MicroRNAs as tiny regulators with big roles

Tiny RNAs, known as microRNAs, have been shown to regulate many components of the body’s cellular machinery. They are called microRNAs because they are only 22 nucleotides in size (compared to the 2200 nucleotide-long messenger RNA). Amazingly, these small non-coding RNAs can turn off the translation of their target genes. They act as control switches by targeting the 3′ untranslated regions of messenger RNAs (mRNA) for translational repression or cleavage, thus resulting in a reduction of protein levels. Because each microRNAs can regulate hundreds of messenger RNAs, there are probably few cellular processes not affected by microRNAs. For instance, microRNAs have recently emerged as playing important roles in a variety of cellular processes, such as heart development, stem cells, insulin secretion, and cholesterol synthesis. MicroRNAs were first discovered in worms more than 20 years ago. For many years, scientists thought that DNA was transcribed to RNA, and then translated to protein. Those proteins are major regulators in the cell. Now, they appreciate that there are more levels of control and a number of non-coding RNAs that regulate the level of cellular components. About one thousand microRNA genes have been discovered in the human genome. This makes the microRNAs one of the most abundant classes of regulatory genes. As a result of the discovery of this new and major level of regulation in the cell, Dr. Andrew Z. Fire and Dr. Craig C. Mello were awarded the 2006 Nobel Prize in Physiology or Medicine.

MicroRNA biogenesis

Unlike other RNAs, the production of microRNAs is quite different. As depicted in figure 1, the generation and activity of microRNAs requires special microprocessors, known as RNA polymerase II, Drosha, Exportin, Dicer, and RISC complex. RNA polymerase II transcribes (reads the microRNA DNA code) primary microRNA transcripts; then the Drosha process transforms primary microRNA into precursor microRNA in the nucleus. For activity and further processing, precursor microRNA are exported into cytoplasm by Exportin. In the cytoplasm, Dicer cuts precursor microRNA and generates mature 22 nucleotide long microRNA. Then, mature microRNA are incorporated into the RNA inducible silencing complex (RISC) where they target messenger RNAs (mRNA), either for degradation or translational repression. Even though there are extensive studies on microRNAs, it is still mostly unknown how microRNAs target specificity is determined and how they target messenger RNAs for mRNA degradation or translational repression. For a functional microRNA in the cell, it is amazing that a series of microprocessors should take place. They recognize different microRNAs as substrates and do their job as they are supposed to. It seems that the existence and regulation of microRNA processing abilities cannot be by mere chance.

MicroRNAs as therapeutics

MicroRNAs are considered “fine tuners” of cellular processes because of their subtle effects on their targets. However, because microRNAs can target a number of genes and genetic pathways, the study of microRNAs and their regulation and role in diseases is highly promising in terms of developing new therapeutic approaches. Treatments by targeting microRNAs using microRNA inhibitors (antisense RNA nucleotides) are under intense study and several of them have been shown to be effective in animal models. A MicroRNA known as miR-122, for instance, has been shown to regulate cholesterol levels. Scientists targeted this liver-specific microRNA by using a microRNA inhibitor and they found that the downregulation of miR-122 resulted in a 40% decrease in cholesterol levels in the blood.

MicroRNAs in cancer therapy

With the discovery of new and better tools to detect and manipulate microRNA levels in cell cultures and tissues, researchers are now attempting to identify the specific features of each microRNA and their role in cancer and other devastating diseases. There are some microRNAs that are highly correlated with cancer formation. Cancer is cellular anarchy characterized by a proliferation of cells without control. A group of miRNAs known as the miR-17-92 family have been found to increase, and their higher levels result in cancer formation as found in some lymphomas and solid tumors. It is believed that better understanding and use of microRNAs or microRNA inhibitors could enable doctors to treat diseases like cancer. In the near future, microRNA studies are also expected to provide early detection of progressive diseases, better markers for cancer initiation, and cancer specific drug selections.

