Messages from STEM and Health Science Scholars
Unity of Actions: Epigenetic Transcription, RNA, LncRNA & Mitochondria
In the 1980s the great hope in medicine was gene therapy and/or precision medicine. As the human genome, once estimated to be a large as 60,000 genes, was whittled down to about 20,000, there was the expectation that particular genes could easily be identified in relation to particular diseases and ailments, and easily turned off. However, of the 20,000 protein coding genes, only about 2000 have been studied in depth, and over 5000 have never been examined. Second, targeting genes is tricky and complex, especially as there are often hundreds, if not thousands of genes involved in a given cellular and organ activity. And even if one is able to correctly isolate and indict a particular gene as being responsible for an illness or disease, once it is turned off or eliminated it is no longer able to participate in other bodily functions. Consequently, as recently reported by Francis Crick Institute, researchers are calling for greater awareness of unintended consequences of CRISPR gene editing, which allows scientists to remove and replace sections of DNA in cells. For example, in a study which examined the role of the OCT4 protein in human embryos during the first few days of development, while the majority of the CRISPR induced mutations were small insertions or deletions, in approximately 16 % of samples there were large unintended mutations.
Genes are also interacting with the environmental heredity, via diet, climate, land masses, and disease. In these respects, DNA tells only a portion of our biological historical nature. In fact, RNA’s long primeval history involves its self-replicating molecule combining amino acids into proteins, its mutations, and it creating a double-helix DNA molecule to store its complex intracellular regulation. When the Human Genome Project was complete in 2001 it was a major surprise that protein-coding genes accounted for as little as 1-2% of the human genome, leading the remainder to be termed as genetic noise or junk DNA. But it is now understood that most of the DNA, while non-protein coding is transcribed into RNA.
Accordingly, epigenetics, which lends insight into the functions of the RNA genome, as per gene expression, and gene splicing to produce an even greater number of proteins, examines how the overall epi-genome interacts within its given environment. More specifically, “Epigenetics is what enables every cell to act differently despite having the same DNA sequence.” In this respect, while cells in the body inherit the same DNA, they don’t use all of it. Instead, they transcribe different regions based on their needs and in response to their environment. Epigenetics regulates this process through various means, such as small chemical modifications to DNA or to the chromatin and histone proteins that package DNA. In fact, at any given moment in the human body, in about 30 trillion cells, DNA is being “read” into molecules of messenger RNA, the intermediary step between DNA and proteins, in a process called transcription.
Transcription begins as proteins called RNA polymerases are recruited to specific regions of the DNA molecules and begin skimming their way down the strand, synthesizing mRNA molecules as they go. During the transcription sequences of nucleotides, introns or In-Coding regions are set aside, and remaining segments called exons are spliced together and introns are removed by RNA splicing, as RNA matures. In other words, the introns are not expressed in the final messenger RNA (mRNA) product, while exons go on to be covalently bonded to one another in order to create mature mRNA. Not surprisingly, considering their long primeval history, research suggests the products of transcription—namely, RNA molecules, regulate their own production through a feedback loop. Too few RNA molecules, and the cell initiates transcription to create more. Then, at a certain threshold, too many RNA molecules cause transcription to draw to a halt.
In molecular biology, a transcription factor is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. For example, animal cells undergo fundamental shifts in gene expression when there are changes in the oxygen levels around them. These changes in gene expression alter cell metabolism, tissue remodeling, and even organismal responses such as increases in heart rate and ventilation. In a similar way, circadian oscillators within individual cells respond differently to entraining signals and control various physiological outputs, such as sleep patterns, body temperature, hormone release, blood pressure, and metabolism. Changes in daylight, temperature, and metabolism are similar types of transcription factors.
MicroRNAs are small RNA molecules that regulate the expression of certain genes through interaction and protein production with mRNA targets. But beyond gene transcription and protein production, RNA also functions as regulators and supervisors of cellular functions, so as to prevent glitches in cellular performance that can lead to defective proteins or insufficient protein production which are involved in most biological functions of the body. For example, a group of proteins, including: A-Raf, B-Raf and C-Raf transmit signals that control proliferation, differentiation, and survival in every cell in the body. Raf proteins, especially B-Raf, when not properly regulated by RNA, are well-known cancer drivers. Hence, Raf’s full name: rapidly accelerated fibrosarcoma. In the normal state, the B-Raf has three parts, including: Mek1, B-Raf, and 14-3-3, whereas the slight difference in the abnormal, which causes the entire protein to get stuck in ON position, is that there an additional B-Raf. As a result of this mutation, the Mek1 continues to send its growth promoting signal, uncontrollably.
