two sequential steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) during import into Saccharomyces cerevisiae mitochondria. To test whether two-step processing is necessary for import and assembly of the iron-sulfur protein into the cytochrome bc1 complex, we mutagenized the presequence of the iron-sulfur protein to eliminate the original MPP site and replace the MIP site with a new MPP site. The mutated presequence is cleaved and forms mature-sized protein in a single step, and the mature-sized iron-sulfur protein is correctly targeted to the outer side of the inner mitochondrial membrane in vitro. Mutant iron-sulfur protein which is processed to mature size in one step complements the respiratory deficient phenotype of a yeast strain in which the endogenous gene for the iron-sulfur protein is deleted. These results establish that mature-sized iron-sulfur protein can be formed by single-step processing and assembled into a functionally active form in the cytochrome bc1 complex in S. cerevisiae.

Amino Acid Sequence↗
PubMed2001

The life cycle of actin patches in mating yeast.

Actin patches are core components of the yeast actin cytoskeleton that undergo redistribution during establishment of cell polarity. Using 4D imaging, we observe the life cycle of actin patches in living yeast for the first time. We observe assembly of actin patches at sites of polarized growth, and disassembly of actin patches concomitant with movement away from those sites. The total lifetime of an actin patch is 10.9+/-4.2 seconds. These findings indicate that actin patches are labile structures, and that the localization of actin patches during establishment of cell polarity occurs by assembly of these structures at sites of polarized cell surface growth. These findings were confirmed and extended by analysis of myosin I proteins and their receptor, verprolin, proteins implicated in actin assembly in yeast. Deletion of type I myosins or their receptor has no effect on the velocity of actin patch movement. However, these mutants show a 65% reduction in number of patch movements and a three-fold increase in patch lifetime. Finally, the actin patch resident proteins Abp1p, fimbrin, and Arp2p show normal association with actin patches in myosin I and verprolin mutants. However, cofilin accumulates in abnormal 'bars' of G-actin in myo3Delta,myo5Delta and vrp1Delta strains, and Las17p/Bee1p is not associated with actin patches in vrp1Delta strains. These findings imply a multi-step process for actin patch assembly. Early events in this process, including assembly of Abp1p, fimbrin and Arp2p with F-actin, can occur throughout the cell and do not require myosin I proteins or their receptor. Later events in this process are myosin I-dependent, and are required for assembly of actin patches at sites of polarized cell surface growth.

Actins↗
PubMed1983

Freeze-fracture analysis of plasma membranes of isolated astrocytes from rat brain.

Plasma membranes of mature rat astrocytes separated by differential centrifugation have been reported to be intact, based on electron microscopic examination of thin plastic sections. However, the effects of the separation procedure on the internal structure of the plasma membranes are unknown. The degree of membrane integrity is of concern to us since our goal is the separation of astrocytic plasma membranes and characterization of the specific intramembranous particle groups called assemblies. We have taken advantage of the astrocyte membrane-marker, the assembly, in order to monitor, by freeze-fracture, the identity of the separated astrocytes and the integrity of their cell membrane. Since some processes of an astrocyte contain assemblies whereas other processes of the same cell do not, it was also necessary to determine if processes with assemblies were separated by this technique. Astrocytic cell membranes were also examined to determine if trypsinization or the mechanical disruption steps of the separation affected the intramembranous particles. Freeze-fracture of the plasma membranes revealed that the particles were rearranged resulting in patches of clumped intramembranous particles and areas of bare membrane. The assemblies were rearranged rather than lost from the membrane since they could be identified among the clumped particles. More astrocytic plasma membranes contained non-clumped, normally distributed particles in the trypsin treated fractions. The non-trypsinized fractions had more damaged astrocytes with aggregated intramembranous particles and much more cellular debris. We interpret the findings for the non-trypsinized astrocytes as due to greater mechanical stress placed on the cells during tissue disruption. Trypsin treatment lessens this stress, thereby, tending to preserve the normal distribution of intramembranous particles.

Animals↗
PubMed1991

The bacterial flagellum and flagellar motor: structure, assembly and function.

The bacterial flagellum is a complex multicomponent structure which serves as the propulsive organelle for many species of bacteria. Rotation of the helical flagellar filament, driven by a proton-powered motor embedded in the cell wall, enables the flagellum to function as a screw propeller. It seems likely that almost all of the genes required for flagellar formation and function have been identified. Continuing analysis of the portions of the genome containing these genes may reveal the existence of a few more. Transcription of the flagellar genes is under the control of the products of a single operon, and so these genes constitute a regulon. Other controls, both transcriptional and post-transcriptional, have been identified. Many of these genes have been sequenced, and the information obtained will aid in the design of experiments to clarify the various regulatory mechanisms of the flagellar regulon. The flagellum is composed of several substructures. The long helical filament is connected via the flexible hook to the complex basal body which is located in the cell wall. The filament is composed of many copies of a single protein, and can adopt a number of distinct helical forms. Structural analyses of the filament are adding to our understanding of this dynamic polymer. The component proteins of the hook and filament have all been identified. Continuing studies on the structure of the basal body have revealed the presence of several hitherto unknown basal-body proteins, whose identities and functions have yet to be elucidated. The proteins essential for energizing the motor, the Mot and switch proteins, are thought to exist as multisubunit complexes peripheral to the basal body. These complexes have yet to be identified biochemically or morphologically. Not surprisingly, flagellar assembly is a complex process, occurring in several stages. Assembly occurs in a proximal-to-distal fashion; the basal body is assembled before the hook, and the hook before the filament. This pattern is also maintained within the filament, with monomers added at the distal end of the polymer; the same is presumably true of the other axial components. An exception to this general pattern is assembly of the Mot proteins into the motor, which appears to be possible at any time during flagellar assembly. With the identification of the genes encoding many of the flagellar proteins, the roles of these proteins in assembly is understood, but the function of a number of gene products in flagellar formation remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