MicroRNAs as cancer drug boosters

The most straightforward application of microRNA research has been cancer chemotherapies. The potential of use of microRNA applications to increase the effectiveness of current cancer drugs seems highly likely. Companies and universities are looking for microRNA partners to increase the effects of drugs like Taxol, which is currently used in chemotherapy. Taxol, for example, currently works for about 30% of lung cancer patients. But, if we can find a microRNA partner with that drug to make it 40%, it will mean saving thousands of lives. This is a hopeful sign for the future of cancer treatment. On the other hand, it is known that in the case of any chemotherapy, there are unwanted side effects. Although use of higher dose of drug will kill more tumors, the side effects of this drug will cause other issues. Discovery of partners like microRNAs that boost the effectiveness of cancer drugs or decrease side effects can help to treat more patients or help them overcome unwanted side effects.

Taking microRNAs to the heart of the matter

Heart diseases represent the primary cause of death in developed countries. Recent studies have identified microRNAs associated with heart diseases, including cardiac hypertrophy, heart failure (inability of the heart to pump sufficient blood to the organism), and myocardial infarction (the death of the cardiac muscle resulting from interruption of the blood supply). Mir-1 expression levels, for example, are low in human heart disease and it is known to regulate Hand2, a protein required for the growth of heart muscle cells. The levels of another microRNA, called miR-21, have consistently increased through cardiac stress and have been shown to regulate cardiac growth as well. Importantly, miR-133 is believed to repress cardiac hypertrophy, thus the use of synthetic miR-133 molecules is possible as a therapeutic for patients with pathological hypertrophy. However, more studies to understand heart-associated miRNAs are needed in order to have clinical trials for the treatment of heart diseases.

Figure 2. MicroRNAs in the heart. Recent studies have identified microRNAs that are associated with heart diseases, including arrhythmic heartbeat (Arrhythmias), cardiac hypertrophy (enlarged heart), septation defect, and cardiac muscle overgrowth (myocyte hyperplasia).

Micromanaging insulin secretion

MicroRNAs are also associated with the onset of diabetes. Diabetes affects about 23.6 million people in the United States. It can lead to serious health issues and even early death. Diabetes is marked by high levels of blood glucose (also called blood sugar). Complications of the disease are due to defects in insulin production and insulin action. Insulin is among the major regulators of sugar levels in the blood. The human genome contains a number of microRNA genes, whose functions are only beginning to come to light. One such microRNA, miR-375, is already implicated in the secretion of insulin from pancreatic cells, thus it represents a novel pharmacological target for the treatment of diabetes.

The mentioned cases above are examples of the tiny RNAs which regulate cellular processes. The loss of the control in such a small component of the cellular machinery can lead to serious problems, like cancer. To use a metaphor, the regular and healthy government of a state does not allow for the presence of multiple governors. Similarly, regulatory tiny RNAs require a controller who knows how the human body works at the macro and micro levels. This forces us to consider that whomever is controlling the human body must be all sustaining and all knowing. With each new scientific breakthrough, the wisdom of creation becomes more and more apparent. The field of miRNAs is a young research area. New discoveries about microRNAs have brought us new hopes for novel therapies to human diseases. However, future discoveries are required before these therapies can be used in a clinical setting.

Resources

• Qur’an: The Family of Imran 191 and The Cow 255.
• Caldas & Brenton. “Sizing up microRNAs as cancer genes”. Nature, 2005.
• Scott M. Hammond. “MicroRNA therapeutics: a new niche for antisense nucleic acids” Trends in Molecular Medicine, 2006.
• Rooij et al. “Toward MicroRNA–Based Therapeutics for Heart Disease” Circulation Research, 2008.
• National Diabetes Statistics, 2007. Retrived from http://diabetes.niddk.nih.gov/DM/PUBS/statistics/
• ScienceDaily. Not ‘Junk DNA’ After All: Tiny RNAs Play Big Role Controlling Genes. 2007.
• Callis & Wang. Taking microRNAs to heart. Trends in Molecular Medicine. 2008.
• Poy et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature,2004.
• Average mRNA length: B. Lewin, Genes 5, Table 2-2. Oxford University Press.
• MicroRNA biogenesis figure: http://content.nejm.org/content/vol359/issue25/images/large/14f1.jpeg

