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Analysis of Small RNA Species: Phylogenetic Trends
Published in S. K. Dutta, DNA Systematics, 2019
Mirko Beljanski, Liliane Le Goff
Although the participation of 5S rRNA as a structural component of 50S ribosomes has been demonstrated, no particular activity was assigned to this RNA. However, it has been shown that 5S rRNA isolated and purified from rabbit reticulocyte ribosomes exhibits a strong inhibitory effect on the translation of mRNA in a cell-free system. Used in nanogram quantities, 5S rRNA provokes a substantial polysomal breakdown in the presence of ATP and polysomes.63,64 It was suggested that 5S rRNA may play a role in the peptidy 1-transferase activity, where the RNA in question may serve as an intermediate acceptor of the growing peptide chain.65 It was also assumed that 5S rRNA contributes to the movement of the ribosome relative to mRNA.66 So far, the biological activity of 5S rRNA has been rather poorly established in comparison to analytical data accumulated for its primary and secondary structure.
rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
A striking feature of the evolution of the rDNA region is the changing relationship between the small-large rDNA gene unit and the 5S genes, suggesting that in lower eukaryotes an important branching occurs with respect to the linkage of these two sets of genes29 (Figure 1). The most primitive state of the gene system would appear to be no apparent linkage of small rRNA, large rRNA, and 5S genes as observed in Thermoplasma acidiphilum.30 In some lower eukaryotes such as yeast the 5S genes are independently transcribed even though they are closely linked. In many lower eukaryotes and all higher eukaryotes examined the 5S genes are in independent arrays; in Neurospora crassa31,32 and Schizosaccharomyces pombe33 the 5S genes are independent of the rDNA region but are not clustered in tandem arrays. The highly favored independence of the repeated arrays of 5S rRNA genes in eukaryotes may reflect a biological role for 5S rRNA which extends beyond a possibly structural role in cross-linking the small and large ribosomal units.34,35 Alternate biological roles for 5S rRNA molecules may have favored the separation of 5S and the other rRNA genes.
Eukaryotic Dna-Dependent Rna Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression
Published in Gerald M. Kolodny, Eukaryotic Gene Regulation, 2018
Trevor J. C. Beebee, Peter H. W. Butterworth
Functions for each class of RNA polymerase have been established, but the problem of reproducing selective expression of specific coding sequences in vitro has been intractable. There are a number of obvious reasons for this. The vast complexity of the eukaryotic genome has tended to restrict attention to those sequences which are heavily reiterated (particularly those for the stable RNAs, tRNA and rRNA). Even in these cases, attempts to reproduce in vitro the in vivo function of the different RNA polymerases has been complicated by our inability to isolate intact DNA duplex, devoid of single-stranded breaks (“nicks”) which constitute pseudopromoters for the polymerases. Small bacteriophage DNAs provided the answer to these problems in studies of bacterial polymerase specificity, but animal viral DNAs have not proved to be the solution in the eukaryotic case. The reader is referred to Chambon’s review article28 where these difficulties are discussed in depth. We shall concentrate on recent experiments concerning the transcription of rRNA and 5S RNA coding sequences in vitro using purified RNA polymerases I and III respectively.
Strategies for targeting RNA with small molecule drugs
Published in Expert Opinion on Drug Discovery, 2023
Christopher L. Haga, Donald G. Phinney
Similarly, oxazolidinone-based antibiotics are known to bind to the peptidyltransferase center (PTC) of the 50S ribosomal subunit, preventing the formation of the initiation complex itself, thus resulting in inhibition of protein synthesis [36]. Oxazolidinone-based antibiotics are characterized by a core structure consisting of an oxazolidone ring with the S configuration of the substituent at C5, linked to an acylaminomethyl group and an N-aryl substituent [37]. The 50S ribosomal subunit is composed of 5S rRNA, the 23S rRNA, and structural proteins. Despite binding to rRNA in the ribosomal complex, the mechanism of action of oxazolidinone-based antibiotics differs greatly from aminoglycoside-based antibiotics and does not affect peptidyl elongation nor translational termination. Oxazolidinone-based antibiotics crosslink the 23S rRNA with other structural ribosomal proteins, binding to the universally conserved U2585 rRNA nucleotide, thereby stabilizing the base in an orientation that induces a nonproductive conformation of the PTC. Importantly, other RNA structures have been shown to efficiently bind to oxazolidinone derivatives and analogs. For instance, oxazolidinone analogs have been shown to bind to the non-ribosomal RNA T-box antiterminator, a key component of the T-box riboswitch [38] responsible for regulating RNA transcription in response to metabolic effector molecules in Gram-positive bacteria.
Ribosomopathies and cancer: pharmacological implications
Published in Expert Review of Clinical Pharmacology, 2022
Gazmend Temaj, Sarmistha Saha, Shpend Dragusha, Valon Ejupi, Brigitta Buttari, Elisabetta Profumo, Lule Beqa, Luciano Saso
Transfer RNA (tRNA) carrying an antisense triplet to decode mRNA and their amino acids are linked to a growing polypeptide chain. The translation process consists of four phases: initiation, elongation, termination, and ribosome recycling [2–6]. Genes responsible for nucleolar organization are present in the short arm of acrocentric chromosomes (chromosomes 13, 14, 15, 21, and 22), the so-called nucleolar organization region (NOR) [7]. Further steps mainly include processing of endo- and exonucleolytic cleavage and modifications, such as pseudouridylation and methylation in rRNAs 18S, 5.8S, and 28S rRNA [8]. RNA Pol III is responsible for the transcription of 5S in the nucleolus, and 5S rRNA genes are organized in clusters of tandem repeat units on chromosome 1 [9]. 5S rRNA is transcribed in the nucleus and remains there before it joins 60S in the form of 5S RNP [10]. Ribosome biosynthesis, including processing three RNA polymerases and activating more than 200 non-ribosomal factors within the nucleolus, requires considerable cellular energy compared to other cellular processes [11,12].
Small RNA-sequence analysis of plasma-derived extracellular vesicle miRNAs in smokers and patients with chronic obstructive pulmonary disease as circulating biomarkers
Published in Journal of Extracellular Vesicles, 2019
Isaac Kirubakaran Sundar, Dongmei Li, Irfan Rahman
To validate a few miRNAs identified in RNA-sequencing data, we performed qPCR analysis of selected miRNA targets. The miRNA-specific primer pairs were obtained from Applied Biological Materials Inc. (ABM, BC, Canada). First, 50 ng of exosomal RNA was reverse transcribed using the miRNA cDNA synthesis kit, with Poly (A) polymerase tailing (ABM, BC, Canada) according to the manufacturer’s instructions. Two microlitres of synthesized cDNA were used as a template for all the qPCR reactions performed with RT2 SYBR Green/ROX PCR master mix using the CFX96 real-time system (Bio-Rad). Differential expression of miRNAs between the exosomal RNA isolated from CSE-treated BEAS-2B or U937 and control cells (without treatment) were expressed as relative abundance/fold change. Fold-Change (2^(- Delta Delta Ct)) is the normalized gene expression (2^(- Delta Ct)) in the Treated Sample (CSE) divided by the normalized gene expression (2^(- Delta Ct)) in the Control Sample (Control). For all the miRNA targets analyzed by qPCR, 5S rRNA was used a housekeeping control, cellular RNA reverse transcribed in a similar manner was used as a positive control, and no template control was used as a negative control. Melting curve analysis was used to confirm the specificity of the amplification reactions.