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Roles of Regulatory RNAs in Biology and Disease

Roles of Regulatory RNAs in Biology and Disease

Abstract The study of RNA has been a developing field over the past few decades. Whereas DNA is the information molecule that codes for everything in an organism, RNA is the vehicle for transformation from encoded information to a direct product. RNA can be coding and non-coding. Coding RNA, or messenger RNA, is the intermediary between DNA and protein synthesis. The non-coding RNA, including ribosomal RNA, transfer RNA, and regulatory RNA, supports the translation of the coded message and regulates the rate product output. Regulatory RNA includes small interfering RNA, microRNAs, PIWI-interacting RNAs, small nucleolar RNAs, small nuclear RNAs, and long noncoding RNAs. Because regulation is necessary for a wide variety of processes, regulatory RNAs are involved in many systems and diseases, like pathway activation and inactivation, organism development and differentiation, DNA repair, phylogeny, cancer, infectious disease, immunological disease, diagnostics, and toxicity. Regulatory RNAs are a huge focus for new research and therapies.
Introduction RNA is critical to a cell’s ability to form proteins as part of the central dogma. RNA is more flexible and unstable compared to DNA; it is often single-stranded or double-stranded, is prone to hydrolyzation thanks to a hydroxyl group on its 2’ carbon, and it can bind to itself, other RNAs, and DNA (7). RNA can either code for proteins or have a its own functionality as an RNA. Types of RNA include coding RNA, also known as messenger RNA, noncoding RNA, also known as ribosomal RNA, transfer RNA, and regulatory RNA. Regulatory RNAs are a subclass of noncoding RNAs that can still influence an organism’s genomic expression and control (5). If an RNA is complementary to another RNA and interferes with its function, it is considered an antisense RNA (1).
Figure 1: The types of RNA found in an organism. RNA can be broken down into coding and non-coding RNA. Coding RNA is referred to as messenger RNA. Non-coding RNA can be broken down into ribosomal RNA, transfer RNA, and regulatory RNA. Regulatory RNAs can be broken down into small interfering RNA, microRNAs, PIWI-interacting RNAs, small nucleolar RNAs, small nuclear RNAs, and long noncoding RNAs.
Types of Regulatory RNAs According to a book written by Mallick and Ghosh, the main types of regulatory discovered so far are small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and long noncoding RNAs (lncRNAs) (5).
siRNA stands for small interfering RNAs, and like the name suggests, can bind to mRNA sequences and inhibit translation through a process called RNA interference, or RNAi (7). In RNA interference, an endonuclease that is part of the RNase III family called Dicer cleaves long double-stranded RNA into siRNA around 20 bases long, which can then combine with other proteins to induce RISCs, or RNA-induced silencing complexes (8).
Dicer is also involved in the generation of microRNAs, or miRNAs; miRNAs interact with messenger RNAs, usually in the 3’ untranslated region, to induce mRNA degradation or translation inhibition (7). miRNAs are a very common type of ncRNA, may regulate up to 30% of an organism’s gene expression, are typically around 22 bases in length, and may be encoded by up to 1% of an organism’s genome (7). Although miRNAs have 22 bases, they have a 5 base long region towards the 5’ region of the miRNA that is directly responsible for the recognition of a 3’ untranslated region (2). miRNAs are made by the following steps: first, the primary miRNA transcript binds to the proteins Drosha and Pasha, which processes them into 70-80 bases long hairpin sections (7) of pre-miRNAs, which then binds to exportin-5 to be carried out of the nucleus (6). Then, Dicer cleaves the pre-miRNA into 22 to 24 nucleotides-long segments of double stranded RNA (6). The double-stranded segments are unwound by RNA helicase (7) and the miRNA is incorporated into the argonaut (AGO) subunit of RISCs and become functional (6). Typically, microRNAs can be found in introns of genes or be expressed independently of other genes (2).
