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

The first is the preservation of characteristic, long term data imprints that describes the development of an organism in the stable DNA sequences.

Second is the storage of medium term epigenetically featured data that is carried a couple of generations further down the cellular level. Epigenetic information is not stored within nucleotide sequences but in the chemical modifications of these sequences (like the methylation of repeated strings of GC dinucleotide).

Third is the storage of data generated as a result of dynamic interactions between proteins, RNA and DNA in order to adapt to the events and changes during cellular life cycle in the form of nucleoprotein or DNA-protein complexes.

The data generation and storage capacity of DNA in three different hierarchical levels and time periods demonstrates that genome plays a plethora of roles in cellular activities and heredity. Formatting of genome for its generation and storage of data is carried out via DNA sequences of various features. Genomic system is composed of repeating DNA sequences. DNA sequences (satellite) function as a marker as they repeat numerous times in various frequencies. Genome includes genomic folders similar to that of computer systems. These genomic folders, also known as the epigenetic index of genomes, are responsible for the remodeling of chromatin and the coordinated control of genomic functions. Repeating DNA sequences play a critical role in replication of genome (making a copy of DNA), dispersal of copied DNA into daughter cells and construction of support systems that enable organization of chromatins.

It is possible to better understand genomic functions in relation to examples such as memory sticks and hard drives that are used in electronic information systems. The difference between a genome as a basic data-information storage medium from a hard disc is that it can be replicated as required by its nature and these replicas can be transferred to daughter cells. Following examples could be given to illustrate that a genome gains function only when it interacts with various data processing modules in the cell.

1. A copy of genome is produced by cellular DNA replication system
2. Correct localization of each genome copy towards daughter cells is only possible when chromosome segregation system works (the centrosomes and microtubules)
3. The central transcription system is responsible for the copy of data from DNA to RNA. Different gene expression patterns are developed via regulation of transcription time and level with the help of transcription factors and a web of cell signalization.

Very intricately organized genomic system structures are designed through the successive combination of protein encoding sequences, signals distributed in various places and repeating DNA sequences. Formatting of genome resembles formatting of computer programs. Various repeated serial commands of computer software are used to allocate addresses to files independent of the original data contained; different computer systems use different signals and structures to manage programs. In a similar fashion, diverse living species often utilize repeating DNA sequences and chromosomal structures to organize the encoded information and to format their genomes.

Diversity and variation of repeating DNA sequences are building blocks that are constructed into different genomic system structures. Genomes of different organisms bear characteristic system morphology just like computers with various operating systems and hardware. For instance, animal cells are created as a good model to take and incorporate foreign DNA into their genomes. Genetic data transfer among organisms of the same kind is referred to as “vertical gene transfer” whereas transfers between different species, genuses and classes are called “horizontal gene transfer.” Mobile DNA sequences like transposons are very effective horizontal gene transfer agents.

Cellular differentiation and morphogenesis (formation of tissue and organ from cell) is not programmed completely in the primary structure of the DNA sequence. Components of modular programs are encoded in a flexible way and a continuous renewed and recombined arrangement is enabled when needed.

The reason behind creation of different organisms from a single genome is this utilization of such genomic structure. Metamorphosis, that is the development of different organisms like invertebrates such as a caterpillar and a butterfly, is a good example of this feature.

Two organisms from the perspective of the same genomic protein and RNA codes can be considered as two different species. Different genomic structures and repetition of sequences among different organisms are distinctive criteria for the identification of species since these features can lead to mismatch of reproductive cells, different expression patterns of genetic code sequences, and may cause ecological diversity as well. That is why repeated DNA sequences are very important in studying parental relationships. Today, microsatellite DNA as repeated DNA sequences are used to configure biological relations among individuals in forensic sciences. Plant species vary in respect to the repeated sequences in centromeres in their chromosomes; these variations are used for identification of species. Main determinants of genomic system structure are diversity, frequency, and genomic localization of repeated DNA sequences. To explain this with examples, we could say that successively repeated sequences at centromeres, telomere repetitions and transcription, packing of chromatin, repeated sequences that are spread throughout genome in charge of cellular functions like nucleus localization are the main elements of the genome system structure. Genome is a single integrated system that is controlled closely and remotely via communication webs that use repeated sequences.