In order to fully grasp the complexity of these types of RNA, let’s take pancreatic cancer as an example, as it is one of the most widespread of cancers due to the fact that the natural rate of cell replication is one of the highest in any of the human organs, and since replication involves careful copying and transcription of genetic structures, errors and mutations are inevitable. Accordingly, an integrated analysis was performed of miRNA and mRNA expression data to explore the deregulation of miRNA and mRNA and regulatory processes underlying pancreatic cancer. Combining mRNA and miRNA expression data with miRNA, target predictions were constructed to infer new miRNA regulation activities in pancreatic cancer. The results show 42 differentially expressed miRNAs, 1376 differentially expressed mRNAs were identified by combining three expression profiles of miRNA and mRNA separately in pancreatic cancer, and 146 miRNA target genes were found in the gene list of integrated mRNA expression profiling based on bioinformatics prediction. These findings may provide new insight into the knowledge of molecular mechanisms of pancreatic cancer and the development of novel targeting therapies, but also indicate the multiple levels of complexity.
Long Non-Coded RNA Complexities & Unknowns
On yet another level, in addition to the complex and dynamic functions of RNA, research provides some insight into the potential roles of the thousands of lncRNAs that are not translated into any proteins which are common in mammals and have mystified scientists for decades. Technically, long non-coding RNAs are loosely described as non-coding protein-coding transcripts of more than 200 nucleotides which promote and inhibit gene expression via a variety of mechanisms, and like protein coding mRNA, the majority of Lnc-RNA’s are transcribed by RNA polymerase. However, epigenetics reveals that all cells of the immune system rely on an integrated and dynamic gene expression program that is controlled by both transcriptional and post-transcriptional mechanisms, which in turn, are regulated by lncRNAs. In this respect, as proteins might be conceived of workers in a factory, DNA is the supplies and materials, and RNA are the managers and supervisors; and as will also be seen—lncRNA can be considered Upper Management.
Moreover, with lncRNA also independently performing activities of proteins and enzymes, and its circular shape directly communicating with the immune system, researchers wonder how its specific sequences provide other new kinds of instruction for the cell. In fact, the field of epigenetics is witnessing a burgeoning paradigm of lncRNA-mediated control of gene expression and the differentiation and function of innate and adaptive cell types. Next-generation sequencing also provides a more complete picture of the composition of the human transcriptome indicating that much of it contains poorly understood non-protein-coding transcripts. In sum, the human genome may transcribe over 100,000 lncRNAs, mediating their functions through interactions with proteins, RNA, DNA, or a combination of these, and are implicated in a variety of disease states, such as cancer.
Mitochondria & LncRNA
Mitochondria is a molecule capable of releasing energy very quickly, but beyond being the bodily ‘batteries’ in providing the needed energy for our cells to function by converting the food that we eat into ADP, mitochondria’s function is affected by the internal and external roles of Nuclear Factor kappa B, which maintains mitochondrial structure or activity. Accordingly, mitochondrial metabolism is fully dependent on factors encoded by the nuclear DNA, including many proteins synthesized in the cytosol and imported into mitochondria via established mechanisms. Nuclear factor kappa B is an ancient protein transcription factor and considered a regulator of innate immunity, and its signaling pathway links pathogenic signals and cellular danger signals organizing cellular resistance to invading pathogens. The Nuclear Factor kappa B also plays a role in cell death, as Howard Chang at Stanford notes that his earlier research revealed that the aging process was not one of decay and deterioration, but rather a metaphorical on/off switch at the biochemical level, organized, directed by a key regulator, namely, Nuclear Factor Kappa B.
While it has been well documented that our adrenals glands convert cholesterol and release cortisol, causing a bodily response via mitochondria in all cells & organs, there are now correlations between neurologically processed psychological stress and deterioration of mitochondria cells & organs. In fact, to maintain homeostasis of the entire cell, an intense crosstalk between mitochondria and the nucleus, mediated by encoded noncoding RNAs (lncRNAs), as well as proteins, is required. Mitochondria-associated lncRNAs have also newly been discovered to work in concert with transcription factors and epigenetic regulators to modulate mitochondrial gene expression and mitochondrial function. There also occurs a culmination of an overproduction of Reactive Oxygen Species ROS—with the end result of cell toxicity. These negative effects of ROS from oxidation, have been offset by supplementing the subjects with antioxidant enzymes in the gut. In this respect, emphasis is placed on the involvement of lncRNAs in cancer metabolism, as tumor cells rely heavily on modifications of mitochondrial functioning as an essential component for sustained tumorigenesis and cancer progression. Due to their key role in cancer progression, they represent potential targets of innovative lncRNA-based treatment strategies.