Figure 2: miRNA Synthesis; First, the primary miRNA is cleaved into pre-miRNA by Drosha and Pasha; then Exportin-5 transports the pre-miRNA through the nucleus into the cytoplasm. Next, Dicer cleaves the pre-miRNA into 22-24 nucleotide segments, helicase separates the double-stranded RNA into single-strands, and the miRNA binds to the AGO subunit of the RISC complex and becomes functional.
piRNAs, or PIWI-interacting RNAs, are the most recently discovered subsection of regulatory RNAs and were identified in 2006 (5). piRNAs bind to a subset of AGO proteins called PIWI proteins to specifically silence the expression of transposons in germ cells and protect against mutation (6). However, it also is found in brain and other tissues beyond germ cells, which suggests more functions than protecting offspring from genetic mutation (6).
Small nucleolar RNAs, or snoRNAs, are commonly found in other gene’s introns, are localized to the nucleolus in 60 to 100 base sequences, and are involved in nucleoside modification and cleavage reactions in mRNAs (7). snoRNAs bind to proteins to form complexes called snoRNPs; these complexes are either have a consensus box H/ACA or consensus box C/D, each of which help with pseudouridylylation and 2’-O-methylations, respectively (7).
Small nuclear RNA’s, or snRNAs, are involved in mRNA splicing and assembling the spliceosome (6). snRNAs U1 and U2 recognize the 5’ splice site and branch point, which allows U4, U5, and U6 to adhere to the site, displace U1, and guide the splice (6). snRNAs are typically between 100 and 200 nucleotides long (7).
Long non-coding RNAs, or lncRNAs, are a sort of catch-all grouping for regulatory enzymes that are longer than 200 bases long and are not a part of the aforementioned groups (6). Because of this, there is a lack of a cohesive singular purpose these regulatory RNAs perform, and there have been questions on the biological relevance of these RNAs (5). lncRNA are a upcoming research field for researchers interested in regulatory RNAs.
Most regulatory RNA is noncoding; however that does not mean that coding RNA is incapable of having regulatory functions. Some mRNAs may carry riboswitch sequences within them that can regulate translation by inducing different conformations in the mRNA in response to signals detected by the riboswitch region (4).
Obviously, the field of study regarding RNA regulation of the cell is still rapidly expanding, elucidating the intricacies of the human genome. As such, there may be unknown types of regulatory RNAs that scientists have yet to identify. Regulatory RNAs cover a diverse field of functions which makes them an important aspect to keep current with.
Biological Roles Because there is such a wide variety of regulatory RNAs, there is also a large variety in their respective functions. Examples of regulatory RNA functions can vary from development and differentiation, to epigenetic roles, to RNA modification, to evolution, and to inheritance (6).
The expression of regulatory RNAs may influence when specific genes are expressed or not expressed. For example, the expression of the lncRNA Braveheart triggers the expression of protein meSP1, which helps differentiate cardiovascular tissue during development (2). On the contrary, regulatory RNAs can inhibit expression of particular genes as well. The first microRNA, lin-4, was discovered in 1993 while studying C. elegans and inhibited the lin-14 gene by being antisense to several sites in the 3’ untranslated region of the lin-14 gene; the LIN-14 protein is only expressed in the first larval phase, and otherwise is kept unexpressed and degraded as an mRNA by the miRNA lin-4 (2).
Another function regulatory RNA can perform is the regulation of DNA repair mechanisms. microRNAs help regulate the base excision repair pathway through the expression of miRNA-16, miRNA-34c, miRNA-199a which regulates UNG2, an uracil-DNA glycosylase (9). miRNA-155, when overexpressed, inhibits hMSH2, hMSH6, and hMLH1, repressing mismatch repair pathways, which can help lead to an increase in mutagenesis (9). In nucleotide excision repair pathways, inhibition of RAD23B can occur with the overexpression of miRNA-373, leading to an increase in accumulation of UV induced damage (9).
Regulatory RNAs have significant roles in both prokaryotic and eukaryotic organisms. In S. aureus, antisense microRNAs bind to and regulate proteins controlling virulence (4). In strains with more antisense RNAs, there was a lower virulence (4). In bacteria, ncRNAs may regulate growth in response to stress as well; researchers have identified a set of ncRNAs, RsaA through K, which appear to help modulate metabolism and growth in S. aureus strains. Archaea have had ncRNA identified as well as homologous genes in the argonaut family, suggesting functional similarities to eukaryotes (10).