While explaining the Qur’anic concept of the Manifest Record (36:12) Bediuzzaman Said Nursi, the great renovator of Islamic thought in Turkey in the twentieth century, wrote that the Manifest Record expresses one aspect of Divine knowledge that is related “more to the past and future than to the present. It is a book of Divine Destiny that contains the origins, roots, and seeds of things, rather than their flourishing forms in their visible existence” (30th Word, Second Aim).

Inspired from this view, a seed can be considered as a tiny adorned form of Divinely creative command as programs and indexes and as a determinant for those programs and indexes in the organization of an entire tree. Since the Manifest Record book, as a title of Divine knowledge and command, observes the past and the future rather than the present, the genome of a grain or a seed acts like a library and an archive in which the future and past of an organism is written.

Sequences encoding different information in DNA

Different information types corresponding with various DNA sequences exist in the genome. These DNA sequences that were considered junk for a long time because they were not coding proteins, have in fact been found to be responsible for an amazing array of functions in genomic structure. Some of these sequences include:

1. Group determining sequences that enable coordinated or successive expression of genes,
2. Sequences acting as a marker in charge of initiation and termination during transcription of DNA to RNA ,
3. Signal sequences responsible for conversion of primary immature RNA, sequences into smaller functional RNA molecules,
4. Transcription control sequences that determine the expression frequency of genes,
5. Sequences that identify and mark the initiation regions for intensification and remodeling of chromatins,
6. Sequences that make binding regions which affect the relocation of genome in nucleus or nucleolus,
7. Sequences that target regions where covalent DNA modification (methylation) with functional groups like methyl takes place,
8. Sequences that control and identify the regions responsible for initiation of DNA replication,
9. Sequences that make the structures which enable completion of replication at terminal ends,
10. Sequences at the segregation points that enable equal distribution of copied DNA molecules into daughter cells and centromere sequences,
11. Sequences responsible for guidance during repair of DNA bound errors and damages,
12. Start point sequences used for repackaging of genomes,

Recurring sequences exist in the genomes of many organisms and shows great structural diversity. Recurring elements function as an initiator or terminator for heterochromatin regions. Furthermore they form an important scaffold and binding spots for folding of DNA structure. As if they carry out the job of an architectural mold in specific shaping of genome to be packed into a very limited area. The ratio of repeating sequences in genome (60-90%) is much more than sequences that are encoding proteins and RNA (10-40%). To explain it with an example, chromosomes in human genome are made up of packages of protein-DNA such as heterochromatin and euchromatin. Heterochromatin regions usually make up the regions with no transcription whereas euchromatin regions feature DNA transcription.

The ratio of protein encoding sequences to the entire human DNA is approximately 1.2%. Around 43% of euchromatin regions are composed of recurring and mobile DNA elements. 18% of heterochromatin region is also made of satellite (dense repeating sequences) and mobile DNA elements. Therefore almost 50% of human genomic DNA is composed of these repeating DNA sequences. In bacteria however, these only make up around 5-10% of the genome. These sequences were described as parasitic and junk individual DNA structures up until today and still continues to be described thus by many researchers and scientist. Nevertheless, even today, mobile DNA elements and repeating sequences are accepted as genomic parasites. Recent advances in the last ten years that have demonstrated this is not true, have instead revealed the vital importance of repeating sequences in genomic functions.

Repeating DNA sequences affect chromatin (dense pack of DNA and protein) structure in two ways. Irregular repeating DNA sequence copies contain binding regions for proteins that organize DNA. Heterochromatin (darker since it is densely packed chromatin) inhibits transcription and recombination, delays replication, and generally blocks the reading of information in DNA sequences that contain genetic coding. Heterochromatin regions are distributed throughout the chromosome. Because of this, presence of regions with coupled successive repeated sequences triggers heterochromatin formation.