A particularly interesting class of all these non-coding RNAs comprises riboswitches. Riboswitches are structured mRNA elements that regulate gene expression upon binding of a specific small metabolite. These biosensors were first discovered in 2002 in bacteria. Later, it was shown that plants, green algae and fungi also posses riboswitches. Although, only one type of riboswitch is found in plants and none has beeen detected in mammals yet, metabolite-sensing riboswitches are commonly used for the regulation of fundamental biochemical pathways in bacteria. Riboswitches help cells monitor the environmental conditions and determine if a compound is present at sufficient levels or not. Based on this decision, production, degradation or transport of the related metabolite is either turned on or off.

Architecture of a Riboswitch

A standard riboswitch is divided into two parts: a ligand-binding domain and a gene expression domain (Figure 1). Small molecular metabolites bind to the ligand-binding domain, which is called aptamer, of riboswitches with astonishing specificity. For instance, purine riboswitches differentiate guanine from adenine by at least 10,000-fold based on the identity of a single pyrimidine (thymine or cytosine) that binds to the ligand (1). When a ligand binds to aptamer, conformational changes of the RNA’s structure occur at the gene expression domain. Eventually, this leads to the modulation of gene expression. Some of the riboswitch mechanisms to regulate the expression of genes include formation of stem-loops, lollipop-like RNA structures, which lead to the blocking of transcription or translation, which are the processes where proteins are synthesized using the mature mRNA, self cleavage or mRNA destabilization (Figure 2).

Classes of Riboswitches

The numerous distinct classes of riboswitches discovered so far are differentiated by their ligands and remarkably, the same class of riboswitches can control gene expression through different mechanisms in various organisms. For example, a riboswitch that recognizes and binds thiamin pyrophosphate (TPP), a derivative of vitamin B1, is called as TPP riboswitch (2). In plant cells, when TPP levels are high, excess TPP binds to a TPP riboswitch located in one of the TPP biosynthesis genes. As a result of TPP binding, conformation of that RNA segment changes, which leads to the formation of an unstable mRNA product which degrades quickly. Although, a very little amount of stable mRNA is also produced, it is not enough for the protein synthesis of an important component of TPP biosynthesis. Therefore, TPP biosynthesis can not be completed and consequently TPP levels drop in the cell. On the other hand, when TPP levels are low in plants, more stable mRNA is formed because there is not enough TPP that can bind to the riboswitch and cause structural changes which will result in the formation of unstable mRNA. The stable mRNA produced in the absence of excess TPP is used for the protein biosynthesis of TPP metabolism successfully and thus TPP levels increase in the cell. Unlike plants in bacteria, when TPP concentration is high, riboswitches down-regulate expression of thiamin biosynthesis genes by either blocking the formation of any type of mRNA (both stable and unstable) or preventing the mRNA process rather than destabilizing the mRNA. Some of the other riboswitches that bind vitamin derived compounds are adenosylcobalamin, the coenzyme form of vitamin B12, and flavin mononucleotide, a biomolecule produced from vitamin B2, riboswitches. There are also riboswitches that can bind to amino acids such as lysine riboswitch. The cyclic diguanylate (c-di-GMP) riboswitch is the first known example of an RNA that binds a second messenger, which carries signals from receptors on the cell surface to target molecules inside the cell (3). Purine riboswitches selectively recognize guanine or adenine and regulate purine biosynthesis and transport, which is important for DNA/RNA synthesis. Another interesting type of riboswitch is the glmS riboswitch. It modulates gene expression by undergoing self-cleavage when there is a sufficient concentration of glucosamine-6-phosphate, an important amino sugar. The spectrum of ligands that can be recognized by riboswitches also includes a metal ion, as well. Mg(2+) riboswitches control Mg(2+) transportation when cells are grown in high Mg(2+) environments. In brief, the growing list of studies on riboswitches shows how novel mechanisms are which they use to regulate the gene expression of many fundamental metabolic pathways.

