In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly ( A denoviruses (AdVs) are nonenveloped DNA viruses consisting of an icosahedral capsid of ϳ90-nm diameter and an inner nucleoprotein core containing a linear double-stranded DNA genome of ϳ36 kbp (1-3). The major structural component of the capsid is the hexon trimer that is present in 240 copies. On the outer surface of the capsid at each of the 12 vertices, fiber proteins are anchored to the penton base. A number of minor capsid proteins on the outer and inner surfaces of the virus particle help to stabilize the capsid (4). The DNA is directly associated with the core proteins, including protein X, the "terminal protein," which is covalently linked to the 5= DNA termini, protein VII, and protein V, which connects the core to the outer capsid.AdV enters the cells by receptor-mediated endocytosis during which the virion becomes partially uncoated (3). Uncoating involves a series of events, culminating with endosomal membrane lysis by protein VI, which allows access of the particle to the cytosol (5). The partially disassembled capsid is then translocated along microtubules to the nucleus using the dynein/dynactin motor complex (6, 7). AdV then interacts with the nuclear envelope (NE) at nuclear pore complexes (NPCs) (8), and the viral genome is translocated into the nucleus by means of nuclear import receptors and/or histone H1 (8-10).NPCs are evolutionarily conserved large protein complexes of ϳ100 MDa spanning the NE that mediate trafficking into and out of the nucleus. Although small molecules passively diffuse through the NPC, macromolecules larger than ϳ20 to 40 kDa are transported in an active manner. This pathway is mediated by cellular transport receptors, including the karyopherin beta family that facilitates the translocation of most proteins and certain RNAs (11,12). NPCs are formed by ϳ30 proteins, which are thought to be present in multiples of 8 copies (13). A third of all nucleoporins (Nups) contain intrinsically disordered regions enriched in Phe-Gly (FG) repeats. The FG repeat domains directly bind karyopherins (12) and play an essential role in trafficking of

Like seed plants, liverworts synthesize and accumulate a myriad of isoprenoid compounds. Using antibodies raised against several isoprenoid biosynthetic enzymes, we investigated their intracellular compartmentation by in situ immunolocalization from Marchantia polymorpha. The enzymes examined were deoxy-xylulose phosphate synthase, geranyl diphosphate synthase, farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, monoterpene synthase, geranylgeranyl diphosphate reductase, phytoene synthase, and phytoene desaturase. Our results show that liverwort oil bodies, which are organelles bound by a single unit membrane, possess isoprenoid biosynthetic enzymes similar to those found in plastids and the cytosol. We postulate that oil bodies play a dynamic role in cell metabolism in addition to their role as sites of essential oil accumulation and sequestration. The occurrence of such enzymes in different cellular compartments might be due to multiple targeting of gene products to various organelles.

Most plant mitochondria messenger RNAs (mRNAs) undergo editing through C-to-U conversions located mainly in exon sequences. However, some RNA editing events are found in non-coding regions at critical positions in the predicted secondary and tertiary structures of introns, suggesting that RNA editing could be important for splicing. Here, we studied the relationships between editing and splicing of the mRNA encoding the ribosomal protein S10 (rps10), which has a group II intron and five editing sites. Two of them, C2 and C3, predicted to stabilize the folded structure of the intron necessary for splicing, were studied by using rps10 mutants introduced into isolated potato mitochondria by electroporation. While mutations of C2 involved in EBS2/IBS2 interactions did not affect splicing, probably by the presence of an alternative EBS2′ region in domain I of the intron, the edition of site C3 turned out to be critical for rps10 mRNA splicing; only the edited (U) form of the transcript was processed. Interestingly, RNA editing was strongly reduced in transcripts from two different intronless genes, rps10 from potato and cox2 from wheat, suggesting that efficient RNA processing may require a close interaction of factors engaged in different maturation processes. This is the first report linking editing and splicing in conditions close to the in vivo situation.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to4 transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited afp9 mRNA is translated.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (aVp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vro system devised to follow the editing of wheat mitochondrial a4p9mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in viro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.Until recently the protein-coding sequence of a mRNA appeared to be inviolable. However, recent data show that in several instances disparate types of RNA modification can change the coding capacity of mRNA (1). Thus, the mitochondrial genes of a variety of plants undergo posttranscriptional changes at multiple sites creating a U residue in the RNA where a C was found in the genome (2-4). Editing of plant mitochondrial RNA alters the coding sequence in mRNAs, leading to amino acid changes as well as to the emergence of termination or initiation codons (5). Plant mitochondrial RNA editing seems to be somewhat simpler than the phenomenon observed in trypanosome mitochondria (6) but is remarkably similar to the editing of a single residue in the apolipoprotein B mRNA observed in some animal tissues (7, 8). The mechanism by which the C-to-U change operates in plant mitochondria is unknown, although an in vitro system has been described in the case of apolipoprotein B mRNA modification (9). A model has been proposed for RNA editing in kinetoplastid mitochondria involving "guide" RNA molecules transcribed from maxicircle or minicircle mitochondrial DNA (10).In studying the organization and expression of protein genes essential for wheat mitochondrial function, our laboratory has mapped and sequenced several wheat mitochondrial genes (11)(12)(13)(14). We showed by cDNA sequencing that eight C-to-U changes occurred in wheat mitochondrial atp9 mRNA (15, 16). Minor forms of atp9 cDNA with partial or overedited sequences were also found. Partial protein sequencing showed that edit...

No abstract

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to4 transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited afp9 mRNA is translated.

Biopsies from patients show that hepadnaviral core proteins and capsids -collectively called core -are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that e, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.