In fruit flies, placement of protein encoding loci required for eye pigmentation near the heterochromatin blocks in centromeres (phenomenon of position effect) is provided via organization of chromosomes and thus, formation of phenotypic characters are inhibited. The “phenomenon of position effect” is convincing evidence that genome is a major system which is integrated with composition of partially repeating DNA sequences. When heterochromatin amount is increased in XYY male fruit flies, reorganized pigmentation of eye expression decreases. In XO males, when heterochromatin amount decreases, inhibition becomes severe. Changes in levels of protein which binds to special heterochromatin specific DNA regions generate opposite effects. Decrease in these proteins reduces or suppresses “phenomenon of position effect.” Surplus synthesis of these proteins also enriches this effect.

Repeating DNA sequences play an important role in the transfer of genome into daughter cells. For instance, they function in formation of the centromeres as chromosomal binding regions for microtubules, during gamete formation as linear terminals of chromosomes are replicated, and during chromosomal matching. Distribution of repeating sequences plays a major role in configuration of genomic functions. Each genome has genomic system structure that is shaped dependent on the amount of repeating DNA sequences to a major extent.

Going back to Nursi’s explanation of the Manifest Record, we can draw a parallelism between the book of the universe and the book of revelation, the first of which shows us that certain sequences in the genome are repeated for significance and necessity, just as many verses are repeated frequently in the Qur’an with nuances to refer to different meanings, benefits, and purposes, opening a wider space for many interpretations.

A genome is not only a book that contains protein and RNA codes, but also has a complex system structure with many functions for cellular vitality. The most needed sequences are those that are repeated more frequently. They are not pieces of junk DNA as predicted, they are jewels Divinely constructed.

References

• Shapiro J. A. 2001. “Genome Formatting for Computation and Function :Genome Organization and Reorganization in Evolution: Formatting for Computation and Function.” Presented at a symposium on “Contextualizing the Genome,” Ghent University, Belgium, November 25 – 28, 2001 (Ann. N.Y. Acad. Sci., in press)
• Shapiro, J.A. 2005. “A 21st century view of evolution: genome system architecture, repetitive DNA, and natural genetic engineering.” Gene 345, pp: 91–100.
• Shapiro J. A. and Sternberg R. V. 2005. “Why repetitive DNA is essential to genome function.” Biol. Rev., 80, pp. 1–24. Cambridge Philosophical Society.

They are tiny, and there are billions of them inside you. Tiny machines, one thousand times thinner than a hair strand but strong enough to carry all kinds of material within your cells. Yes, there is a complex army of tiny machines inside your body performing an amazing array of functions while you sit at home sipping your tea.

Your heart is beating. Its lifelong duty is to pump blood to tissues to deliver essential nutrients. Transportation of nutrients continues from blood vessels to cells and then into subcellular compartments. Inside of a cell, there is a need for sophisticated biomachines which are responsible for transport. Did you know that you were equipped with minuscule motors that transported cargos in your cells? Or about cellular highways where molecular cargos are transported?

There are various proteins called “motors” in the cell. They can convert chemical energy to mechanical energy to produce force and motion in the cellular highways.¹ Amazingly, molecular motors are much superior to man-made motors in terms of energetic efficiency by hydrolyzing ATP to fuel enzymatic reactions. These molecular motors include rotary motors, polymerization motors, nucleic acid motors and cytoskeletal motors.

Rotary motors

Bacterial flagellum, used for swimming, acts as a propeller and uses a rotary motor. It has been suggested that this motor is similar to Fo motor found in FoF1-ATP synthase. FoF1-ATP synthase takes part in the conversion of chemical energy in ATP to proton gradient, or vice versa. This chemical reaction involves mechanical rotation of parts of the complex.

