Significant Amount Of RNA Research Articles

RNA: Nuclear Glue for Folding the Genome.

A significant amount of RNA is present in the nucleus of mammalian cells but only a small proportion of it is destined for the cytoplasm and subsequent translation, leaving much RNA to associate with chromatin. Historically, nuclear RNA was thought to interact with proteins to form a filamentous nuclear matrix, but this idea became less popular as more dynamic models of chromatin behaviour became more prevalent. Using new molecular and imaging approaches it is becoming clear that RNA should be considered an integral component of nuclear organisation; it is transcriptionally responsive and interacts with abundant nuclear RNA-binding proteins. We suggest that these protein/RNA structures form a dynamic nuclear mesh that can regulate interphase chromatin structure.

Purification and Characterization of RNA Allied Extracellular Tyrosinase from Aspergillus Species

Production of L-DOPA, an anti-Parkinson's drug, using biological sources is widely studied in which tyrosinase is known to play a vital role. Tyrosinase is an omnipresent type 3 copper enzyme participating in many essential biological functions. Understanding properties of tyrosinase is essential for developing useful tyrosinase-based applications. Hence, extracellular tyrosinase from Aspergillus flavus UWFP 570 was purified using ammonium sulphate precipitation and DEAE ion exchange chromatography up to 8.3-fold. Purified protein was a riboprotein in nature containing significant amount of RNA which was confirmed colorimetrically and by electrophoresis. Removal of RNA reduced the activity and altered the conformation of tyrosinase as suggested by spectroflurometric results. Optimum pH and temperature of this 140 kDa protein were 7 and 40 °C, respectively. Copper sulphate and magnesium chloride enhanced the activity whereas in contrast FeCl₃ inhibited the activity completely. Purified tyrosinase transformed L-tyrosine (5 mM) to L-DOPA within 5 h.

Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding

Recently, a novel uracil-DNA-degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil-DNA-degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA-UDE) revealed essential differences. UDE cannot be converted into RNA-UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real-time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA-assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA-dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA-processing enzyme.

Proof-reading signal accuracy of gene expression by binary differential display

Differential display (DD) is commonly used for identifying differentially expressed genes. However, each cDNA species identified by DD must be verified so a "real difference" can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary differential display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5'-base matches and the other with additional mismatches at the 5'-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of differentially expressed genes discovered by DD.

The Multifaceted Human Transcriptome

A large amount of experimental data collected over the last decade has shown that genomic organization is very complex and has highlighted the fact that the current set of gene annotations does not fully capture this complexity. Furthermore, the transcriptome complexity has expanded in several directions. First, there is a significant amount of RNA that can be detected in a cell that originates from outside the exons of annotated genes, representing a multiplicity of novel exons of protein coding genes and stand alone transcripts. Second, exons of annotated and unannotated transcripts separated by large genomic distances can be joined together in chimeric transcripts. Third, individual compartments within the cell differ in the amount of un‐annotated transcription with nucleus and polyA‐ transcriptomes being highly enriched in un‐annotated transcripts. Fourth, the short RNA component of the transcriptome is vast and underappreciated. These results suggest that the functional space of the human genome is not limited to the annotated exons of protein coding genes and is likely to produce a vast amount of non‐coding RNAs. What are the implications of such genomic architecture in terms of increased information content, transcriptional complexity, evolution and disease states?

Large quantity of ribosomal RNA exists extracellularly in mouse spleen.

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen 'supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green-Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that 'thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.

Synthese Poly(A)‐haltiger RNA in auswachsenden Sporen von Bacillus subtilis

AbstractThe synthesis of poly (A) ‐containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls‐labelled with 3H‐uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)‐containing RNA was detected. The amount of poly(A)‐RNA during the outgrowth and first cell division remains constant. Besides poly(A)‐RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)‐contaning RNA during outgrowth of B. subtilis spores.

Distribution of the globin gene in active and inactive chromatin fractions from friend erythroleukemia cells

Stimulation of the T3C12 clone of Friend erythroleukemia cells with 1.2% dimethyl sulfoxide (DMSO) results in progressive increase in the concentration of globin mRNA sequences in the total cellular RNA of treated cells, as measured by nucleic acid hybridization employing a globin cDNA probe. The greatest increment in the content of globin RNA occurs between 30 and 40 h after addition of DMSO. Globin cDNA was also used to measure the concentration of globin-specific sequences in the DNA and RNA of transcriptionally active and inactive chromatin fractions prepared from these cells by the DNase II-MgCl 2 procedure of Gottesfeld et al. [16]. Essentially equal concentrations of globin sequences are present in the DNA isolated from active and inactive chromatin fractions of cells grown in the presence of 1.2% DMSO for 50 h (the time of initiation of hemoglobin synthesis). Furthermore, there are no significant differences in the globin gene concentrations between the active chromatin fractions from DMSO-treated and control cultures at either 50 or 120 h after initiation of DMSO treatment. However, chromatin-associated RNA isolated from the active chromatin of cells synthesizing maximum amounts of hemoglobin (120 h) contains a higher concentration of globin sequences than RNA from the active chromatin of control cells. Chromatin fractions from untreated cells also contain a significant amount of RNA which hybridizes to the globin cDNA probe. These observations suggest that both transcriptional and post-transcriptional control mechanisms are involved in hemoglobin gene expression in T3C12 erythroleukemia cells.

An autoradiographic study of tritiated uridine incorporation into the larval ovary of Xenopus laevis.

The in vitro incorporation of 3H-5-uridine into the germinal and somatic cells of the larval ovary of Xenopus laevis has been studied using both light and electron microscope autoradiography. Incubation for only one hour in the presence of precursor revealed that the follicle cells are highly active in rRNA synthesis, whereas substantial oogonial nucleolar labeling was not detected for several hours. Semi-quantitative analysis of high-resolution autoradiograms indicated that the density of silver grains associated with "nuage" in oogonia was almost 4-fold greater than the surrounding cytoplasm. This strongly suggests that a significant amount of RNA is associated with "nuage" at this stage of Xenopus oogenesis, in addition to its well documented protein composition. "Pulse-chase" experiments further suggest that the nuage-associated RNA is stable for at least 24 hours. These results are discussed (within the limitations imposed by the methodology) both in relation to other studies on the composition of nuage in a wide variety of germinal cell types and in the light of growing speculation that nuage and germinal granules are synonymous.