These points regarding mitochondria and its RNA regulators being involved in a vital and deeper inter-bodily physical sense of connection are compelling, and also suggest that one very fundamental job of sleep—perhaps underlying a network of other effects—is to regulate the ancient biochemical process of oxidation, by which individual electrons are snapped on and off molecules in service to everything from respiration to metabolism. Sleep, the researchers imply, is not solely the province of neuroscience, as previously asserted by Hobson in Nature: “Sleep is of the Brain, by the brain and for the brain”—but something more deeply threaded into the biochemistry that knits together the animal kingdom. Sleep loss alters metabolism in humans, as noticeable in changes in the microbiome, and there are related connections to diabetes and metabolic syndrome and other chemical processes. Just what the findings mean still needs to be explored, but they suggest that sleep is vitally important to the body’s regulation of oxidation, particularly in the gut.
In keeping with my scientific writing submission dealing with cancer research, titled: “Unity of Actions: Epigenetic Transcription, RNA, LNCRNA & Mitochondria,” which is an excerpt from a longer journal article, I am attaching a short reflection on the long-standing personal intellectual process that led to its creation. As this process was particularly dramatic, I have chosen to categorically list the stages of its development in terms of a five-act play.
My first memory of the foreboding “C” word took place when my mother and her sister came home from the doctor’s office, after hearing that their father’s case was terminal. He was dying of lymphoma. Their emotional cries lingered in the air like sour milk. We had intimations of his fate, earlier on. I remembered my grandfather sitting in the rocker on the porch, with a blanket covering his frail body, with him moaning a low murmured moan as the cancer had now overtaken his entire body.
In contrast, he had been a virile man, even in his seventies, who had tendered to a flower and vegetable garden, grew his own grapes, made his own wine, baked bread, and cooked all the family’s meals. There were now no nutrients to revive him.
Heidegger speaks of the un-thought thought, referring to those experiences that are in some way known to the individual, but about which the individual is unable to think; and in that preverbal sense, I had been preoccupied with development of cancer, even as a child. With a mutational strain of genetic eye turn (strabismus) running on my father’s side, amid my sister and I, along with two cousins, I was dealt with the worst blow of crossed eyes, requiring surgery at ages 3 and 7.
Hence, when I met with Howard Gardiner a few years ago at the Radcliffe Institute, after corresponding with him regarding my writing of poetry, he asked me why I was studying and teaching cancer research, so I told him of my existential torments as a child. To my surprise, he took off his glasses and gestured to his own long-lasting turn in one of his eyes. I was taken back by the fact that I had not noticed it in prior viewings of his videos and photographs. I also realized why I had felt a need to meet with him, beyond my interest in his theory of multiple intelligences.
Kierkegaard centers much of his philosophical thought around the notion of the subjective thinker: how one considers a topic deeply from an intellectual point of view and is also driven by an unspeakable passion. Such a restless meditative state had permeated my mind and came to fruition during my college years, as I embarked on an introspective philosophical search into the conceptual motifs of self, truth, and meaning, in both Western and Eastern traditions. This in turn, led to a more instrumental and methodological study in the social and physical sciences, and the teaching of a writing across the disciplines course, called: “Brains, Genes, and Lingo.” Students who had been haunted by family cancers of their own, contributed to a new sense of gravitas and urgency, leading to the development of a course which exclusively centered on cancer research.
But the cause(s) of cancer remain elusive. Like Freud’s replacement of singularly pointed theory of etiology with that of over-determinism, disease caused by multiple causes, cancer research reveals numerous paradoxes, ambiguities, and complex mysteries yet to be unraveled. In this regard, I have offered a particular section of my writing on the nature of cancer research, which deals with the dimensional connections between DNA, RNA, long non-coding RNA, and mitochondria. But in terms of completing my dramatic narrative, it should be noted that new research into the nature of mitochondria provides insight into both strabismus, and cancer development.
During the heyday of single-celled life about 2 billion years ago, the forerunners of mitochondria were bacteria that found a niche inside larger cells, providing them energy. This symbiosis was so beneficial that it likely powered the evolution of multicellular organisms. As a relic of their bacterial origins, mitochondria still carry their own small genome, separate from the cellular genes in the nucleus. Strabismus is associated with deletions or point mutations of mitochondrial DNA, as tissues with a high metabolic demand such as the retina, heart, and skeletal muscle are commonly affected.
As per mitochondria’s connection to cancerous developments, mitochondria’s function is affected by the internal and external roles of Nuclear Factor kappa B, which maintains mitochondrial structure or activity. Nuclear factor kappa B is also an ancient protein transcription factor and considered a regulator of innate immunity, and its signaling pathway links pathogenic signals and cellular danger signals organizing cellular resistance to invading pathogens. In the other words, when functioning properly, mitochondria is the farmer who ensures the fox stays out of the chicken coop, but if it is malfunctioning, the strike of fatal disease is often forthcoming.
