  |
AsMA NewsScience & Technology Watch
Keeping You Informed of the Latest ********** Drs. Kupfer and Burian present a summary of micro-RNAs and the methodologies in which it is employed in physiology studies. MiRNA and protein transcription factors provide powerful tools used to understand gene expression regulation. ********** MicroRNA: The Newest Player in Gene Expression Regulation Doris M. Kupfer, Ph.D. and Dennis Burian, Ph.D. Most gene expression studies have focused on changes in transcript levels as a reflection of the response of the cell to stress or disease. DNA microarrays and quantitative polymerase chain reaction (qPCR) are popular, well-validated technologies for monitoring regulation of gene expression at the transcription level. Microarrays can screen large numbers of genes—including the complete transcriptome for sequenced organisms, humans as well—for relative levels of expression. qPCR is an exquisitely sensitive method of determining the precise relative or absolute levels of mRNA present in a sample. Even recognition of the role of alternative splicing has been taken into account with the newest generation of commercially available exon microarrays and probes to account for this additional complexity in regulation. MicroRNAs (miRNA), an exciting and newly discovered gene expression paradigm and the methods to assay their levels, are opening up a new world of gene expression level discovery that will be added to our arsenal of tools to discover markers important in aerospace medicine. It was proposed as early as 1969 that RNAs might regulate which genes were turned on or off (2). This theory languished due to the discovery of protein transcription factors. However, in 1993 a group at Dartmouth Medical School published the first animal RNA silencing report ushering in recognition of post-transcriptional regulation through non-coding RNAs (6). The study showed that a C. elegans non-coding small RNA from the gene lin-4 was responsible for negative regulation of a second gene, lin-14 and that the regulation was through binding of the Lin-4 small RNA molecule via an anti-sense sequence to the lin-14 3’ UTR. Over the next few years RNA silencing pathways were discovered in a broad array of organisms including plants, fungi, insects and mammals. A common feature of all, a dsRNA intermediate, was determined by Nobel Prize winners Fire and Mello (4). The observation that introduction of an artificial dsRNA complementary to a gene of interest can result in artificially activating a silencing pathway and lead to the destruction of the targeted mRNA has led to research on developing RNA interference tools in mammals. Lin-4, mentioned above, was the first characterized micro-RNA (miRNA). These are a large class of endogenous RNA silencing molecules found in plants and animals which are evolutionarily conserved, non-protein-coding RNAs. miRNAs are involved in post-transcriptional gene silencing by inhibition of protein translation or targeting transcripts for degradation if there is perfect homology with their mRNA. These small RNAs (19-30nt) are processed from much larger stem-loop precursor transcripts from genes which are not their targets (see Fig. 1 [pdf document] for steps in miRNA biogenesis). Roughly 25% are from intronic regions, with the remainder from clustered intergenic or antisense transcribed regions. miRNAs have been found in all tissues examined, including blood, and have been shown to be involved with a surprisingly wide range of functions including early development, cell proliferation and death, apoptosis, fat metabolism, cell differentiation, organogenesis, and hematopoietic lineage differentiation. Cancer, viral disease, and neural development have also been connected with the activities of miRNAs (1,8). Target prediction algorithms indicate that each miRNA potentially regulates multiple genes and there appears to be multiple miRNA binding sites in many target genes. It appears that at least 30% of human genes may be targets for regulation by miRNAs. miRBase (http://microrna.sanger.ac.uk/), (5) is a public access database registry of all published miRNA sequences and their predicted gene targets. Release 10.1 lists 5,395 entries, with 541 human miRNA sequences. Greater than 230 of these have been experimentally verified in human. The remaining sequences have been verified in zebrafish or are obvious homologs to verified mouse and rat miRNAs. All of these findings suggest an enormous post-transcriptional regulatory circuitry controlled by miRNA acting in a combinatorial fashion, which is in addition to the well-known protein transcription factor regulation tapped by current technology. The extent of gene expression controlled by miRNA appears to have the potential to be at least as global as that regulated by protein transcription factors and seems likely to be interwoven with it. It makes sense then, to address the role of miRNAs in any new study of gene expression regulation. Detection of miRNAs is complicated by their small size and sequence conservation, i.e., miRNAs may differ by a single nucleotide. Recently, modified RNA isolation protocols which retain molecules < 500 nt have been developed and are available commercially. At least one method has been used successfully to isolate total RNA containing miRNAs from blood, a key point since this is the most available tissue for human physiological studies. A variety of methods now have been adapted for use to profile the expression of miRNAs. These include Northern blots, oligonucleotide macroarrays, qPCR-based amplification, bead-based arrays, and spotted DNA microarrays (3). Commercial products which include specific probes based primarily on miRBase for characterized miRNAs as well as additional predicted small RNAs are now commercially available for all of these methods. Innovative approaches have been used to optimize array and qPCR techniques for miRNA. There are available several oligo-based microarrays optimized for miRNA binding. Techniques used for the arrays include using primer extension methodology for increased specificity or primers containing oligos with optimized amounts of linked nucleic acids (LNAs) which increase the Tm and stability of hybridization products. Panels of LNA or standard primers are available for custom spotting, in commercial spotted arrays or coupled to fluorescently labeled beads. Optimized approaches to qPCR include polyadenylation to increase yield of reverse transcriptase (RT) products and stem-loop primers to optimize RT and specificity for mature miRNAs. Macroarrays based on successful qPCR technology are or will soon be available in a 96-well format. Most commercial applications access miRBase and use additional sequences generated by prediction algorithms. Complete mouse, rat, and human panels are available and often can be found in a combined prearrayed format as well as individually for qPCR application. A search of the web will show these and other technologies are accessible now and that new techniques are under development for application to research and potential diagnostics. miRNA regulation of post-transcriptional gene expression promises to be as significant as the well-studied transcriptional regulatory pathways. Two factors make study of miRNA regulation on a relatively large scale possible now; the improvement of RNA isolation technology to allow unbiased isolation of total RNA including miRNA and the adaptation of the powerful microarray and qPCR technologies for use with miRNA. These techniques, along with the establishment of a well-regarded public database, have facilitated the rapid development of readily available, reliable commercial arrays and kits which are accessible and affordable. The over 1,000 miRNA publications that can be found in the PubMed database for 2007 suggest that this is an active area of research ready for application in human physiological studies. References
********** The AsMA Science and Technology Committee provides the Watch as a forum to introduce and discuss a variety of topics involving all aspects of civil and military aerospace medicine. Please send your submissions and comments via email to: barry.shender@navy.mil. Watch columns are available at www.asma.org in the AsMA News link under Journal. |
 |
 |