Regulatory RNAs have many functions during the development of an organism. Zebrafish generate miRNA-430 to bind to maternal mRNA immediately after the mid-blastula transition; miRNA-430 is nonspecific and recognizes about 40% of the maternal mRNA 3’ untranslated regions (2). Regulatory RNAs can influence overall cell processes, as seen with miRNA-1 and miRNA-133, which can influence the rate of cell division in cardiac muscle (2).
Regulatory RNA sequences can also provide phylogenetic evidence for evolution. Like conserved genes such as BMP Hox, Pax, and Wnt family genes, certain miRNAs are conserved across organisms as well, like miRNA-12 found in the gut cells of animals and miRNA-124 found in the central nervous systems of animals with nervous system differentiation (2).
Regulatory RNAs may regulate inflammation by inhibiting and activating certain pathways. miRNA-146a targets IRAK-1 and TRAF6 to affect the TLR signaling pathway (9). Following treatment with lipopolysaccharide, lung cells began to express miRNA-214, miRNA-21, miRNA-223, and miRNA-224 (9). In addition to influencing inflammation, regulatory RNAs are also responsible for controlling transcription factors responsible for T and B cell differentiation; for example, miRNA-150 targets c-myb in mature B cells but not immature B cells (9). siRNAs can be recognized by toll-like receptors like TLR3, TLR7, and TLR8 (11).
Roles in Disease Overexpression of regulatory RNAs can result in disorders and disease. For example, when there is trisomy of chromosomes, such as with Down’s syndrome, there can be an increase in the expression of the microRNAs on the chromosome. As seen with the overexpression of miRNA-155, the increase in microRNA expression leads to the suppression of transcription factors necessary for neural and cardiac development (2). On the contrary, inhibition of the expression of regulatory RNAs seen in different diseases and disorders can pose a problem as well. In multiple myeloma cell lines, the expression of miRNA-342 and miRNA-363 is significantly lower than in healthy cell lines, and the protein it inhibits, Runx2, which promotes bone metastasis, is expressed at much higher levels (3). When the cells were transfected with miRNA-342 and miRNA-363 mimics, the expression of Runx2 was reduced (3). Targeting regulatory RNAs may be a tactical strategy for discovering new treatment pathways, especially in the area of cancer.
Researchers have been looking into the possibility or using miRNAs to differentiate tumor cells to remove some of their stem cell qualities (2). By increasing the expression of miRNAs that are considered tumor suppressors or methylation regulators, they hope to make the tumor cells have differentiated phenotypes so they are easier to treat (2). In addition, miRNAs can act directly as oncogenes and tumor suppressor genes. A cluster of 6 miRNAs, miRNA-17-92, is the direct target of oncogenic c-MYC transcription factors; additionally, it has been shown to be upregulated in lung, colon, pancreatic, prostate and breast cancers as well as lymphomas (9). When tumor-suppressive miRNAs are dysregulated, cancerous tissue can grow; when let7 is under expressed in cancer tissues, its target, the oncogene RAS, is able to be overexpressed (9).
Regulatory RNAs can be used as a diagnostic tool for disease. Studies have shown that specific changes in miRNA expression can be used to indicate the presence or absence of drug-induced liver toxicity (9). In rats, acetaminophen toxicity was shown to decrease miRNA-298 and miRNA-370 expression, both of which target oxidative-stress related enzymes, as soon as six hours after treatment with acetaminophen (9). It may be possible to use certain levels of miRNAs in the blood serum or urine associated with specific diseases as biomarkers; this is advantageous because miRNA meets the qualifications for a biomarker: high tissue specificity, availability, stability, detectable early, easy to measure, and associated with a known mechanism (9). miRNAs are highly tissue specific; miRNA-124 expression is quantifiable in brain tissues 270 to 24000-fold higher than in other tissues (9). Diseases can cause the release of miRNAs into the blood at different stages (9). miRNAs have been shown to be resistant to changes in pH and temperature, and they are easy to quantify using polymerase chain reaction (9).
Understanding regulatory RNAs can also be beneficial for public health and containing infections. Given that the binding of microRNAs to bacteria can affect the virulence of the bacteria, it may be possible to target the virulence of an infectious species to mitigate the harmful effects of the strain instead of trying to kill the pathogens (4).
