Differences in the level of plastid-specific tRNA's in chloroplasts and etioplasts of phaseolus vulgaris

Comparison of the chromatographic profiles of chloroplast and etioplast leucyl-tRNA's and valyl-tRNA's shows that the levels of plastid-specific tRNA species are relatively higher in the chloroplasts. This suggests that light can stimulate the synthesis of plastid-specific tRNA's in higher plants.     

The procollagen type III, alpha 1 (COL3A1) gene first intron expresses poly-A+ RNA corresponding to multiple ESTs and putative miRNAs

The mouse COL3A1 first intron is 9684 bp. RNA's of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly‐A RNA from fetal mice and total RNA from suckling and adult mouse intestine using ³²P‐labeled, anti‐sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655–7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNA's was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. “BLAST” analysis indicated that the mouse first intron 5 prime sequence has 94–100% identity to 13 mouse ESTs. These mouse first intron EST's lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron EST's have significant identity to known miRNA, mature sequences, mmu‐miR‐466f‐3P, mmu‐miR‐1187, and mmu‐miR‐574‐5P as well as others. Predicted targets for mmu‐miR‐466f‐3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development. J. Cell. Biochem. 112: 541–547, 2011. © 2010 Wiley‐Liss, Inc.     

Differentiation of chick embryo cerebral hemispheres: a critical evaluation of a bulk preparation of neuroblasts and spongioblasts

Abstract— A critical evaluation of a previously described method of bulk preparation of neuroblasts and spongioblasts from chick embryo cerebral hemispheres furnished the following results.

• 1 A major loss of total RNA occurs during the preparation of the cell fractions.
• 2 The loss occurred mainly during the dissociation of the tissue and the following centrifugations.
• 3 The missing RNA's were found in the supernatants after cell centrifugation at low speed.
• 4 There was a selective loss of transfer RNA amounting to 70–80 per cent relative to ribosomal RNA.
• 5 The relative proportions of the 29, 18 and 5s ribosomal RNA's were constant, but there was a decrease in their proportion relative to heavy-molecular weight, rapidly-labelled nuclear RNA.
• 6 The ribosomal RNA's in the low-speed supernatant could be resedimented at 35,000 g whereas most transfer RNA could not.

The results are consistent with a loss of fragments of cytoplasm containing ribosomes and with additional extraction of transfer RNA during the preparation.     

Synthesis of the small molecular weight monodisperse nuclear RNA's in contact-inhibited cell cultures

The small molecular weight monodisperse nuclear RNA's are synthesized in contact-inhibited cultures of 3T3 cells. The level of synthesis of these RNA's, and of ribosomal and transfer RNA's, appears to be only 8–20% of that observed in growing cultures. The synthesis of all of these relatively stable RNA species may thus be coordinately controlled.     

Nonviral Interferon Inducers

Interferon production can be stimulated by a great variety of microbial and nonmicrobial agents other than viruses. The nonmicrobial inducers can be divided into polyanions, mitogens, and a miscellaneous category including the various endotoxins and antibiotics. The polyanions appear to require a stable, high molecular weight backbone and a high density of free anionic groups whether they are polynucleotides, plastics, or polysaccharides. Mitogen-induced interferon appears to be but one of a constellation of substances produced following lymphocyte transformation. The process of transformation can be stimulated either by specific immune recognition or non-specifically by phytohemagglutinin. Synthetic polynucleotide inducers are active; the thermostable, double-stranded RNA''s are much more active than the double-stranded DNA''s or 1-, 3-, or 4-stranded RNA''s. Some success has been obtained with potentiation of nucleotide inducers through the use of polycationic substances, complexing with a polysaccharide, concurrent administration of a metabolic antagonist, or substitution of phosphate by thiophosphate in the polynucleotide backbone. The stages in the interaction of interferon stimulating RNA and cells can be divided into three steps: first, binding to cell surface, next, a temperature dependent "recognition" step, and finally, degradation and utilization of monomers in cellular RNA synthesis; the critical recognition site has not yet been determined. The vast majority of cell-associated polynucleotide remains at the surface of the cell. Information from animal models resembling human diseases suggests that certain of these nucleotide inducers may have clinical usefulness in therapy or prophylaxis.     

Regulation of ribonucleic acid synthesis in growing bacterial cells. II. Control over the composition of the newly made RNA

Studies have been made by base-ratio analysis and DNA-RNA hybridization of the pulse-labeled, nascent RNA in cells of Escherichia coli growing exponentially in minimal-glucose and Casamino acids-supplemented media, as well as during shift-ups or shift-downs between them. It is found that the cell varies the fraction of the newly made RNA given over to the synthesis of ribosomal and (presumably) transfer RNA under these different conditions. Furthermore, for cells in exponential growth or during shift-up, these values are in good agreement with the amount of “stable” RNA made as defined in the kinetic experiments in the accompanying paper. Thus under these conditions, there is no need to postulate the existence of a mechanism involving the rapid destruction of otherwise stable RNA's, a situation which may occur in shift-down or other growth conditions.     

