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MicroRNAs are natural, single-stranded, small RNA molecules that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. In contrast to the identification and validation of many miRNA genes is the lack of experimental evidence identifying their corresponding mRNA targets. The most fundamental challenge in miRNA biology is to define the rules of...
Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified...
The in situ detection of microRNAs (miRs) expression offers several challenges. It would be advantageous to have a method which can be used in paraffin embedded, formalin fixed tissue to be able to access the large data bank of archival material. Further, it would be helpful if one could differentiate between precursor and mature, active forms of the miR. In this review, two different methods for...
After 12 years from its first application, microarray technology has become the reference technique to monitor gene expression of thousands of genes in the same experiment. In the past few years an increasing amount of evidence showed the importance of non-coding RNA (ncRNA) in different human diseases. The microRNAs (miRNAs) are one of the groups of ncRNA. They are small RNA fragments, 19–25 nucleotides...
Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides—and tens of millions of small RNAs—from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from...
Distinct classes of small RNAs, 20–32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable...
This report presents computational methods of analysis of cellular processes, functions, and pathways affected by differentially expressed microRNA, a statistical basis of the gene enrichment analysis method, a modification of enrichment analysis method accounting for combinatorial targeting of Gene Ontology categories by multiple miRNAs and examples of the global functional profiling of predicted...
microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to...
In some living organisms the 20 aa-tRNA species participating in protein synthesis are not charged by a complete set of 20 aminoacyl-tRNA synthetases. In prokaryotes, the deficiency of asparaginyl- and/or glutaminyl-tRNA synthetases is compensated by another aminoacyl-tRNA synthetase of relaxed specificity that mischarges the orphan tRNA and by an enzyme that converts the amino acid into that homologous...
Selenocysteinyl-tRNA Sec , cysteinyl-tRNA Cys , glutaminyl-tRNA Gln , and asparaginyl-tRNA Asn in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNA-dependent modifying enzyme. The in vitro characterization of these modifying...
In addition to their key role in protein biosynthesis, aminoacyl-tRNA synthetases have other biological functions that appeared during their long evolutionary development. In mammalian cells, specific members of this family of enzymes are also procytokines that, upon conversion, are active cytokines in pathways for angiogenesis, and thereby connect translation to control of blood vessel development...
Transfer RNA (tRNA) plays a pivotal role in protein synthesis within cells, where it is recognized by one cognate aminoacyl-tRNA synthetase, in competition with the remaining non-cognate synthetases, and esterified with an amino acid. For many years the levels of tRNA aminoacylation, in a given population of cellular RNA, have been analyzed using methods that include northern analysis and/or oxidation...
Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or “charging”) specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase...
In some bacteria Lys-tRNA Lys is used both in translation and for the specific addition of Lys to phosphatidylglycerol in the cytoplasmic membrane. This reaction is catalyzed by the membrane protein MprF, and the lysyl-phosphatidylglycerol formed contributes to the resistance of these bacteria to various cationic antibacterial molecules. Obtaining proteins and reconstituting an in vitro system...
The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic...
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