Many immunological disorders can be influenced by regulatory RNAs as well. Disorders like rheumatoid arthritis, psoriasis, asthma, idiopathic pulmonary fibrosis, and inflammatory bowel disease have all been associated with irregularly upregulated or downregulated miRNAs. For example, in psoriasis, upregulation of miRNA-146a targets IRAK-1 and TRAF6 to stimulate the TNF-? pathway, which increases inflammation causing psoriasis (9).
Conclusion Regulatory RNAs are a major portion of genomic regulation. Much of the research being done focuses on the impacts of microRNAs on different functions, however, siRNAs, piRNAs, snoRNAs, snRNAs, and lncRNAs all play important roles in the day to day function of cells in the body. Regulatory RNAs are involved with organism development, differentiation, DNA repair, bacterial virulence, phylogeny, cell processes, and pathway inhibition and activation. Since there is such a diverse spread of functions, regulatory RNAs have a wide variety of implication in disease. miRNAs play an increasingly large role in regulating cancer in addition to affecting other diseases like immunological disorders. It can play a role in public health, diagnostics, and pathway regulation. Studying regulatory RNAs is integral to the future of scientific study and treatment, and it is important to keep current with the plethora of ongoing research being produced.
Bibliography Eguchi, Y., Itoh, T.,

Antibiotic Resistance in Livestock

Antibiotic Resistance in Livestock

Abstract
For the past 70 years, antibiotics have been used in the feed of commercially farmed animals. This was done to prevent diseases and infections from spreading between the animals. The use of antibiotics on livestock also promotes growth, and this all helps meat prices stay affordable. The problem with using antibiotics is that their overuse has led to antibiotic-resistant bacteria that are extremely difficult to treat and can be deadly. Antibiotic-resistant bacteria are easily spread throughout the environment when animal waste is used as fertilizer. People can also contract antibiotic-resistant bacteria from eating contaminated meats. In this review, the reasons why antibiotics are used in livestock will be discussed. This review will also discuss the dangers of antibiotic-resistant bacteria and possible methods on how antibiotic resistance will be reduced.
Introduction
Without the use of antibiotics farms, there would be an increase in the number of animal deaths, and the growth rate would slow down. The agriculture industry has become dependent on antibiotics to keep up with the demand for food. But this does not justify the overuse of antibiotics. Antibiotic resistance is one of the most pressing issues that global health organizations are facing. The overuse of antibiotics in the livestock industry has contributed a great deal to this problem. Every passing year resistant strains grow stronger, and infections that used to be easily treated with simple antibiotics have become deadly and untreatable. The struggle to control this pandemic has been going on for decades with little to no results. This paper will discuss how and why antibiotics are used in animal feed. This will also discuss how the overuse of antibiotics has led to the increase of antibiotic resistant bacteria, and the dangers that it possess to people.
Why Antibiotics Are Used for Raising Livestock
Antibiotics have been used in livestock since the 1950s. Commercial farm animals are usually kept in crowded, stressful environments that make them susceptible to contract numerous diseases and infections that could lead to the loss of many animals [1]. Typically, antibiotics are used in livestock feed to prevent diseases and infections that would lead to death in the live stocks. Another reason antibiotics are used for raising livestock is that it promotes growth in the animals that are treated [2]. It has also increased the feed conversion ratio because some medicines can illuminate certain types of bacteria in the intestinal tract. This allows the animals to produce more muscle and fat quicker than untreated livestock. For example, the addition of antibiotics in pig feed has increased the average weight gain 3.3-8.8% [3]. With a growing world population, farmers have to resort to methods such as using antibiotics to meet the ever-growing demand. Since medicines improve the survival, and growth rate of livestock allows meats and animal products to be more affordable to consumers. Without the addition of antibiotics to livestock feed, there would be a higher percentage of food animals that would die from diseases and infections. For example, since Europe has banned the use of antibiotics in livestock feed, Denmark which is the world leading exporter of pork has seen the numbers of farmed pig’s death increase by 25 percent. They have also seen an increase in the number of newborn pigs dying from illnesses with 21% out of the 32 million piglets not making it [3].
