miRNA

Small RNA Transfection: How Small Players Can Make a Big Impact

Posted on by Simon Moe

When looking at small aspects of living things, especially cells, it can often be difficult to fully grasp the magnitude of regulation employed within them. We first learn the central dogma in high school biology. This is the core concept that DNA makes RNA and RNA makes protein. Despite this early education, it can be lost on many the biological methods that are employed to regulate this process. This regulation is very important when one considers the disastrous things that can occur when this process goes askew, such as cancer, or dysregulated cell death. Therefor it is very important to understand how these regulatory mechanisms work and employ tools to better understand them.

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The Five Steps to miRNA Profiling

Posted on by Kelly Grooms

MicroRNAs (miRNAs) are small, non-coding RNAs that play a role in regulating cancer by acting as both tumor suppressors and oncogenes. Ranging in size from 18–25 nucleotides, miRNAs function in feedback mechanisms to regulate many cellular processes including cell proliferation, apoptosis, cell signaling and tumorigenesis (1).

Not surprisingly, dysregulation of miRNA expression can have serious repercussions. For example, miRNAs are dysregulated in almost all human cancers (1). Because of the potential to influence cancer growth and development, there is growing interest in miRNA profiling to identify possible biomarkers for cancer diagnosis or prognosis, as well as potential therapeutic targets (1).

Growing interest in miRNAs as both biomarkers of disease and therapeutic targets drives the need for fast and effective methods for miRNA profiling. Profiling miRNA targets follows a relatively simple workflow: sample selection, RNA extraction, RNA QC and quantitation, RNA profiling and data analysis (2,3). So what happens at each step?

Five steps of miRNA profiling
The Five Steps of miRNA Profiling

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microRNA: The Small Molecule with a Big Story

Posted on by Sara Klink

Introduction

miR-34 precursor secondary structure. The colors indicate evolutionary conservation. Ppgardne [GFDL (http://www.gnu.org/copyleft/fdl.html)

RNA molecules have become a hot topic of research. While I was taught about messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA), many more varieties have come into the nomenclature after I graduated with my science degrees. Even more interesting, these RNAs do not code for a protein, but instead have a role in regulating gene expression. From long non-coding RNA (lncRNA) to short interfering RNA (siRNA), microRNA (miRNA) and small nucleolar RNA (snoRNA), these classes of RNAs affect protein translation, whether by hindering ribosomal binding, targeting mRNA for degradation or even modifying DNA (e.g., methylation). This post will cover the topic of microRNAs, explaining what they are, how researchers understand their function and role in metabolism, cancer and cardiovascular disease, and some of the challenges in miRNA research.

What are microRNAs? MicroRNAs (miRNAs) are short noncoding RNAs 19–25 nucleotides long that play a role in protein expression by regulating translation initiation and degrading mRNA. miRNAs are coded as genes in DNA and transcribed by RNA polymerase as a primary transcript (pri-miRNA) that is hundreds or thousands of nucleotides long. After processing with a double-stranded RNA-specific nuclease, a 70–100 nucleotide hairpin RNA precursor (pre-miRNA) is generated and transported from the nucleus into the cytoplasm. Once in the cytoplasm, the pre-miRNA is cleaved into an 18- to 24-nucleotide duplex by ribonuclease III (Dicer). This cleaved duplex associates with the RNA-induced silencing complex (RISC), and one strand of the miRNA duplex remains with RISC to become the mature miRNA.

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The Promise of miRNAs as Therapeutic Agents in Treating Disease

Posted on by Nicole Sandler

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.

Basic research on miRNA is making its way into the clinic.
Research on miRNA is making its way into the clinic.

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.

More recently a new possibility is being explored: can miRNA be manipulated to interfere with disease progression? Continue reading “The Promise of miRNAs as Therapeutic Agents in Treating Disease”

A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation

Posted on by Promega

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.

mirnaThe 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”

MicroRNAs as Circulating Biomarkers

Posted on by Radhika Ganeshan

12097693_lMicroRNAs (miRNAs) are short strands of RNA averaging between 19-24 nucleotides in length that were first discovered in C.elegans and subsequently shown to exist in species ranging from algae to humans (1). Speculated to be merely “junk” more than a decade ago, miRNAs have emerged as powerful regulators of a wide array of cellular processes because of their influence on gene expression at the posttrancriptional level. Dysregulation of these miRNAs is also associated with life-threatening conditions such as cancer and cardiovascular disease, which points to a potential use of miRNAs in diagnosis and treatment. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, protected from degradation by inclusion in lipid or lipoprotein complexes. This opens up the possibility to exploit miRNA as a useful diagnostic tool in clinical samples. Continue reading “MicroRNAs as Circulating Biomarkers”

How MicroRNAs Have a Big Effect on Genetic Regulation

Posted on by Terri Sundquist
miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.
miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

Some of us scientists who have been around for a while still think about RNA molecules falling into three categories: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). However, I have revised my outdated RNA classification scheme as scientists discover exciting new classes of RNAs that do some fairly amazing things. For example, in the early 1980s, Thomas Cech discovered ribozymes, RNAs that have catalytic functions (1), and in the early 1990s, researchers began to take interest in short noncoding RNAs that act as a genetic regulators, the first of which was discovered in C. elegans (2). RNA is no longer simply a biological middleman between DNA and protein. These ephemeral nucleic acid molecules play a much bigger role of cellular physiology and gene regulation than we had previously ascribed to RNA.

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Insight into DICER1 Revealed in Macular Degeneration Research

Posted on by Maria Perr

A scene as it might be viewed by a person with macular degeneration.
I was sad to learn that a friend of mine was diagnosed with age-related macular degeneration (AMD) at the age of 62. Doctors told him that the blurriness he was experiencing in the center of his field of vision (see photo)  was a classic symptom of AMD. Millions suffer from this chronic condition that is now the leading cause of blindness in people 60 and older. This debilitating eye disease is caused by the degeneration of the macula, the central portion of the retina important for reading and color vision.
 
There were encouraging findings into the etiology of AMD in the March 11 issue of Nature by Kaneko et al., entitled “DICER1 Deficit Induces Alu RNA Toxicity in Age-Related Macular Degeneration”. The authors not only proposed a molecular mechanism leading to AMD, but also described a new function for the role of the microRNA processing-enzyme, DICER1. Continue reading “Insight into DICER1 Revealed in Macular Degeneration Research”

The Ongoing Legacy of the Human Genome Sequence

Posted on by Sara Klink

When the first draft sequence of the human genome was announced, I was a research assistant for a lab that was part of the Genome Center of Wisconsin where I created shotgun libraries of bacterial genomes for sequencing. Of course, the local news organizations were all abuzz with the news and sought opinions on what this meant for the future, including that of the lab’s PI and oddly enough, my own. While I do not recall the exact words I offered on camera, I believe they were something along the lines of this is only the first step toward the future of human genetics. Ten years later, we have not fulfilled the potential of the grandiose words used to report the first draft sequence but have gained enough knowledge of what our genome holds to only intrigue scientists even more.

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