RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.     

Direct Visualization of a Transmembrane Channel Associated with Ribosomes

Examination of directly frozen rough endoplasmic reticulum (ER) of retinal pigment epithelial cells by freeze-fracture and freeze-substitution revealed distinct paired transmembrane proteins associated with membrane ribosomes. Ribosomal subunits on intact ER membrane are directly visualized for the first time, providing a global view of the structure of the ribosome and the corresponding structures on the ER membrane. The ribosomal intersubunit cleft appears to be continuous with a cleft between paired transmembrane proteins that extends into the lumen of the ER. This continuous cleft may be the path taken by nascent polypeptides.     

Identification of a Heterogeneous Nuclear Ribonucleoprotein-recognition Region in the HIV Rev Protein

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9–14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions.     

Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Demonstration of Ribosomes in Mesosomes Associated with Bacillus subtilis Protoplasts

The physiological differences between Bacillus subtilis (ATCC 6633) cells derived from a glucose-salts-yeast extract (GSY) medium and those of cells from tryptose broth permitted the identification of variables in protoplasting environments which noticeably affected the clarity of mesosomal ribosomes. They were the sucrose and magnesium ion concentrations and the type of buffer used. The environment suitable for conversion of GSY cells to the protoplast state was a 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, containing 0.6 M sucrose and 0.03 M MgCl(2). Branched mesosomal tubules and a unique organization of vesicles were detected in thin sections and in negative stains of the specimens. Ribosomes were demonstrable in the extruded structures associated with protoplasts that had been prepared according to four fixation schedules and embedded in either of two epoxy plastics. Adjustments in the fixation schedules improved the clarity of the large bodies of protoplast cytoplasm to a degree equivalent to that of their dangling appendages.     

Stage-Specific Expression and Localization of MENT, a Nuclear Protein Associated with Chromatin Condensation in Terminally Differentiating Avian Erythroid Cells

Polyclonal antibodies have been raised against a nonhistone protein (MENT) which has been previously shown to be associated with the repressed chromatin of mature chicken erythrocytes and to promote the in vitro condensation of chromatin of immature erythrocyte nuclei. Here we report that the expression pattern of MENT closely follows chromatin condensation in maturing arian erythrocytes of definitive and primary lineages. Accumulation of MENT correlates more strongly with chromatin condensation than does accumulation of histone H5. In addition to being present in erythrocytes, the protein was also found in neutrophil nuclei and an immunofluorescence reaction was observed with embryonic (nucleated) thrombocytes. MENT was not detected in other chicken tissues (brain, liver, testis). In intact erythrocytes, MENT immunofluorescence was found in foci close to the nuclear periphery, while in isolated, decondensed nuclei, the fluorescence signal was uniformly distributed. In neutrophil nuclei, containing approximately 10 times more MENT than adult erythrocytes, intense staining associated with the peripheral heterochromatin was observed. These findings are discussed in regard to a possible mechanism for chromatin condensation by MENT.     

A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5′ end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.     

Ribosomes and Ribosomal Protein from Neurospora crassa I. Physical, Chemical, and Immunochemical Properties

Ribosomes from Neurospora crassa, initially characterized by ultracentrifugal and immunochemical analyses, have been used to prepare ribosomal protein for physical, chemical, and immunochemical study. The acrylamide gel disc electrophoretic profiles of Neurospora ribosomal protein exhibit a degree of heterogeneity comparable to what has been observed in other systems. Only by chemical modification or by aggregation of the protein do alterations in the profile become apparent. Disulfide-bond formation appears to play a role in the aggregation of ribosomal protein to complexes of S_20,w = 200. The aggregation can be prevented by alkylation of −SH groups, and protein treated in this fashion has a subunit molecular weight of about 20,000 as determined by equilibrium centrifugation. Finger-printing of tryptic peptides indicates that more than one unique sequence of amino acids must be present in ribosomal protein, although gross primary structural heterogeneity is questioned. Antigenic heterogeneity is much less apparent; only a few precipitin bands are resolved by immunodiffusion tests, although complete reactivity of total ribosomal protein is suggested by quantitative precipitin analysis. The antigenically active ribosomal protein components appear to reside in at least two fractions; one is removed readily from the ribosome by CsC1 treatment. Ribosomal protein of N. crassa possesses antigenic determinants present in E. coli ribosomal protein as judged by spur formation in immunodiffusion tests.     

Fraction of Ribosomes Synthesizing Protein as a Function of Specific Growth Rate

The fraction of ribosomes engaged in protein synthesis in Escherichia coli growing at specific growth rates ranging from 0.3 to 1.2 h(-1) was estimated from measurements of nascent protein/ribosome and of polyribosomes. Both measurements showed that the fraction of ribosomes synthesizing protein increased with specific growth rate, from 30 to 70% over the range studied. Polyribosome measurements made at different specific growth rates in four E. coli strains showed no significant differences between strains. The increase in the fraction of polyribosomes had begun within 30 s after a shift-up from glucose-minimal medium, and was complete within 2 to 5 min. These results indicate that the function of ribosomes is regulated.     

Protein Kinase Activity Associated with the Extracellular and Occluded Forms of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Protein kinase activity is associated with both the extracellular and the occluded forms of Autographa californica nuclear polyhedrosis virus, a baculovirus. Serine and threonine are the predominant amino acids phosphorylated by the kinase activity associated with both viral forms; no phosphotyrosine was detected. The addition of calcium, cAMP, or cGMP has no apparent effect on the amount of phosphorylation or the substrates phosphorylated.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

Pdro,a Protein Associated with Late Endosomes and Lysosomes and Implicated in Cellular Cholesterol Homeostasis

Background

Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content.

Methodology/Principal Findings

Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY.

Conclusions/Significance

LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.     

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.     

The Nuclear Envelope Protein,LAP1B,Is a Novel Protein Phosphatase 1 Substrate

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.