Antibiotic Resistance dangers
More than 70% of all the antibiotics produced in the U.S. is used for livestock. This overuse has led to bacteria becoming more resistant to antibiotics [4]. This overuse is a result of natural selection, random mutations, horizontal transfer of immune genes, and the inheritance of genes. A chronic undertreatment in food animal farming has promoted the emergence of immune genes. This is a consequence of low levels of antibiotics added to the feed of livestock. Animals are also given more antibiotics when they show signs of illness. For example, dairy cows are usually given penicillin if they show symptoms of mastitis. Pigs and poultry are given tetracycline when they show symptoms of raspatory diseases. Fish farms and orchards use various forms of tetracyclines and other antibiotics when there is a suspected fungal or bacterial outbreak present [5]. As the antibiotics kill off the bacteria that are sensitive, a few resistant cells remain. These cells then reproduce and horizontally transfer immune genes to surrounding bacteria. As a result, antibiotic resistance has increased due to the overuse in farms along with the overuse in people.
Antibiotic-resistant bacteria have the potential to be one of the most dangerous infections. The number of patients that have been hospitalized in the U.S. with infections from antibiotic-resistant bacteria has increased 359% from 1997 to 2006 with, 37,005 cases in 1997, and 169,985 in 2006 [6].

Figure 1. Methods of Contamination. This diagram produced by the CDC show how antibiotic resistant bacteria can spread and infect people and animals [7].
The latest reports conclude that every year in the U.S. there are over 2 million people who contract antibiotic-resistant bacteria with 23,000 deaths on average [8]. There are many ways to contract an antibiotic-resistant bacterium. A common way is by consuming undercooked animal meats that are contaminated with animal waste. Since most of the resistant microbes live in the intestines when the animals defecate, they are essentially infecting their surroundings. For example, cow feces contain millions of microorganisms including antibiotic-resistant bacteria that have the potential to affect humans and other species when used in fertilizers. Researchers from Yale where able to extract 80 genes from bacteria from within the cow’s intestines that are responsible for making the bacteria antibiotic resistant. This concluded that cow manure used for fertilization is spreading antibiotic resistant gene through the environment that will eventually infect soil, crops, and groundwater before making its way to humans. Figure 1. Illustrates how antibiotic resistant bacteria spreads throughout the environment through people and animals [9]. Antibiotic resistant bacteria spread fast just like any other bacteria. For example once one animal in a commercial farm gets infected with a antibiotics resistant microbes it can spell disaster for all the surrounding animals costing farmers millions every year.
Salmonella and Campylobacter are the two most common bacteria that people tend to contract, and resistant strains for both have been on the rise. For example, Newport 9 is an antibiotic-resistant strain of Salmonella that has been increasing in frequency. This strain is called Newport 9 because it is resistant to nine antibiotics including ceftriaxone which is known to kill most bacteria. The Newport 9 strain originated from Salmonella typhimurium which is found in livestock. Treatment is difficult for antibiotic-resistant bacteria due to the lack of pharmaceutical companies producing new medications to help combat the growing problem of antibiotic-resistant microbes [4].
Efforts to Reduce the Impact of Antibiotic-Resistant Microbes
Efforts have been made in the past to attempt to battle the rising numbers of antibiotic-resistant bacteria. The first attempt was formed in 1978 The FDA tried to ban the use of antibiotics in some agriculture practices, and the pharmaceutical companies resisted these regulations claiming there wasn’t sufficient evidence that the use of antibiotics in livestock is causing antibiotic-resistant bacteria. Since then the evidence and knowledge of bacterial resistance have grown. In 2000 the FDA, CDC, and USDA sighed a draft to coordinate an 11-step federal response to the growing issues of antibiotic resistance [5]. In 2016 the O’Neil commission published a list of recommendations to reduce the use of antibiotics worldwide. The goal was to put in place a global organization that would educate the public about the dangers of antibiotic-resistant bacteria [10]. Its purpose was to also reduce the unnecessary use of antibiotics in livestock farming in hopes of reducing the number of resistant bacteria that make it out into the environment.
