When researchers first identified a new family of seemingly non-functional “junk” RNA molecules, it’s unlikely they could have predicted the power and promise of these nucleic acids. The small, non-coding, single-stranded RNAs – typically 21-25 base pairs in length – were first discovered over 20 years ago in C. elegans, yet they were quickly found to be ubiquitous in species from worms to flies to plants to mammals. The role of these novel RNAs in the regulation of developmental pathways in worms, coupled with their prevalence, inspired researchers to better understand their significance.
We now know that miRNAs (for microRNAs) serve as post-transcriptional repressors of gene expression by targeting degradation of mRNA or interfering with mRNA translation. While small, each can have a big effect; a single miRNA can regulate dozens to hundreds of distinct target genes. They’ve been implicated in a variety of critical cellular processes such as differentiation, development, metabolism, signal transduction, apoptosis and proliferation.
Tissue-specific expression patterns revealed that specific miRNAs are enriched in mammalian tissues including adult brain, lung, spleen, liver, kidney and heart. More compelling was the identification of abnormal miRNA expression in tumorigenic cell lines. It’s no wonder that this growing family quickly became ripe for exploration in disease development.
Within only a few years, a rapidly expanding body of research supported the theory that miRNA expression may indeed play a role in the development of human diseases including cardiovascular disease, cancer, diabetes, cystic fibrosis, and liver disease. Investigations into the expression of miRNAs in cardiovascular disease, in particular, have demonstrated not only their value as disease markers, but also how their dysregulation is linked to disease processes.
Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.
The accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.
Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.
Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control. Continue reading “A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation”
MicroRNA (miRNA) is a group of small (approximately 18–24 nucleotide) single-stranded, non-coding RNAs that function in negative regulation of gene expression.
Lest that non-coding part make miRNA sound inconsequential, read on. While discovery of miRNA is relatively recent, miRNA is some ancient and seriously important gene regulatory material.
Identification of miRNA was published in late 1993 by Lee, Feinbaum and Ambros regarding their work with the worm, C. elegans.
miRNA has been studied in plants, mammals and even viruses, where miRNA functions to repress mRNA expression through base-pairing to complementary sequences in mRNA. This binding can silence the mRNA by several mechanisms, including cleavage of the mRNA, shortening of the poly(A) tail and interference with translation efficiency. Continue reading “miRNA: An Ancient, Small and Important Gene Regulatory Element”
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