Polymerization and nucleic acid motors

Polymerization motors take role in polymerizations and these polymerizations generate forces for repulsion (Actin or microtubule polymerization), or separation of clathrin buds from plasma membrane (Dynamin).

DNA and RNA synthesis also involves the use of molecular motors such as RNA polymerase (RNA synthesis from DNA), DNA polymerase (DNA synthesis), Helicases (separation of double stranded DNA prior to DNA or RNA synthesis), Topoisomerases (removal of supercoiling of DNA), RSC, SWI/SNF, and SMC proteins (Chromatin remodeling and chromosome condensation). Moreover, there are specific viral DNA packaging motors that pack tightly viral DNA into capsids. separation of double stranded DNA prior to DNA or RNA synthesis), Topoisomerases (removal of supercoiling of DNA), RSC, SWI/SNF, and SMC proteins (Chromatin remodeling and chromosome condensation). Moreover, there are specific viral DNA packaging motors that pack tightly viral DNA into capsids.

Cytoskeletal motors

Dyneins, kinesins and myosins denote the three major classes of molecular motor that moves along cytoskeletal structures. Myosin is among the most prominent of motor proteins that takes role in muscle contraction. Kinesin operates on microtubules (long tubes composed of dimers of the protein tubulin, arranged to form 13 parallel tracks) to move cargos inside the cells away from the nucleus (toward positive end of microtubules) and play essential roles in the formation of spindle apparatus and axonal transport. Dynein is also known to transport cargo but in the opposite direction to Kinesin, towards the cell nucleus (toward minus end of microtubules). In addition, dynein is required to beat cilia and flagella.

How molecular motors move

Myosin and kinesin are structurally similar in terms of being dimeric with two motor heads, two legs, and a common stalk. The head regions control the forward movement by binding itself to actin or microtubule filaments. Movement is facilitated by the consumption of ATP by ATPase sites. It is fascinating how these motors translate chemical energy into motion and still be different to the movement of cars. There are different proposals as to how molecular motors move, such as walking (hand-over-hand model), inchworm model, and biased diffusion model.

The-hand-over-hand model suggests that ATP binding induces a conformational change in the forward head movements and keeps fixed, thus leading to the movement of the rear head forward and vice versa. This model, which is also known as the walking model, is similar to upright walking where one foot moves forward while other stay fixed, and vice versa. On the other hand, the inchworm model suggests that only forward head movements use ATP and leads while the other head follows. Studies on the Myosin VI with shorter legs suggested a biased diffusion model. In the diffusion model, the motor moves randomly to the next binding site in a forward direction. In order to find out which mechanism used by molecular motors, scientists measured how much of the head moves following staining with a fluorescent dye. Since molecular motor movements are so small (5-10 nM), optical traps and cantilever probes (>100 μm) were not useful to watch head movements. By increasing both photostability and brightness of organic dyes, Dr. Yildiz at UC Berkeley was able to measure head movements down to 1.5nM scale.

Kinesin: A molecular motor that walks

Kinesins are among microtubule-based motors recently shown to walk like a mountain climber by swapping its two motor units (analogous to feet) in a hand-over-hand mechanism rather than an inchworm mechanism. This recent discovery sheds light on how kinesin moves its cargos such as membrane components, messenger RNA, signaling moleculers, and others along microtubules. In addition, as suggested by findings of Dr. Yildiz, kinesin demonstrates an asymmetric walking where motor heads alternate with slow and fast steps. Further studies using advanced microscopy techniques (called FIONA) which allow nano scale detection of movement down to 2nM resolution demonstrated delicately that processive kinesin motor takes about 8 nM steps (eight-billionths of a meter) for each ATP molecule consumption with alternating 16-nm and 0-nm steps. Furthermore, kinesin is attached to the microtubule while it waits for ATP between steps. Since kinesin is used for long distance cargo transport on relatively big highways of a cell, it elegantly demonstrates a processive motor that reliably travels in a coordinated manner. Of course, not all motors will be moving like kinesin.