In sum, what was very, very inward and personal, has become very societal and scientific, leading to new and surprising connections, relationships, and understandings of self and truths about the human body and its functioning in larger contexts, and to what mysteriously lies on the edge of human understanding.
Perhaps, above all, this passionate intellectual quest, about that which I had always known something, but had not been able to fully think about, would not have been possible without the tools of language and the creative writing process.
- The Novina Dana Farber Lab also notes that a major challenge for genome editing is that such “editors” have many unintended off-target effects including promiscuous binding of Cas9 to loci with high sequence similarity to the intended target site and unspecific enzymatic activity of editors leading to unwanted modifications. The lab recently described a novel tool for targeted DNA methylation by tethering a “split-fusion” methyltransferase to an endonuclease-deficient mutant Cas9. Its split-fusion approach minimizes off-target effects by ensuring that enzyme activity is specifically reconstituted at the targeted locus. ↵
- Niakan, Kathy. “Researchers call for greater awareness of unintended consequences of CRISPR gene editing,” Cambridge University Research, April 9, 2021. ↵
- Zimmer, Carl. She Has Her Mother’s Laugh, 2018. ↵
- Greer, HMS assistant professor of pediatrics at Boston Children’s Hospital. ↵
- Crick has aptly noted that the flow of information from DNA to RNA to protein is fundamental to all planetary life forms. Nurse, Paul. What is Life?, 135. ↵
- Through a series of modeling and experiments at the lab bench, researchers have confirmed their hypothesis that the effect of RNA on transcription is due to RNAs molecules’ highly negative charge. It was predicted that initial low levels of RNA enhance, and subsequent higher levels dissolve, condensates formed by transcriptional proteins. Because the charge is carried by the RNAs’ phosphate backbone, the effective charge of a given RNA molecule is directly proportional to its length, insuring its accuracy. ↵
- LncRNA plays a vital role in cells, tissues and organs, including: physiologic cellular processes, genomic imprinting, inactivation of chromosome X, maintenance of pluripotency, formation of different organs via changes in chromatin, transcription, and translation. BBA, Gene Regulatory Mechanisms, 2020. ↵
- Chang, Stanford CheM-H, 2019. ↵
- While traditional biochemical methods to define RNA protein interactions are limited by low throughput and are biased towards identifying the most abundant RNA-protein interactions, the Novina Lab has developed technologies integrated into a platform that can efficiently define lncRNA function by systematically identifying their associated proteins. For example, non-coding RNA binding to transcription factors or histone-modifying enzymes implicates those RNAs in transcription or locus control, respectively. Moreover, by screening fragments of a disease-relevant lncRNAs against the human proteome, their platform can define the RNA sequence and structural determinant that specify protein interaction. ↵
- During the heyday of single-celled life about 2 billion years ago, the forerunners of mitochondria were bacteria that found a niche inside larger cells, providing them with energy. This symbiosis was so beneficial that it likely powered the evolution of multicellular organisms. As a relic of their bacterial origins, mitochondria still carry their own small genome, separate from the cellular genes in the nucleus,"Social mitochondria, whispering between cells," Quanta Magazine, 7, 2021. ↵
- Bola De Paepe, Steve Lefever, and Pieter Mestdagh. “How long noncoding RNAs enforce their will on mitochondrial activity: regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer,” Current Genetics volume 64, 2018, 163–172. ↵
- As per stress, as noted by Sapolsky at Stanford University, human beings, unlike other species, have difficulty turning off the fight or flight response. ↵
- Blumenthal-Perry et al. “Retrograde signaling by a mtDNA-encoded non-coding RNA preserves mitochondrial bioenergetics, Communications Biology, volume 3, article number 626, October 30, 2020. ↵
- As per the issue of ROS, we now have the complete atomic structure of MnSOD via neutron diffraction of crystals and how it guards against oxidative stress caused by an imbalance between production and accumulation of oxygen-reactive species. This is done by the manganese superoxide dismutase/antioxidant enzyme, which protects the mitochondrion by a mechanism involving electron and proton transfers to lower ROS levels in mitochondria (University of Nebraska Medical Center and Department of Energy, Oak Ridge National Laboratory, April 6, 2021). ↵
- A non-exhaustive list of lncRNAS, including ANRIL, AS cmt-RNA, H19, HOTAIR, LincRNA-p21, MALAT1, RMRP, SAMMSON, and VL30, which have emerged as potent regulators of mitochondrial metabolism, in Cell, June 4, 2020. ↵
- Yijing Zhao, Lemeng Sun, Rachel R. Wang, Ji-Fan Hu, Jiuwei Cui. “The effects of mitochondria-associated long noncoding RNAs in cancer mitochondria: new players in an old arena,”Critical Reviews in Oncology/Hematology, volume 131, November 2018, 76–82. ↵