Applications for Riboswitches as drug targets and chemosensors

Riboswitches are powerful and essential components in all three domains of life which are bacteria, archaea (a group of single-celled microorganisms) and eukaryotes ( organisms whose cells contain complex structures inside membranes such as plants and fungi) functioning as intracellular biosensors and regulators. They regulate gene expression in a highly efficient, precise and fast way. Therefore, they can be engineered for various applications.

First of all, riboswitches are excellent candidates as drug targets since they control many important bacterial and fungal genes. Chemical analogs that mimic the actual ligands of the riboswitches can be designed to silence or regulate the expression of defective genes responsible for disease development or even kill certain bacterial pathogens by turning off the genes that are involved in fundamental metabolic pathways. A great example of this strategy is presented by the Breaker laboratory at Yale University. The Breaker group chose guanine-binding riboswitches as targets for the development of novel antibacterial compounds. They have designed several guanine analogues and tested their ability to be bound by the riboswitch and repress bacterial growth (4). They have been able to inhibit the bacterial growth by inducing guanine riboswitch action. Their approach could be used to discover new antibacterial compounds that specifically target other riboswitch classes.

In addition to being drug targets, riboswitches can open new frontiers in bioremediation, bionanotechnology, and synthetic biology. In 2007 Shana Topp and Justin P. Gallivan from Emory University demonstrated that Escherichia coli can be reprogrammed to detect, follow, and precisely localize to a completely new chemical signal by using a synthetic riboswitch (5). They suggest that the bacteria with synthetic or mutated riboswitches could be used to follow and degrade pollutants in soil or target small-molecule signals of disease. Overall, their work to equip the bacteria which can autonomously follow chemical signals, degrade, synthesize or release compounds can help scientists invent new technologies in bioremediation, drug transport, and synthetic biology.

Furthermore, riboswitches are ideal candidates for use in analytical devices and techniques. Their binding features make them suitable in all specific analytical applications in which selective recognition is required. Therefore, these powerful molecular tools can be utilized in analytical chemistry, molecular biology and biosensor technology (6).

As a result, living systems utilize riboswitches to detect the concentrations of small-molecule metabolites and to modulate the expression of related genes via numerous elegant mechanisms. The use of genetic engineering of riboswitches holds enormous potential for inventing new applications to sense and destroy pathogens, deliver drugs, perform bioremediation, detect chemicals and many others that might have significant impacts on our lives. It is also remarkable that only a couple of decades ago most of the non-coding RNAs were assumed to be useless, and are called junks since they do not provide any information for protein synthesis. Yet riboswitches by themselves are enough to prove that God has created everything with a purpose. He is All-Wise and he does nothing in vain. Riboswitches are not useless at all. They are such complex, and perfectly working systems that they can serve humanity as well-designed on/off switches in numerous applications.

Safiye Arslan is a research fellow in the area of biological chemistry and lives in Nevada.

References

1. Gilbert SD, Reyes FE, Edwards AL, Batey RT. 2009. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs. Structure 17, 857-868

2. Wachter A, Tunc-Ozdemir M, Grove BC, Green PJ, Shintani DK, Breaker RR. 2007. Riboswitch control of gene expression in plants by splicing and alternative 3′ end processing of mRNAs. Plant Cell 19, 3437-3450.

3. Kulshina N, Baird NJ, Ferré-D’Amaré AR. 2009. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch. Nature Structural & Molecular Biology 16, 1212-1217.

4. Kim JN, Blount KF, Puskarz I, Lim J, Link KH, Breaker RR. 2009. Design and antimicrobial action of purine analogues that bind Guanine riboswitches. ACS Chemical Biology 4, 915-927.

5. Topp S, Gallivan JP. 2007. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society 129, 6807-6811.

6. Mairal T, Ozalp VC, Lozano Sanchez P, Mir M, Katakis I, O’Sullivan CK. 2008. Aptamers: molecular tools for analytical applications. Analytical and Bioanalytical Chemistry 390, 989-1007.