Dynein moves through uncoordinated stepping of ring domains

Another motor protein involved in long distance cargo transport is dynein. Dynein is a staggering giant which is much bigger and complex than kinesin and myosin motors. There are about 15 types of dyneins known to take role in cilia and flagella movement and 2 cytoplasmic forms. Cytoplasmic dynein is a homodimeric AAA+ (ATPases associated with cellular activities) motor that transports toward the microtubule minus end, acting opposite to kinesin. FIONA assay demonstrated that the heads moving processively but independently. This mechanism is quite different from the hand-over-hand stepping of kinesin and myosin, for dynein’s steps are not strictly coordinated and highly variable. Most of the time, dynein heads move alternatively with variable head-to-head distance of about 5-50nM. Each head of dynein mostly does not pass each other.

Elegant design, efficiency in transportation and being part of the living system makes molecular motors in the cells superior to man-made motors. Molecular motors travel on cellular highways in the cellular microcosm in the manner of dutiful officials of a king traveling in his domain in security via the fastest modes of transportation and easily cross provincial boundaries, demonstrating more evidently that the Sovereignty of the Eternal King is limitless. Indeed, the signs of His Dominion are reflected by each and every entity from the microcosmic world to macrocosmic universe.

Note

1 Cellular highways are composed of microtubules, microfilaments and actin filaments. Myosin moves along microfilaments through interaction with actin, but dynein and kinesin move along microtubules through interaction with tubulin

References

• DeWitt MA et al. Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains. Science. 2012 Jan 13;335(6065):221-5. Epub 2011 Dec 8.
• King SM. AAA domains and organization of the dynein motor unit. J Cell Sci. 2000 Jul;113 ( Pt 14):2521-6.
• Wilhelm J. Walter & Stefan Diez. A staggering giant. Nature. Vol 482. 2 February 2012.
• Molecular motors and Motor proteins. Retrieved from Wikipedia on 3/31/2012.
• Yildiz et al. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization. Science 27 June 2003:Vol. 300 no. 5628 pp. 2061-2065
• Yildiz et al. Kinesin Walks Hand-Over-Hand. Science 30 January 2004: Vol. 303 no. 5658 pp. 676-678

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.

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At the same time as these events are happening almost simultaneously, information from my eyes and my finger is being sent to the brain so that my finger will move in the way I expect it to. For example, if the path of my finger’s movement is somehow blocked, my brain can redirect it.

However, the event described above is not that simple. Starting from the neurons and continuing until we reach the muscles, every element that acts during this process displays extraordinarily complex alterations at both cellular and molecular levels.

Consider muscle cells, since they are moderately well understood. Upon arriving at the muscle cell, the electrical impulse causes the voltage-sensitive calcium channels in specific compartments within the cell to open and release calcium into the cell. You might remember from high school biology that muscle contraction is the result of two proteins (myosin and actin) sliding over each other. Normally, actins are masked by proteins known as tropomyosin. During the waiting period, therefore, the interaction between myosin and actin, which leads to contraction, cannot occur. This is why the muscle cell releases calcium, for when calcium is free in the cell, it binds to tropomyosin and enables it to move. As a result, actin is free to interact with myosin.1

After that, millions of molecules containing energy, known as ATP, bind to millions of myosin proteins, and the muscle contracts. When the contraction ends, the freed-up calcium is stored once again in specific compartments. When calcium is not present, tropomyosins again mask the actin proteins, and millions of muscle cells revert to their initial position, ready to respond to another contraction.

I realize that all of this is hard for the average reader to understand. However, the events that take place are even more complicated.