CRISPR-Cas9 is a genome-editing tool that has massive potential for reducing the impact of antibiotic-resistant bacteria. CRISPR-Cas9 can be used as an artificial immune system designed to protect bacteria from foreign nucleic acids that would cause the bacteria cell to become resistant to antibiotics. CRISPR-Cas9 is also capable of removing specific genes that allow bacteria cells to be resistant to antibiotics. This is possible because two important molecules which are Cas9, and guide RNA (gRNA). The Cas9 enzyme performs like a pair of scissors and cuts the two strands of DNA at the desired position where the genome with be taken out or placed. The guide RNA is responsible for finding the target sequence location and binding the Cas9 enzyme. Even though there is a lot of potential for CRISPR-Cas9 to battle antibiotic resistance in bacteria, CRISPR-Cas9 still needs to be studied more intensively. This is because CRISPR-Cas9 has only been used on cloned E. coli cells, and in the real-world application, it would be much more difficult to carry out.
Conclusion

Despite advancements in understanding of antibiotic resistance it will be an issue that will be dealt with for a while. This review has gone over the reasons why antibiotics are used, and the dangers it brings to the public. Many organizations are raising awareness to the issue. Maybe one day CRISPER-Cas9 can help reverse the resistance in microbes but until then educating people and farmers on how to responsibly use antibiotics will be the most effective step in battling antibiotic resistance.
Works Cited

[1]. Hao, Haihong et al. “Benefits and risks of antimicrobial use in food-producing animals” Frontiers in microbiology vol. 5 288. 12 Jun. 2014, doi:10.3389/fmicb.2014.00288
[2]. Landers, Timothy F et al. “A review of antibiotic use in food animals: perspective, policy, and potential” Public health reports (Washington, D.C. : 1974) vol. 127,1 (2012): 4-22.
[3]. Cox, Louis Anthony, et al. “Routine Use of Antibiotics in Food Animals Increases Protein Production and Reduces Prices [with Reply].” Clinical Infectious Diseases, vol. 42, no. 7, 2006, pp. 1053–1054. JSTOR, JSTOR, www.jstor.org/stable/4463771.
[4]. Schmidt, Charles W. “Antibiotic Resistance in Livestock: More at Stake Than Steak.” Environmental Health Perspectives, vol. 110, no. 7, 2002, pp. A396–A402., www.jstor.org/stable/3455499.
[5]. MLOT, CHRISTINE. “Antidotes for Antibiotic Use on the Farm: As Pathogen Resistance Spreads, Researchers Look for Alternatives to the Heavy Use of Antibiotics in Food Production.” BioScience, vol. 50, no. 11, 2000, pp. 955–960. JSTOR, JSTOR, www.jstor.org/stable/10.1641/0006-3568(2000)050[0955:afauot]2.0.co;2.
[6]. Mainous, Arch G., et al. “Trends in Hospitalizations with Antibiotic-Resistant Infections: U.S., 1997-2006.” Public Health Reports (1974-), vol. 126, no. 3, 2011, pp. 354–360. JSTOR, JSTOR, www.jstor.org/stable/41639372
[7]. “National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 26 Jan. 2018, www.cdc.gov/narms/faq.html.
[8]. Ronald R Marquardt, Suzhen Li; Antimicrobial resistance in livestock: advances and alternatives to antibiotics, Animal Frontiers, Volume 8, Issue 2, 7 June 2018, Pages 30–37, https://doi.org/10.1093/af/vfy001
[9]. Faden, Mike. “Antibiotic Resistance in Dairy Cow Manure.” Frontiers in Ecology and the Environment, vol. 12, no. 5, 2014, pp. 263–263. JSTOR, JSTOR, www.jstor.org/stable/43187786.
[10]. O’Neil, J. 2016. Tackling drug-resistant infections globally: final report and recommendations. The review on antimicrobial resistance. https://amr-review.org/publications (accessed 2018). 84pp.
[11]. Pursey, Elizabeth et al. “CRISPR-Cas antimicrobials: Challenges and future prospects” PLoS pathogens vol. 14,6 e1006990. 14 Jun. 2018, doi:10.1371/journal.ppat.1006990

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