RanGTP-binding protein NXT1 facilitates nuclear export of different classes of RNA in vitro

To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopus extract-dependent manner. U1 snRNA export requires a 5' monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.     

Visualizing aquatic bacteria by light and transmission electron microscopy

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.     

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and approximately 15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.     

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.     

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.     

The molecular mechanisms of messenger RNA nuclear export

In eukaryotic cells, the nuclear membrane creates a barrier between the nucleus and the cytoplasm. Whereas RNA synthesis occurs in the nucleus, they mostly function in the cytoplasm; thus export of RNA molecules from the nucleus to the cytoplasm is indispensable for normal function of the cells. The molecular mechanisms involved in each kind of cellular RNA export is gradually understood. The focus of this review will be mRNA export. mRNAs are multiformed. In order to ensure that this variety of mRNA molecules are all exported, cells are probably equipped with multiple export pathways. A number of proteins is predicted to be involved in mRNA export. Ascertaining which proteins play crucial roles in the pathways is the key point in the study of mRNA export.     

Molecular classes of heterogeneous nuclear RNA in sea urchin embryos.

Nucleotide sequences within a phage genome can be detected in individual phage plaques by in situ hybridization with complementary RNA sequences. Results with phage A and a derivative having 10% of its DNA deleted indicate that sequences 500 to 1000 base-pairs long should be detectable with confidence.     

Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy

In studies of dynamic cellular processes, it would be ideal to be able to combine the capability of in vivo fluorescence video microscopy with the power of resolution of electron microscopy (EM). This article describes an approach based on the association of these two techniques, by which an individual intracellular structure can be monitored in vivo, typically through the use of markers fused with green-fluorescent protein, and then analysed by EM and three-dimensional reconstruction methods, resulting in a 'snapshot' of its fine structure at any chosen time in its life cycle. The potential of this approach is discussed in relation to various aspects of cell biology and especially to the question of the morpho-functional organization of the intracellular membrane trafficking pathways.     

Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy.

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.     

MPF localization is controlled by nuclear export.

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.     

Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes

Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA. We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy. A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers. Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe. The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542). The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531). Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.