Optimal alphabets for an RNA world

Experiments have shown that the canonical AUCG genetic alphabet is not the only possible nucleotide alphabet. In this work we address the question ''is the canonical alphabet optimal?'' We make the assumption that the genetic alphabet was determined in the RNA world. Computational tools are used to infer the RNA secondary structure (shape) from a given RNA sequence, and statistics from RNA shapes are gathered with respect to alphabet size. Then, simulations based upon the replication and selection of fixed-sized RNA populations are used to investigate the effect of alternative alphabets upon RNA''s ability to step through a fitness landscape. These results show that for a low copy fidelity the canonical alphabet is fitter than two-, six- and eight-letter alphabets. In higher copy-fidelity experiments, six-letter alphabets outperform the four-letter alphabets, suggesting that the canonical alphabet is indeed a relic of the RNA world.     

Various molecular aspects of cytology and embryology

Our present knowledge of the cell structure, which is largely based on electron microscopy, is compared with what was known a few decades ago, when only light microscopy was available to the cytologist. The importance of cytochemical methods for the detection and localization of macromolecules (nucleic acids, proteins) is stressed. But it is pointed out that further analysis, with biochemical techniques, was required in order to understand the actual mechanisms of macromolecule synthesis in the cell (in particular, the relationships existing between nucleic acids and protein synthesis). The importance of genetical analysis in simple systems such as viruses and bacteria for the development of ‘molecular’ biology is then emphasized: in particular, the work of Avery identifying the ‘transforming principle’ with DNA, of Beadle leading to the ‘one gene, one enzyme’ theory, of the virologists who demonstrated that it is the nucleic acid component of viruses which carries the genetical information, have been of fundamental importance for the development of modern biology. No less important has been the work of the X-ray crystallographers (Crick and Watson, Perutz, Kendrew, etc.) who established the fine structure of nucleic acids and of proteins. A brief review and a schematic representation of present ideas regarding the control exerted by DNA on the synthesis of specific proteins are then given: the main characteristics of the different kinds of RNA's, their interactions for the formation of polysomes, the role of the latter in protein synthesis, the main principles of the genetic codes, are briefly summarized. But cells are, in many respects, more complicated than bacteria. The concepts of molecular biology cannot be applied to cell differentiation without a recognition of the greater complexity of animal and plant cells. They represent, however, a most useful and powerful guide for research in that area: for instance, many aspects of morphogenesis in the unicellular alga Acetabularia and in amphibian eggs can be explained on the assumption that messenger RNA's are produced by the nucleus and stored, in a stabilized form, in the cytoplasm during days or even weeks. This stability of messenger RNA's in eggs and algae is at variance with their short life in bacteria. The behaviour of non-nucleate fragments of Acetabularia is surprising in many respects: they are the site, not only of the synthesis of specific proteins, but even of RNA and DNA net synthesis. Such a synthesis of macromolecules, in the absence of the nucleus is probably linked to the presence of the chloroplasts in this alga: they contain DNA, can synthesize RNA and proteins, and can even increase in number in the absence of the nucleus. The presence of large amounts of DNA in the cytoplasm of many animal eggs raises a number of questions and might account for the extremely important role of the cytoplasm in the very early stages of embryonic development. It is concluded that none of the great problems of cell biology will be solved without the help of the techniques and the theoretical ideas which have been so fruitful for the simpler systems used by the molecular biologists.     

Characterization of the direct physical interaction of nc886, a cellular non-coding RNA, and PKR

We have recently shown that nc886 (pre-miR-886 or vtRNA2-1) is not a genuine microRNA precursor nor a vault RNA, but a novel type of non-coding RNA that represses PKR, a double-stranded RNA (dsRNA) dependent kinase. Here we have characterized their direct physical association. PKR’s two RNA binding domains form a specific and stable complex with nc886’s central portion, without any preference to its 5′-end structure. By binding to PKR with a comparable affinity, nc886 competes with dsRNA and attenuates PKR activation by dsRNA. Our data suggest that nc886 sets a threshold for PKR activation so that it occurs only during genuine viral infection but not by a minute level of fortuitous cellular dsRNA.     

Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.     

NMR of RNA - Structure and interactions

RNA was shown to have a more substantial role in the regulation of diverse cellular processes than anticipated until recently. Answers to questions what is the structure of specific RNAs, how structure changes to accommodate different functional roles, and how RNA senses other biomolecules and changes its fold upon interaction create a complete representation of RNA involved in cellular processes. Nuclear magnetic resonance (NMR) spectroscopy encompasses a collection of methods and approaches that offer insight into several structural aspects of RNAs. We review the most recent advances in the field of viral, long non-coding, regulatory, and four-stranded RNAs, with an emphasis on the detection of dynamic sub-states and in view of chemical modifications that expand RNA's function.     

Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5''-terminal genomic RNA fragment.

The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(p10), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(p10)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(p10), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved.     

Denaturation of RNA with dimethyl sulfoxide

The denaturation of single-stranded and double-stranded RNA's in solutions with varying proportions of dimethyl sulfoxide has been followed by changes in absorbancy, optical rotation, and—with a double-stranded form of bacteriophage of MS2 RNA— infectivity for bacterial spheroplasts. By these criteria the RNA's studied, including the synthetic polynucleotide rG:rC, are completely denatured at room temperature in high concentrations of this solvent. In lower concentrations, the T_m of the RNA preparation is decreased only slightly as the dimethyl sulfoxide concentration is raised until a critical concentration is reached. The T_m falls sharply with small further increases in dimethyl sulfoxide concentration. Sedimentation studies can be conducted directly in these media. The determination of sedimentation velocity in 99% dimethyl sulfoxide containing 0.001M EDTA provides a reliable estimate of RNA molecular weights.     

U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.