Expressions like “ATP binds to myosin” and “calcium is stored in compartments” are, in fact, simplified ways of explaining a highly complex event. Since there is a reason for everything, our cells should contain something that is performing these functions and carrying out such events. If we expand this problem to its limits, we will have to understand that each cell contains a large set of rapid and specific chemical reactions that occur constantly and yet do not interfere with one another. Based on current scientific knowledge, we can say that enzymes conduct almost all reactions in a cell, and that DNA has all the necessary information to produce enzymes. Enzymes are protein molecules that speed up and regulate all of the reactions that take place in a cell. If there were no enzymes, the reaction that a cell carries out in seconds could only be completed in thousands of years, and consequently, life as we know it would not exist. Life requires that the correct enzyme be found in the correct place and at the correct concentration.

Based on this, let’s revisit the above example. When the electric impulse reaches the muscle cell and calcium ions are released, this and every external and internal signal is conveyed to the DNA through a mechanism that we are just beginning to appreciate: signal transduction. Later, RNA is produced in those regions of the DNA that are responsible for producing the enzymes that enable the cell to give the appropriate answer (RNA helps DNA to produce enzymes). The synthesis of the enzyme is regulated at various checkpoints, such as during RNA production or RNA translocation out of the nucleus by other enzymes.2 ATPase, one of the many enzymes produced, makes it possible to use ATP, while another enzyme makes sure that the ATPases are in the correct location in the cell. Meanwhile, in order to sustain life, thousands of other enzymes conduct various reactions at the correct time and place. Therefore, when I move my finger, the number of active elements increases enormously.

Let’s look at the finer points of the cell. Using a simple calculation, in which each number is much smaller than the actual number used, if we assume that one million cells perform some kind of action from the reception of the first impulse in the brain until the time the muscle contracts, and if we calculate that one thousand reactions occur in each of these cells, this means that one billion reactions are performed for the simple action of moving a finger. One billion reactions, in just one second. And at the same time, my heart is beating, new blood cells are being produced, my eyes are sending visual information to my brain, my kidneys are filtering my blood, my lungs are exchanging old air with new, fresh air, my digestive system is supplying the necessary nutrients to my blood stream, and much, much more. Moreover, all of these are continually taking place. The fact that all of these actions are occurring, again based on a very rough and simple calculation, means that maybe one trillion reactions are occurring every second. As a result, a person might feel that it is quite possible, at any instant, for this perfect machine-the human body-to fall apart.

Realizing this, one might actually find it hard to believe that he or she is really alive. For example, I would never believe that such a machine would work if I did not have the empirical knowledge that it does work. How, for example, can I believe that I can produce one trillion reactions every moment and never confuse one with another, that it takes one billion reactions to move my finger, and that one trillion gears are working by themselves without making any mistakes?

With this idea in mind, I see the following lines in Epitomes of Light: “Also, since a building that contains every kind of artwork and riches cannot exist without having been built by someone, the existence of this universe is intimately connected with the existence of the Builder. If someone thinks carefully, it is impossible to accept one without the other.”3 Upon reading these words, I start to realize that all of these gears are not working by themselves, but rather that every second all of the trillion gears are being regulated by the One for whom nothing is difficult.

Suddenly, I remember that whenever the names of God are recited, I hear the name al-Hayy right next to al-Qayyum: God is He besides Whom there is no god; He is al-Hayy (the Ever-Living), al-Qayyum (the One Who sustains and protects all that exists) (Qur’an 2:255). Putting al-Qayyum next to “life” indicates, at least to me, that every living being is kept alive at each instant by al-Qayyum. If His control over each person’s existence were to be lost for even one second, one trillion gears would become irreversibly mixed up and the body would fall apart instantly. While thanking God for all that He has given me, I realize that I cannot thank Him enough for even one gear.

References

1. Harvey Lodish et al., Molecular Cell Biology, New York: Scientific American Books, c1995, 1027-29.
2. Lewin, Benjamin, Genes VI, Oxford, NY: Oxford University Press, 1997, 847.
3. Nursi, S., Epitomes of Light: Mathnawi al-Nuriya: The Essentials of the Risale-i Nur, Kaynak A.S., 1999.