Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Regulation of the export of RNA from the nucleus

Transport of macromolecules across the nuclear envelope is an essential activity in eukaryotic cells. RNA molecules within cells are found complexed with proteins and the bound proteins likely contain signals for RNA export. RNAs microinjected into Xenopus oocyte nuclei are readily exported, and their export can be competed by self RNA but not by RNAs of other classes. This indicates that the rate-limiting step in RNA export is the interaction of RNAs with class-specific proteins, at least when substrate RNAs are present at saturating levels. Export of host mRNAs is inhibited following infection by some animal viruses, while the export of viral RNAs occurs. The HIV-1 RNA-binding protein, Rev, mediates the export of intron-containing viral RNAs that would normally be retained in nuclei. This requires a nuclear export signal (NES) within Rev and an element within the RNA to which Rev binds. In yeast, heat shock causes accumulation of poly(A)(+)RNA within nuclei but heat-shock mRNAs are transcribed and exported efficiently. This requires elements within heat shock mRNA that probably interact with a cellular protein to facilitate RNA export. In these cases, the proteins that recognize critical sequences in the RNAs probably direct the RNAs to an RNA export pathway not generally used for mRNA export. This would circumvent the general retention of most poly(A)(+)mRNAs following heat shock in yeast and the need for complete splicing of viral mRNAs that travel through the normal mRNA export pathway.     

Nonrandom packaging of host RNAs in moloney murine leukemia virus

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA(Met) were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Psi- mRNAs and some other host RNAs may be specifically excluded from assembly sites.     

mRNA-decapping enzyme from Saccharomyces cerevisiae: purification and unique specificity for long RNA chains.

An enzyme that hydrolyzes one PPi bond of the cap structure of mRNA, yielding m7GDP and 5'-p RNA was purified from Saccharomyces cerevisiae to a stage suitable for characterization. The specificity of the enzyme was studied, using both yeast mRNA and synthetic RNAs labeled in the cap structure. A synthetic capped RNA (540 nucleotides) was not reduced in size, while as much as 80% was decapped. Yeast mRNA treated with high concentrations of RNase A, nuclease P1, or micrococcal nuclease was inactive as a substrate. The use of synthetic capped RNAs of different sizes (50 to 540 nucleotides) as substrates showed that the larger RNA can be a better substrate by as much as 10-fold. GpppG-RNA was hydrolyzed at a rate similar to that at which 5'-triphosphate end group were not hydrolyzed.     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

Substrate specificity of the exonuclease activity that degrades H4 histone mRNA

We have investigated the substrate specificity of an exonuclease that degrades human H4 histone mRNA, using synthetic RNA templates incubated in a cell-free mRNA decay system (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Five RNAs that lacked poly(A), including histone, were degraded rapidly in vitro. Polyadenylated histone mRNA was degraded at least 10-fold more slowly than unmodified histone mRNA. Double-stranded RNA and DNA were very stable. Single-stranded DNA was degraded approximately 20-fold more slowly than single-stranded, non-polyadenylated RNA, and RNA with a 3' phosphoryl group was degraded more slowly than RNA with a 3'-hydroxyl group. Uncapped RNAs were degraded rapidly in the unfractionated system but were stable in reactions containing a ribosomal high salt wash extract. Therefore, the exonuclease activity released from ribosomes by high salt extraction was separated from the enzyme(s) that degraded uncapped RNAs.     

Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

An in vitro system for the editing of apolipoprotein B mRNA

A novel form of RNA editing generates two forms of apolipoprotein B (apo-B) mRNA by converting C at nucleotide 6666 to U or a U-like base. We have established an in vitro system for the editing of apo-B mRNA using synthetic RNAs and S100 extracts from rat hepatoma cells. Editing was detected by a sensitive primer extension assay and confirmed by DNA sequencing. The in vitro editing activity is specific and sensitive to proteinase K. Apo-B100 RNAs were synthesized in vitro from deletion mutants spanning nucleotide 6666. Synthetic RNAs containing 2383, 483, and 55 nucleotides of apo-B mRNA sequence were edited in vitro with similar efficiency, but an RNA containing 26 nucleotides was not edited.